High-Throughput Analysis of Cytochrome P450 Inhibition in Intact Human Hepatocytes

Importance of the Topic

Efficient in vitro assessment of cytochrome P450 (CYP) inhibition in human hepatocytes is essential to predict potential drug–drug interactions early in drug discovery. This reduces late-stage failures and guides safe dosing strategies.

Objectives and Overview of the Study

This study aimed to develop and validate an ultrafast workflow using the Agilent RapidFire High-Throughput Mass Spectrometry System coupled to an Agilent 6490 Triple Quadrupole Mass Spectrometer. Six key liver CYP isoforms were profiled using probe substrates and 15 known inhibitors across seven concentrations to generate dose–response curves and determine IC50 values.

Methodology and Used Instrumentation

A rapid 13-second per sample analysis was achieved on:

• Agilent RapidFire 365 high-throughput sampling system
• Agilent 6490 Triple Quadrupole mass spectrometer
• Agilent MassHunter Qualitative Analysis Software B.05.00
• Agilent MassHunter Quantitative Analysis Software B.05.00

Cryopreserved human hepatocytes from ten donors were thawed, pooled, and incubated with specific probe substrates (e.g., tacrine for CYP1A2, midazolam for CYP3A4). Inhibitors were tested in triplicate at final concentrations from 0 to 200 µM. After 60 min incubation, reactions were quenched with acetonitrile containing internal standards.

Main Results and Discussion

Ultrafast analysis delivered over 275 samples per hour, with isoform-selective and dose-dependent inhibition observed consistent with literature values. Key findings include:

• Robust IC50 determination for each inhibitor–isoform pair, e.g., ketoconazole IC50 < 0.27 µM for CYP3A4, quinidine IC50 < 0.27 µM for CYP2D6.
• Exceptions such as lack of CYP2C8 inhibition by gemfibrozil, suggesting a requirement for pre-incubation to allow formation of active glucuronide.
• Minimal inhibition of CYP2C9 and CYP2D6 by 1-aminobenzotriazole, highlighting selectivity differences.

Benefits and Practical Applications of the Method

The ultrafast workflow offers significant advantages:

• High throughput reduces bottlenecks in ADME screening.
• Intact hepatocyte model preserves transporters and cofactor environment, improving physiological relevance.
• Rapid IC50 profiling supports early decision-making in lead optimization.

Future Trends and Potential Applications

Further developments may include multiplexed assays to simultaneously assess multiple metabolic pathways, integration with automated compound logistics, and adaptation to other in vitro ADME endpoints such as transporter inhibition and metabolite profiling.

Conclusion

The combination of Agilent RapidFire sampling and triple quadrupole MS enables reliable, high-throughput CYP inhibition screening in human hepatocytes with analysis times below 13 seconds per sample. This approach enhances early detection of drug–drug interaction risks and accelerates the drug development pipeline.

References

1. Miller VP. High-Throughput In Vitro ADME Analysis with Agilent RapidFire/MS Systems: Cytochrome P450 Inhibition. Agilent Technologies Application Note 2011;5990-9184EN.
2. Danker K, Schlicht KE. Optimization of Ultrafast CYP3A4 Inhibition and Multiplexed CYP Inhibition Assays Using the RapidFire High-Throughput Mass Spectrometry System. Agilent Technologies Application Note 2013;5991-3693EN.
3. Miller VP. SPE-MS analysis of absorption, distribution, metabolism and excretion assays: a tool to increase throughput and streamline workflow. Bioanalysis. 2012;4(9):1111–1121.
4. Li AP. Human hepatocytes: isolation, cryopreservation and applications in drug development. Chem Biol Interact. 2007;168(1):16–29.