CYP Inhibition | Kinact/KI

About the Assay

The CYP3A4 (Midazolam) k_inact/K_I assay is designed to quantify the time-dependent inhibition (TDI) potential of compounds against the major drug-metabolising enzyme CYP3A4 using human liver microsomes. This assay evaluates the irreversible or quasi-irreversible inhibition that occurs when compounds form metabolite–enzyme complexes, ultimately affecting drug disposition and potential drug–drug interactions. Midazolam, an FDA-recommended probe substrate for CYP3A4 inhibition studies, is used to monitor enzyme activity, while LC–MS/MS detection ensures high analytical sensitivity and specificity. The approach allows precise determination of kinact, the maximal rate of enzyme inactivation, and K_I, the inhibitor concentration at half-maximal inactivation rate. Together these parameters produce the k_inact/K_I ratio, an important metric for ranking irreversible inhibitors and assessing clinical relevance. Human liver microsomes, pooled across mixed gender donors, ensure biological relevance and translational value. This assay supports drug discovery teams by revealing potential CYP3A4 liabilities early, enabling optimisation of compound design before progressing to more costly studies. As CYP3A4 is responsible for metabolism of over half of marketed drugs, the ability to identify inhibitory liabilities early enhances safety assessment and reduces the risk of late-stage clinical failure.

Protocol Summary

The validation followed a structured process beginning with preparation of test compounds in DMSO and serial dilution to generate a suitable concentration range. Diluted solutions were introduced into pre-incubation plates, after which human liver microsomes were thawed, diluted and added. After an initial equilibration period, NADPH was added to initiate pre-incubation, with staggered timing ensuring consistent termination of all reactions. In parallel, Midazolam substrate solutions were prepared and added to a separate incubation plate. At the conclusion of each pre-incubation time point, a small aliquot was transferred to the incubation plate, initiating the secondary incubation and allowing residual CYP3A4 activity to be quantified. Reactions were stopped by addition to cold acetonitrile, promoting protein precipitation. Following centrifugation, supernatants were diluted with internal standard solution and analysed by LC–MS/MS. Activity values were then used to derive Kobs values via linear regression across time points at each inhibitor concentration. These Kobs values were subsequently fitted by non-linear regression to derive k_inact and K_I parameters. Replicate analysis and comparison with literature controls ensured assay robustness and reproducibility.

Validation Results

Validation of the CYP3A4 (Midazolam) k_inact/K_I assay demonstrated robust performance, strong reproducibility, and alignment with established literature values. Mifepristone was used as the positive control to benchmark the system, generating nine independent determinations across the validation exercise. The resulting k_inact, K_I and k_inact/K_I metrics showed high consistency and fell within expected ranges, demonstrating the suitability of the assay for reliable assessment of time-dependent inhibition. The mean k_inact value obtained for Mifepristone was 0.0871 min⁻¹ in line with expectations. K_I was similarly aligned, with a measured mean of 3.17 µM. The resulting k_inact/K_I ratio was 29.0 min⁻¹ mM⁻¹, again comparable to literature. Coefficients of variation across nine assays were low for k_inact (14.2%), K_I (32.7%) and k_inact/K_I (18.7%), demonstrating tight clustering and analytical reproducibility. Together these metrics confirm that the assay reliably quantifies CYP3A4 enzyme inactivation kinetics and is suitable for screening compounds. The strong concordance with literature further supports the scientific validity of the methodology.

¹ Zimmerlin, A., Trunzer, M., & Faller, B. (2011). CYP3A Time Dependent Inhibition Risk Assessment Validated with 400 Reference Drugs. Drug Metabolism and Disposition, 1039-1046.