The publication of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) M12 guidance in May 2024 marks a milestone that introduces a unified science-based foundation for the design and interpretation of drug interaction (DDI) studies, addressing divergence among regional requirements from the US Food and Drug Administration (FDA), European Medicines Agency (EMA) and Pharmaceuticals and Medical Devices Agency (PMDA). This regulatory convergence is driven by the recognition that human hepatocytes are not only supportive data but are the central drivers of clinical trial strategy, enabling integrated evaluation of pharmacokinetic interactions mediated through metabolic enzymes and transporters using neutral terminology.
Standardization of In Vitro Study Design
The central priority of the guidance is to harmonize the design of in vitro studies to reduce interlaboratory variability and facilitate data reproducibility through explicit procedural requirements for validated assays. To ensure data integrity, ICH M12 describes the critical quality attributes: the mass balance in the test should be at least 80% to rule out underestimation of the DDI potential due to nonspecific binding, and in the enzyme induction study, the threshold of viable cells should be not less than 70%, leading to an adequate physiological relevance of the mRNA changes and avoiding confounding the experiment with cytotoxicity.
Comprehensive In Vitro Assessments
Primary human hepatocytes (PHH) are regarded as the ‘gold standard’ due to the full complement of phase I and II enzymes, as well as endogenous transporters, enabling the assessment of complex mechanisms not accessible to subcellular fractions (HLM). Assessment of precipitant potential involves calculating reversible inhibition using static models and specific thresholds for intestinal inhibition of CYP3A, and a critical update of the scaling factor for time-dependent inhibition (TDI) from 50 x Cmax,u to 5 x Cmax,u, therebysignificantly reducing the rate of false-positive findings. Furthermore, the guideline necessitates an assessment of the inhibitory potential of metabolites according to the 10%/25% rule (if the AUC of the metabolite is > 25% of the AUC of the parent drug or ≥ 10% of the total substance in systemic circulation) and a thorough evaluation of a panel of transporters (P-gp, BCRP, OATP1B1/3, OAT1/3, OCT2, MATE1/2-K) following the unified criterion Iu, max/IC50 ≥ 0.1.
Risk-Based Approach to DDI Assessment
The ICH M12 risk-based approach includes prioritization of studies based on pharmacology and anticipated co-medications, with physiologically based pharmacokinetic (PBPK) modeling serving as a cornerstone for in vivo quantitative predictions. Experimental quantitative modelling, as part of systems pharmacology and toxicology, offers opportunities to enhance our understanding of biological, pharmacological, and toxicological processes via integration of the pharmacokinetic and the pharmacodynamic (PK/PD) information streams with other data sources. Utilization of PBPK models provides an opportunity to seamlessly combine information on fraction metabolized (fm) and inhibition parameters to more accurately predict the exposure of “victims”, which allows for dose adjustments that are substantiated or even for dismissing the need for conducting clinical trials based on robust in vitro data obtained from 3-D spheroids or tricultures that can maintain stable activity for weeks.
No Requirement for Animal Validation
The ICH M12 guidance codifies the abandonment of validation of induction and inhibition potential in animals, with the emphasis on new approach methodologies (NAMs), including human organoids and microphysiological systems. This is aligned with global trends in the reduction of animal use (3Rs) and the FDA Modernization Act 2.0, as human cell models enable direct observation of human-specific bioactivation and metabolism pathways, which are often not possible to sufficiently model in other biological species due to substantial interspecies differences in the expression of CYP-enzymes and transporters.
LC/MS-MS and PCR in Induction Studies
The ICH M12 methodology for enzyme induction assessment is based on the use of in-adhesion hepatocyte culture, where quantitative PCR (qPCR) for CYP1A2, 2B6, and 3A4 mRNA levels is the primary assay. However, LC-MS/MS for functional enzyme activity remains an indispensable verification method in situations where mRNA determination is not feasible or when discordance in tissue-specific induction is detected.
Alignment with EMA and FDA Guidelines
Harmonization at Step 4 of the ICH M12 enables the progression away from fragmented regional guidances, such as the FDA and EMA, toward a single global standard for integrating experimental thresholds for recommended substrates and probes and mathematical models of risk evaluation. Implementation of the guidance by regulatory bodies (FDA from 08/2024, EMA from 11/2024), offers developers the opportunity to generate a single data package for the global market applying harmonized methods for assessment of reaction phenotyping (enzyme mapping), where identification of metabolic pathways contributing >25% to total elimination can now be performed utilizing a single substantiated approach without potential confirmation bias from a second method, as they were advised in the earlier FDA guidances.
At Preci, we provide state-of-the-art in vitro models that align with the latest ICH M12 guidelines, ensuring accurate and reliable DDI assessments. Contact us today to learn more about our innovative solutions and how we can support your drug development efforts.
Written by Yulia Motina