What are the most common in vitro drug-drug interaction study gaps?

Conducting in vitro DDI studies provides early insights into possible adverse interactions that could affect the drug’s safety and efficacy. Identifying and understanding these interactions during the nonclinical phase helps to  

• guide clinical strategy,
• support regulatory submissions, and
• ensure that the new drug can be safely co-administered with other medications.  

Moreover, these studies help optimize dosage regimens and mitigate risks associated with polypharmacy, thereby contributing to overall patient safety and the success of the drug development process.

In 2024, the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) published the final version of the harmonized guideline for drug interaction studies (M12), which prompted a review of all available in vitro (and in vivo) DDI data for multiple new and ongoing drug development programs.  Below are some commonly flagged gaps in these DDI data packages. 

See our blog on ICH M12 to learn more.

The absence of microsomal binding data  

Per ICH M12, you can exclude the risk (“basic method”) for reversible CYP enzyme inhibition if:

Where, K_i,u is the unbound inhibition constant, requiring that the K_i (or IC₅₀) values measured in the in vitro CYP inhibition assay be corrected for loss of drug bound to the microsomal matrix present in the assay (eg, the actual unbound concentration of the drug in the assay). Without this correction, the risk of DDI could be underestimated. Note that you can measure microsomal binding experimentally or predict it using in silico methods.

A similar approach may also apply to in vitro assays using hepatocytes, such as CYP induction, where understanding the actual drug concentration in the assay medium is important.

Figure 1: Theoretical impact of microsomal binding on CYP IC₅₀ values. In this example unbound fraction in microsomes is 0.33. Unbound test article assay concentrations (IC₅₀ = 2 µM; blue dashed line) versus nominal concentrations (IC₅₀ = 6 µM; black line).

The absence of blood partition data

Per ICH M12, the risk for OATP1B1 and OATP1B3 uptake transporter inhibition is determined based on the estimated unbound maximum plasma concentration of the inhibitor at the liver inlet (for oral drugs). This estimation includes “RB” or the blood-to-plasma concentration ratio.

Figure 2. Schematic of a blood-to-plasma partition assay. Source: Mamada, H., Iwamoto, K., Nomura, Y. et al. Predicting blood-to-plasma concentration ratios of drugs from chemical structures and volumes of distribution in humans. Mol Divers 25, 1261–1270 (2021).

A need to refine the DDI risk based on updated cut-off values in the ICH M12 guideline

ICH M12 updated some cut-off values for basic DDI risk assessment compared to previous guidances. Will a negative DDI risk remain so, or be considered positive or inconclusive using the new cut-off values? Will your planned clinical DDI study no longer be required, as your assumed positive in vitro signal is now negative?

Inadequate in vitro assay conditions

• Two appendices of ICH M12 focus on the technical aspects for in vitro evaluation of metabolism and transporter-based DDIs. In short, the in vitro systems should be robust and reproducible. Common gaps leading to potential review issues by regulatory agencies include not confirming drug solubility in assay media, lack of cytotoxicity determination in cell-based assays, inadequate recoveries, stability, and non-specific binding assessments, or failing to correct for these factors.
• Other deficiencies may include use of an inadequate concentration range or lack of sensitivity of positive controls (especially in induction assays), as specified in the guidelines for the various in vitro

Incomplete characterization of major circulating metabolites in humans

• Metabolites can affect the DDI potential of the drug, despite their polarity or being a result of Phase 2 metabolism (e.g., a conjugation reaction). In vitro DDI characterization of a major (at least 10% of drug-related material and ≥25% of parent AUC) human circulating metabolite may be necessary as a substrate if significantly contributing to efficacy, and/or as an inhibitor based on plasma exposure, regardless of the metabolite potency.
• Identification of “new” in vivo major metabolite(s) in human (following the human AME study, for example) may also require additional in vitro phenotyping to identify the metabolic enzyme(s) involved.

In conclusion, in vitro DDI characterization is continuous throughout the life cycle of a small molecule drug development program. Tailor these studies for each new chemical entity. Design them with high data quality that meets the current regulatory guidance(s).

Our Drug Interaction Science Innovation Engine experts can help you design, review, and interpret in vitro DDI studies.