In Vitro Assessments and Drug‑Drug Interactions: Predicting Clinical DDI Risk and Informing Regulatory Decision-Making Under ICH M12

By Rachel Rozakis, PharmD, Senior Clinical Pharmacologist at Allucent and Connor Slattery, PharmD Candidate, Clinical Pharmacology, Modeling and Simulation Intern at Allucent

Introduction

Nonclinical (in vitro) drug-drug interaction (DDI) studies are conducted to assess DDI risk in therapies early in development, before clinical (in vivo) testing. The U.S. Food and Drug Administration (FDA) recommends evaluating key enzymes and transporters in nonclinical studies. The nonclinical study results inform whether an investigational drug should be evaluated further in accordance with ICH guidance. ICH M12, the harmonized guideline on DDI studies, provides recommendations for interpreting enzyme- and transporter-mediated DDI data, with a primary focus on small molecule drugs. Careful interpretation of nonclinical DDI data is critical for identifying potentially clinically relevant interactions, guiding the next steps in DDI risk assessment, and ultimately supporting the selection of optimal dosing regimens for patients.

ICH M12 Guidance and its Origin

Although regional guidelines for DDI investigations have existed for years, inconsistencies among these guidelines made it difficult to ensure uniform design, conduct, and interpretation of DDI studies. To address this, in 2018 the FDA, along with other regulatory agencies, initiated efforts to harmonize a set of recommendations on DDI evaluations. After various steps including release for public consultation and subsequent refinement, the ICH M12 Guidance was implemented in the United States in August of 2024. ICH M12 focuses on DDIs mediated by metabolic enzymes and transporters and is limited to pharmacokinetic (PK) interactions. In vitro DDI evaluations are described within the guidance in three main sections: metabolism-mediated, transporter-mediated, and metabolite-mediated. Each section includes a description of DDI types along with quantitative methods, where appropriate, for estimating a drug’s potential for clinical DDI risk.

In Vitro DDI Study Basics

Important DDI terms include “object”, which is a drug whose action is altered by another drug (often referred to as a “substrate”), and “precipitant”, which is a drug that causes a change in another drug. Precipitants can act as inhibitors (block enzyme/transporter activity) or inducers (increase expression of enzymes/transporters) of concurrently administered medications. Drug developers can use enzyme or transporter-specific in vitro assays to screen for inhibition or induction-mediated DDIs. Drug-metabolizing enzymes, e.g., cytochrome P450s (CYPs), are responsible for biotransformation such as the conversion of drugs into metabolites, which can also contribute to DDIs. Transporters are located throughout the body and regulate uptake and efflux of drugs and endogenous molecules across cell membranes. DDIs involving these enzymes or transporters can have a significant clinical impact on the action of drug. For example:

• If a drug inhibits the CYP3A4 enzyme and a patient is taking a concomitant medication that is a substrate of CYP3A4, increased systemic exposures of the substrate medication may result in toxicity.
• If a drug is a substrate of the CYP1A2 enzyme, and a patient is taking a concomitant medication that is an inducer of CYP1A2, a patient could experience subtherapeutic levels of the CYP1A2 substrate, meaning the drug may have reduced efficacy.
• If a drug induces P-glycoprotein (P-gp), an efflux transporter at the blood-brain-barrier (BBB), and a patient is taking a concomitant CNS drug that is a P-gp substrate, subtherapeutic levels of the CNS drug could result. The CNS medication may therefore have reduced efficacy because it cannot adequately reach its target sites in the brain.

In Table 1, enzymes and transporters involved with drug metabolism and distribution are summarized.

Table 1. Summary of Enzymes and Transporters Relevant to DDI Risk Assessments

ADH = alcohol dehydrogenase, ALDH = aldehyde dehydrogenase, AO = aldehyde oxidase, BCRP = breast cancer resistance protein, BSEP = bile salt export pump, CES = carboxylesterase, CYP = cytochrome P450, GI = gastrointestinal, FMO = flavin-containing monooxygenase, MAO = monoamine oxidase, MATE = multidrug and toxin extrusion protein, MRP2 = multidrug resistance-associated protein 2, NAT = N-acetyltransferase, OATP = organic anion transporting polypeptide, OAT = organic anion transporter, OCT = organic cation transporter, P-gp = P-glycoprotein, SULT = sulfotransferase, UGT = uridinediphosphate-glucuronosyltransferase, and XO = xanthine oxidase.

Metabolic Pathways

Drugs undergo different stages of metabolism referred to as Phase 1 and Phase 2 metabolism. Phase 1 metabolism involves oxidation and reduction reactions primarily mediated by CYP enzymes. Phase 1 metabolism also involves non-CYP enzymes such as alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and monoamine oxidase (MAO). In Phase 2 metabolism, enzymes such as UDP-glucuronosyltransferase (UGT) are responsible for conjugation reactions with drugs or Phase 1 metabolites. Metabolic pathway identification experiments are used to identify the number and structures of metabolites formed from a drug. These studies are conducted early in development as they inform the necessity and design of reaction phenotyping studies.

Reaction Phenotyping

Reaction phenotyping studies identify and quantify which drug‑metabolizing enzymes are responsible for the metabolism of a drug and estimate each enzyme’s contribution to overall elimination. When an enzyme is responsible for ≥25% of the total elimination of a drug, additional clinical characterization is necessary to assess the potential for clinically meaningful DDIs. By identifying metabolic pathways involved in metabolism as well as determining which enzymes are responsible for metabolism, drug developers can better understand how a drug is processed in the body, laying the groundwork for determining what additional DDI studies are needed.

Test Systems

Test systems used for in vitro DDI investigations, including metabolic pathway and reaction phenotyping studies, commonly include human hepatocytes (liver cells) and human liver microsomes (HLMs). Hepatocytes are used because they contain the full set of Phase 1 and Phase 2 drug-metabolizing enzymes. For phenotyping and inhibition studies, it is recommended to use hepatocytes pooled from approximately 5 to 10 donors, whereas induction studies should typically be conducted using hepatocytes from at least 3 individual donors. In contrast, HLMs are a collection of endoplasmic reticulum fragments obtained from liver cells. The endoplasmic reticulum holds many drug-metabolizing enzymes (e.g., CYPs and UGTs) making HLMs another useful test system. When using HLMs for in vitro DDI testing, the guidance recommends pooling HLMs from at least 10 donors. The larger pool helps capture a better representation of expression of the metabolizing enzymes for a population compared to an individual for both hepatocytes and HLMs. Other systems that can be used include liver S9 fraction (supernatant of liver homogenate containing microsomal and cytosolic enzymes) and recombinant systems, which usually express one single enzyme (e.g., a human CYP or UGT enzyme).

Metabolism-Mediated Interactions

A review by Zanger and Schwab reported that the CYP family of enzymes is responsible for biotransformation of 70 to 80% of all clinical medications, necessitating thorough evaluation of potential CYP‑mediated DDIs. A review by Blondet and colleagues reported that UGTs represent the majority of Phase 2 metabolism, accounting for roughly 40% of the Phase 2 conjugation of drugs. However, compared to CYPs, the magnitude of UGT inhibition-mediated DDIs are limited and in vitro methods to evaluate UGT induction are not well established. Therefore, evaluation of DDI risk involving UGT enzymes (as well as other non-CYP enzymes) may not be necessary for some investigational drugs.

Metabolism-Mediated Inhibition

To determine if an investigational drug acts as a precipitant for CYP or UGT inhibition, drug developers must assess the potential for reversible inhibition (RI) or time-dependent inhibition (TDI) across the major CYP enzymes (Table 1). RI is a concentration‑dependent decrease in enzyme activity; when drug levels fall, enzyme activity can fully recover. TDI is an irreversible inhibition of an enzyme and recovery is determined by new enzyme synthesis. UGT inhibition assessment is warranted when direct glucuronidation accounts for the majority of the elimination of the investigational drug. If glucuronidation is not the major elimination pathway, in vitro inhibition assays for UGT inhibition are not needed.

Metabolism-Mediated Induction

To determine if an investigational drug induces CYP enzymes, induction is evaluated in human hepatocytes by quantifying mRNA fold change for CYP3A4, CYP2B6, and CYP1A2. An increase in CYP mRNA may indicate the need for further assessment of induction potential. In induction interactions, a drug binds to protein receptors such as the pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) and causes the increased production of mRNA for more enzymes: CYP3A4, CYP2B6, and CYP1A2, respectively. Drugs that activate PXR and CAR are also likely to induce UGTs; however, the guidance does not have established approaches to evaluate UGT induction.

Transporter-Mediated Interactions

An investigational drug that inhibits or induces a transporter can change the absorption, distribution, and elimination of concomitant medications and potentially cause toxicity or reduced effectiveness. Which effect occurs depends on which transporter is affected and where it is located (e.g, in the gut, liver, kidney, or brain). Drugs also often depend on transporters to reach their intended target at therapeutic concentrations. If a drug is a substrate of one or more transporters, those transporters can significantly influence the PK profile, including bioavailability and elimination. Orally administered investigational drugs that are eliminated through biliary excretion or active renal secretion should undergo in vitro substrate testing for P-gp and/or BCRP transporters. Per the ICH M12 guidance, OATP1B1/OATP1B3 substrate investigation should be considered when the hepatic clearance or biliary excretion reaches ≥25% of total elimination, or when the liver is the pharmacological target of the drug. Likewise, OAT1/OAT3 substrate evaluation should be considered when renal active secretion is ≥25% of systemic clearance.

Transporter Inhibition

The ICH M12 guidance recommends conducting assessments to determine if an investigational drug is an inhibitor of any of the following nine key transporters: P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1, and MATE2-K. The guidance also recommends assessing other transporters, such as BSEP and MRP2, as needed. P-gp is of particular interest as this transporter mediates the efflux of drugs from cells located in many different locations in the body including the small intestine, blood-brain barrier, hepatocytes, and kidney proximal tubule. In a systematic review by Yu and Ragueneau-Majlessi of in vitro transporter inhibition data from New Drug Applications (NDAs) submitted in 2018, 29 out of 42 (69%) approved drugs inhibited at least one transporter, with the largest number of drugs found to be inhibitors of P‐gp.

Transporter Induction

ICH M12 does not provide an established framework for the in vitro evaluation of transporter induction.  However, because P-gp is coregulated with CYP3A, (meaning activation of PXR and/or CAR will increase both P-gp and CYP3A expression), the guidance states that if an investigational drug reduces the AUC of a sensitive CYP3A substrate by 50% or more, a clinical study may be warranted. To determine if a clinical study is needed, drug developers must consider the specific details of the interaction including the likelihood of concomitant use of the investigational drug with P-gp substrates, clinical relevance of the decreased AUC observed, and involvement of additional transporters. Transporter induction can also be investigated in vivo during cocktail studies. During cocktail studies, inhibition and induction are evaluated simultaneously. The investigators select known substrates of transporters and CYP enzymes and administer them to participants along with the investigational drug to measure plasma concentrations of the substrates with and without the investigational drug.

Metabolite-Mediated Interactions

The DDI potential of metabolites with substantial plasma exposure or pharmacologic activity should be evaluated in the same way as the parent drug, but this assessment can usually be done later in development once more is known about the metabolite’s exposure and activity. If the data indicates that changes in metabolite exposure could impact clinical efficacy or safety, the risk of DDIs resulting from altered formation or elimination of that metabolite should be characterized using in vitro phenotyping.

For evaluation of metabolite inhibition, in vitro inhibition assays with the relevant CYP enzyme and transporter panel are recommended when a metabolite’s total AUC is ≥25% of the parent drug’s AUC and accounts for ≥10% of circulating drug-related material. For evaluation of metabolite induction, mRNA-based assays are recommended when the drug is a prodrug or the metabolite is formed extrahepatically; in these cases, the same ≥25% AUC ratio and ≥10% thresholds apply. Based on the in vitro assessments of the metabolite, the determination of whether to conduct further DDI studies follows the same approach as for the parent drug.

ICH M12 Guidance Summary

Based on available in vitro data and clinical information, such as C_max, the ICH M12 guidance includes recommendations on how to assess different types of DDIs to determine whether an investigational drug reaches a defined threshold for clinical DDI risk. For each DDI category, Table 2 provides a summary of the ICH M12 recommendations for determining whether additional DDI assessments should be conducted. Additional details about the components of each calculation are available in the guidance.

Table 2: Summary of ICH M12 Recommendations by DDI Type

The results of in vitro DDI assessments help determine whether additional evaluation through modeling or clinical DDI studies is needed to characterize potential clinical impact.

Physiologically Based Pharmacokinetic (PBPK) Modeling for DDIs

PBPK modeling is an effective tool to help inform drug developers on the potential for DDIs involving the investigational drug using both in vitro and in vivo data to support the model. Results from PBPK models can support regulatory decision-making and can reduce the need for clinical DDI studies.

For CYP-mediated DDIs, PBPK modeling can be used to inform clinical DDI study design, explain PK observations, support the lack of clinical DDI potential, and predict DDI effects under varying dose regimens. For transporter-mediated DDIs, PBPK models can be used to support the initial study design for clinical DDI studies when a DDI is indicated from in vitro testing. Additionally, PBPK models can be used to support PK observations when the investigational drug is an object of transporter-mediated DDIs (e.g., PK changes due to genetic polymorphisms in transporters).

Clinical DDI Studies

Results from in vitro DDI studies and PBPK modeling can help determine if clinical DDI studies are necessary. Clinical DDI studies, typically conducted in Phase 1 of drug development, involve assessing DDI potential of investigational drugs in human participants using known inhibitors/inducers or substrates of enzymes or transporters. Clinical DDI study results often inform labeling recommendations by specifying when to avoid certain drugs or drug classes that may interact with the investigational drug, either because other medications can affect the investigational drug’s exposure or because the investigational drug can affect the exposure of other medications. Knowledge gained from Phase 1 DDI assessments also shapes the design of subsequent trials by indicating whether specific concomitant medications should be permitted or prohibited in trial participants. Ideally, the clinical relevance of potential DDIs should be determined as early as possible in development, whenever practical, for patient safety reasons and to avoid unnecessary concomitant medication restrictions in clinical studies.

Conclusion

Accurate interpretation of in vitro DDI study results is necessary to determine whether additional DDI investigations are needed, either through PBPK modeling or clinical studies. The ICH M12 guideline provides a harmonized framework for the evaluation of potential metabolism-mediated, transporter-mediated, and metabolite-mediated DDIs. Thorough characterization of DDI risk informs the design of clinical trials and eventual drug labeling, allowing medicines to be used in a way that preserves patient safety. Allucent offers expert characterization of nonclinical DDI data to assess clinical DDI potential along with strategic approaches to ensure your investigational drug advances safely and efficiently through development.