Assays Required for Regulatory Drug-drug Interaction (DDI) Studies

With the number of drugs taken concomitantly by the general population increasing over the past decades, the risk of drug-drug interactions (DDI) increases as well. To minimize DDI risk, drug developers need to evaluate their compound as a potential precipitant or object of such interactions. DDI studies are usually conducted following guidance documents by regulatory authorities, that start with a series of in vitro evaluations, based on which in vivo DDI risk can be assessed, and follow-up clinical studies can be conducted if necessary.

The regulatory landscape for drug-drug interactions has undergone a dramatic shift over recent years. After multiple revisions to guidance documents released by US Food and Drug Administration (FDA)^[1], European Medicines Agency (EMA)^[2], and Japanese Pharmaceuticals and Medical Devices Agency (PMDA)^[3], the ICH published their M12 harmonized draft guideline on Drug Interaction Studies^[4] in 2024, which aims to globally harmonize DDI to create a single international reference document. This ICH M12 will ultimately become the standard guidance for Sponsors developing compounds from pre-clinical through final submission for registration. The guideline covers both in vitro and clinical assessment and provides recommendations to support investigations and data interpretation for:

Metabolic enzyme-mediated interactions
Transporter-mediated interactions

Final ICH M12 Guidelines – What’s New?
Explore how the Final M12 guidance differs from its 2022 draft version and the FDA 2020 DDI guidance, and how these changes may impact your program and study design.
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Assessing Metabolic Enzyme-mediated Drug-drug Interactions (DDI)

Reaction phenotyping

A large fraction of drugs undergo enzymatic metabolism in the body, most commonly via enzymes belonging to the CYP450 family. In such cases, the introduction of other drugs that alter the functionality of these enzymes as precipitants may lead to changes in a substrate compound’s metabolism, leading to altered clearance rates and PK. It is therefore important from a DDI perspective to characterize the pathways involved in a drug candidate’s metabolism and identify the enzymes that contribute to and quantify their contribution.

In vitro metabolite characterization and identification studies are usually conducted relatively early in drug development. These results can inform whether metabolic enzyme phenotyping studies are necessary, and how they should be conducted. Regulatory documents recommend approaching phenotyping in a stepwise manner: driven by the type of interaction and starting with the most common enzyme involved in that pathway.

For oxidative metabolism studies, for example, usually start with the most common CYP450 enzymes. If metabolism is not fully accounted for, the studies move on to other CYP450s and different phase I enzymes, such as ADH/ALDH, AO, CES, FMO, MAO, XO, etc. Similarly, if conjugation is observed, depending on the metabolite structure, phase II enzymes should be addressed; for example UGTs in case of glucuronidation, SULTs in case of sulfation, GSTs in case of glucuronidation, etc.

The guidelines recommend clinical DDI studies with enzymes that are responsible for ≥ 25% of drug elimination.

Recommended in vitro assay systems for phenotyping:

Metabolic enzyme inhibition in HLMs or hepatocytes
Individual human recombinant CYP enzymes

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Enzyme Inhibition

Drug-drug interaction studies also need to be conducted to assess a candidate’s effect on major drug metabolizing enzymes. Both so-called direct (or reversible) and time-dependent inhibition should be tested for DDI risk assessment for the seven major CYPs:

CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A

CYP450 enzymes can be inhibited either via direct inhibition or time-dependent inhibition (TDI):

Direct inhibition involves rapid association and dissociation of an inhibitor drug and the enzyme
Time-dependent inhibition, defined as an interaction where the extent of inhibition increases upon incubation of the inhibitor with the enzyme before the substrate is added

CYP450 inhibition assays are usually run using human liver microsomes (HLMs), but depending on the program, other systems, such as hepatocytes or recombinant systems may also be used. It is important, however, to apply an assay setup that allows understanding whether the compound is a reversible or a time-dependent inhibitor. The need for a follow-up clinical drug-drug interaction study or the exclusion of in vitro DDI risk is determined differently for the two types of inhibition.

According to the ICH M12^[4], inhibition of UGT enzymes may also need to be considered if the major elimination route of a candidate drug is via glucuronidation, or in case it will be commonly administered alongside a drug that is cleared via glucuronidation.

Enzyme	Inhibition	Induction
CYP1A2	Yes	Yes
CYP2B6	Yes	Yes
CYP2C8	Yes	Only if CYP3A4 is induced
CYP2C9	Yes	Only if CYP3A4 is induced
CYP2C19	Yes	Only if CYP3A4 is induced
CYP2D6	Yes	No
CYP3A(4)	Yes	Yes

Enzyme Induction

Drugs can not only repress but also induce metabolic enzymes, which carries drug-drug interaction risk. CYP450 enzyme induction is usually achieved via receptor-mediated transcriptional gene activation. This is initiated by binding to one of the nuclear receptors or transcription factors involved in CYP450 regulation: AhR, CAR, or PXR. PXR primarily induces the transcription of the CYP3A and CYP2C families, CAR induces the CYP2B family and AhR induces the CYP1A family.

CYP induction assessment is also performed in a stepwise manner:

First, induction of CYP1A2, CYP2B6, and CYP3A4 is tested
In case CYP3A induction results are positive, a follow-up study of CYP2C (CYP2C8, CYP2C9, and CYP2C19) enzyme induction should be studied either in vitro or clinically

CYP induction assays are run with plated or sandwich-cultured hepatocytes (SCH) with read-outs as fold-change in the mRNA levels of each enzyme, and/or as changes in each enzyme’s specific metabolic activity (except in the case of CYP2C19, where only activity readouts are considered reliable). A drug is considered an inducer, warranting follow-up studies if it increases CYP mRNA levels in a concentration-dependent manner, with a >2x increase at 50xc_max,u.

Visit the CYP450 enzyme interactions page for further details on these assays.

Assessing Transporter-mediated Drug-drug Interactions (DDI)

Similarly to enzymes, drugs may interact with transporters as substrates or inhibitors – resulting in potential object or precipitant roles in drug-drug interactions. While dozens of clinically relevant drug transporters exist, only a few of them are commonly involved in drug-drug interactions. For these, regulatory guidelines provide a specific list of assays to be performed for both substrate and inhibition assays. They also state, however, that depending on the compound's pharmacokinetic properties, the indication and patient population, and chemical structural information, additional transporters may need to be considered.

Both the ICH M12 [4] and FDA [1] guidelines require the same "core" set of transporters to be assessed:

MDR1, BCRP, OAT1, OAT3, OATP1B1, OATP1B3, OCT2, MATE1, and MATE2

A candidate drug needs to be tested as a potential inhibitor of all nine above-listed transporters. For substrate studies, the inclusion in a drug-drug interaction study is more conditional and is driven by the drug's elimination route. While substrate interactions with MDR1 and BCRP efflux transporters need to be studied in almost all cases, the listed uptake transporters are characteristic of a specific elimination route, hepatic or renal, and only need to be assessed in case ≥25% of the compound is actively cleared via that route.

Substrate interactions are determined as an over 2-fold change in compound efflux ratio (ER) or Net ER in bidirectional permeability assays or in cellular or vesicular uptake when compared to the control system. In this case, follow-up DDI studies are required. For inhibition, transporter-specific DDI risk exclusion cut-off values are provided in regulatory guidelines that are driven by the IC_50,u.

	Transporter	Inhibition		Substrate
	Transporter	FDA 2020	ICH 2024	FDA 2020	ICH 2024
Efflux	MDR1 (P-gp)	Yes	Yes	Yes	Yes
	BCRP	Yes	Yes	Yes	Yes
	MRP2	No	No	No	Consider
Uptake	OAT1	Yes	Yes	If ≥25% active renal elimination	If ≥25% active renal elimination
	OAT3	Yes	Yes	If ≥25% active renal elimination	If ≥25% active renal elimination
	OATP1B1	Yes	Yes	If ≥25% hepatic or biliary elimination	If ≥25% hepatic or biliary elimination or target in the liver
	OATP1B3	Yes	Yes	If ≥25% hepatic or biliary elimination
	OCT2	Yes	Yes	If ≥25% active renal elimination	If ≥25% active renal elimination
	MATE1	Yes	Yes
	MATE2K	Yes	Yes
	OCT1	No	No	No	Consider
	OATP2B1	No	No	No	Consider

In addition to the "core" transporters, the ICH M12 guidance^[4] also lists three transporters for "consideration" in substrate studies: the MRP2 efflux, and the OCT1 and OATP2B1 uptake transporters. While the M12 does not describe the conditions when these transporters may be relevant, according to the International Transporter Consortium (ITC), MRP2 and several other MRPs are most commonly involved in the distribution of glucuronide metabolites, OATP2B1 should be addressed if there is hepatic or intestinal uptake that cannot be attributed to more common mechanisms or if the intestinal absorption is faster than it could be explained by passive permeability, while OCT1 should be studied similarly to other hepatic uptake transporters^[5-6]. Cut-off values for these transporters should be deduced from those for the "core" transporters^[4].

Our extensive transporter assay portfolio with experiments set up to meet regulatory requirements covers all transporters required for drug-drug interaction studies, and over 70 additional transporters that may affect absorption, distribution, elimination, or toxicity of certain drugs.

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Frequently Asked Questions (FAQs) About Regulatory Drug-drug Interaction Studies

What is the correct timing for DDI studies?

The ICH M12 guidance states that enzyme and transporter substrate data need to be generated before starting the clinical phase in patients, while inhibition and induction data is required before initiating larger studies in patients. These times may differ between drug development projects. As in the case of oncological drugs, clinical trials usually start in patients right away, while for other indications, patients represent only Phase 2 and later trial populations. The ultimate aim of drug-drug interaction studies is to ensure the safety of trial participants and to avoid restrictions of concomitant medications or subjects who are on concomitant medications in clinical studies. The ICH also states that "timing of non-clinical and clinical studies is dependent on the context and type of product" and indeed, in our experience, DDI data is generated gradually from candidate selection stage and onward in most development programs.
Are drug-drug interaction studies relevant for highly protein-bound drugs?

Regulatory guidelines often use unbound drug fractions in different risk cut-off calculations, accounting for protein binding in the plasma or in the assay system. They do not offer specifications that would call for different approaches depending on protein binding. However, it is stated in the ICH M12 guidance that recovery and unbound drug quantities need to be determined from in vitro assays, and there is also a technical appendix segment dedicated to protein binding. The FDA 2020 DDI guidelines still call for the use of a conservative 1% free fraction for highly plasma protein-bound drugs, while the ICH M12 acknowledges the existence of precise enough measurement methods for determining a <1% free fraction, and allows for the measured results to be used in DDI risk calculations as long as accuracy of the method can be demonstrated. This may reduce the risk of overproducing DDI risk for highly protein-bound compounds.
Do drug metabolites need to be assessed for drug-drug interactions?

Regulatory documents state conditions for investigating metabolites for enzyme- or transporter-mediated drug-drug interactions. According to the ICH M12 guideline, if active secretion is the major elimination pathway of a metabolite with significant target activity (≥50% of total effect) or if it could contribution to off-target (adverse) effects, attempts should be made to identify the transporter(s) involved. Inhibition of enzymes or transporters by a metabolite (active or not) is required if AUC_metabolite ≥25% AUC_parent, while for enzyme induction to be required, the parent also needs to be a prodrug, or metabolite needs to be formed mainly extra-hepatically. These precipitant interactions are usually only studied in vitro if no clinical drug-drug interaction studies are conducted with the parent, otherwise it is assumed that the metabolites and their effects can concomitantly be addressed through in vivo experiments. It is noted in the FDA 2020 guidance that some phase II metabolites can be better substrates or inhibitors of various transporters, which can lead to higher chance of drug-drug interactions than the parent drug. FDA thus recommends that the drug-drug interaction potential of a metabolite as a substrate or inhibitor of major drug transporter should be assessed on a case-by-case basis using the same principles and strategies that are applied for the parent drug.

References

Drug-drug Interaction Studies for Regulatory Submission