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What In Vitro Metabolism and DDI Studies Do I Actually Need?

Though there is no ‘roadmap’ spelling out required studies to achieve regulatory approval for clinical entry, a drug candidate’s metabolism and potential for drug-drug interaction (DDI) are emphasized in recent documents published by FDA, EMA, and PMDA. FDA 2017 guidance highlights the importance and standardization of DDI components of preclinical development, stating “Inadequate studies of DDIs can hinder the FDA’s ability to determine the benefits and risks of a drug and result in restrictive labeling, [PMRs or PMCs], and/or delayed approval.”

The intention of drug-drug interaction studies in a preclinical data package is to prevent patients’ unnecessary exclusion from clinical trial participation by predicting potential for harmful interactions with concomitant drugs through a risk-based approach. Regulatory agencies have published key guidance documents for drug developers to use in planning these critical preclinical assays. For example, in 2017 the FDA published “In Vitro Metabolism- and Transporter-Mediated Drug-Drug Interaction Studies,” which focuses on in vitro experiments to evaluate DDI potential involving drug-metabolizing enzymes and transporters, as well as how in vitro results can inform clinical DDI study planning in a risk-based approach. Specific differences in expectations between the three agencies can be seen in our 2018 poster by VP of Consulting, Dr. Brian Ogilvie, and founder, Dr. Andrew Parkinson, but here we draw from the 2017 guidance mentioned above to outline the basic in vitro studies to support and evaluate drug metabolism- and transporter-mediated DDI of an investigational new drug:

Preliminary assays:

Study: plasma protein binding

Purpose: Determine the percentages of bound and free drug available in the plasma for both target and off-target interactions in humans and preclinical species

In vitro study expectations: “In vitro metabolic and plasma protein binding data for animals and humans and systemic exposure data in the species used for repeated-dose toxicity studies generally should be evaluated before initiating human clinical trials.”1

Study: permeability

Purpose: Measure permeability to predict intestinal absorption and tissue distribution

In vitro study expectations: Investigators measure the permeability of the test article across cell lines (e.g., Caco-2 or MDCKII cells) or an artificial membrane (e.g., PAMPA) and compare with high- and low-permeability controls.

Study: metabolic stability

Purpose: Measure in vitro intrinsic clearance in humans and preclinical species

In vitro study expectations: Test article is incubated with pooled microsomes, S9, plasma or hepatocytes to determine the rate of clearance. Treated pools of microsomes, S9, or hepatocytes are used to compare concentration of parent compound before and after interaction with drug-metabolizing enzymes in determination of whether there is significant metabolism within cells/organelles.

Study: metabolite characterization/identification (Met ID)

Purpose: “Identify the number and structures of metabolites produced by a drug and whether the metabolic pathways are parallel or sequential”

In vitro study expectations: FDA suggests conducting metabolite characterization (Met ID) prior to reaction phenotyping: “Metabolic pathway identification experiments identify the number and structures of metabolites produced by a drug […] data obtained from metabolic pathway identification experiments help determine whether and how to conduct a reaction phenotyping study.” Suggested test systems include human hepatocytes, human liver microsomes, or recombinant enzyme systems.

Metabolism-mediated drug-drug interactions:

Study: reaction phenotyping

Purpose: “Determining if the investigational drug is a substrate of metabolizing enzymes”
In vitro study expectations:

  • Standard: Major cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5)
  • Additional if appropriate: Other CYPs (CYP2A6, CYP2J2, CYP4F2, CYP2E1), Phase I enzymes (MAO, FMO, XO, AO), Phase II enzymes (UGTs)
  • Validated method to monitor substrate loss or metabolite formation
  • Two recognized test systems, sponsor “should use both” with validated, reproducible analytical methods: human liver microsomes (+10 donors), human recombinant CYP enzymes
  • If an enzyme is responsible for >25% of drug’s elimination, clinical DDI study necessary

Study: CYP enzyme inhibition

Purpose: “Determining if the investigational drug is an inhibitor of metabolizing enzymes”

In vitro study expectations: Reversible (direct) and TDI (time-dependent inhibition) for 7 major CYPs (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5). Test systems include human liver microsomes, microsomes from recombinant CYP-expression systems, hepatocytes.

Study: CYP enzyme induction

Purpose: “Determining if the investigational drug is an inducer of metabolizing enzymes”
In vitro study expectations:

  • Standard: Induction potential for major CYPs (CYP1A2, CYP2B6, CYP3A4/5)
  • Additional if appropriate: If positive induction results from CYP3A4/5, follow up with CYP2C family (CYP2C8, CYP2C9, CYP2C19)
  • Test systems include plateable cryopreserved or freshly isolated human hepatocytes or immortalized hepatocytes, from at least three donors

Transporter-mediated drug-drug interactions:

Study: drug transporter inhibition

Purpose: “Determining if the investigational drug is an inhibitor of a transporter”
In vitro study expectations: Recommendations of timing for transporter studies can vary dependent on molecular properties of the compound (“The timing of the in vitro evaluation of each transporter may vary depending on the therapeutic indications of the investigational drug”), and the current guidance suggests different test systems for each transporter type.

  • Standard: P-gp, BCRP, OATP1B1, OATP1B3, OCT2, MATE1, MATE2-K, OAT1, OAT3
  • Additional if appropriate: BSEP, OCT12

Study: drug transporter substrate

Purpose: “Determining if the investigational drug is a substrate of a transporter”

In vitro study expectations:

  • ABC/efflux: P-gp and BCRP (but not recommended for highly-soluble, highly-permeable drugs)
  • SLC/uptake: Hepatic (OAT1B1, OATP1B3) and renal (OAT1, OAT3, OCT2, MATE1, MATE2-K) substrate potential should be evaluated based on ADME (absorption, distribution, metabolism, excretion) data if it suggests hepatic uptake/elimination or renal secretion is significant.

Learn more about our contract research services
Read more about when to perform these studies
Learn more about regulatory expectations for your studies and data
Read more about preparing for and dealing with compounds requiring additional studies
Contact us with questions or feedback or to book your study today

Learn more about:

1 Page 6, M3(R2) “Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals” FDA January 2010
2 Page 25, “Guideline on the Investigation of Drug Interactions.” European Medicines Agency 2012.

About the Authors

Madison (Knapp) Esely-Kohlman received her BS from the University of Missouri – Columbia and is currently SEKISUI XenoTech’s Marketing Communication Specialist, developing scientific content that communicates the value and expertise of internal contract service and test system production teams. Madison joined SEKISUI XenoTech as the Scientific Communications Coordinator in 2019 after serving in similar positions at CropLife America, Bond Life Sciences Center and the University of Missouri CAFNR Office of Communications.
Greg Loewen is the Director of Technical Support at SEKISUI XenoTech. He has worked with XenoTech since 2000 in all aspects of drug metabolism, drug transporter and bioanalysis as an analyst, study director and group leader before shifting to Business Development team. In his current role, he is responsible for scientific discussions with clients to evaluate projects and define outcomes. Greg holds a BS in Chemistry from the University of Montana.

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