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What nonclinical ADME / DDI studies do I need for a successful IND / NDA?

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 2020 finalized guidance for industry 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 an investigational drug and could result in restrictive labeling, postmarketing requirements or commitments, and/or delayed approval until sufficient information on DDIs is available.” 

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 January 2020 the FDA published edits final guidance for industry “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. The finalized guidance comes as an update to the 2017 draft guidance. Differences in expectations between the three primary global agencies (FDA, EMA, PMDA) and specific updates in FDA guidance from 2017 to 2020 can be seen in our 2020 poster put together by Dr. Brian Ogilvie, and founder, Dr. Andrew Parkinson.

FDA approval map

Basic in vitro DDI studies:

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 test systems 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 and identification as early as possible (“We encourage the identification of any differences in drug metabolism between animals used in nonclinical safety assessments and humans as early as possible during the drug development process.”2 ) 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: Validated method to monitor substrate loss or metabolite formation in 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 is necessary.
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)

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, OCT13

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 they suggest hepatic uptake/elimination or renal secretion is significant.


References

  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 6, “Safety Testing of Drug Metabolites.” Guidance for Industry, Food and Drug Administration 2016.
  3. Page 25, “Guideline on the Investigation of Drug Interactions.” European Medicines Agency 2012.

Timing of in vitro and in vivo

When should you be planning nonclinical drug metabolism and DDI studies? Get our advice

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