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:
- Plasma Protein Binding
- Metabolic Stability
- Metabolite Identification (Met ID)
- Reaction Phenotyping
- CYP enzyme Inhibition
- CYP enzyme Induction
- Drug Transporter Inhibition
- Drug Transporter Substrate
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
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.
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.
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:
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
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.
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:
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
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.
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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.
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