In our previous post, “Drug Interaction Studies for NDAs and BLAs“, we introduced the 2012 FDA Guidance titled, “Clinical Drug Interaction Studies – Study Design, Data Analysis, and Clinical Implications” and summarized key points related to labeling.
In this post we will focus on, recommendations regarding in vitro studies of drug metabolism, drug transport, and drug-drug or drug-therapeutic protein interactions conducted in support of new drug applications (NDAs) and biologics license applications (BLAs) regulated by CDER (Center for Drug Evaluation and Research).
In Vitro Studies
In vitro studies, in combination with clinical pharmacokinetic data, can be used to rule out the need for additional in vivo studies, or to provide a mechanistic basis for clinical studies using a modeling and simulation approach. In vitro studies are designed to assess whether the investigational drug (or major metabolite) is a substrate, inhibitor, or inducer for various enzymes or transporters. The guidance includes several decision trees to guide evaluation as well as suggestions about appropriate methods and common models. Key considerations for in vitro studies include the selection of appropriately validated methods, the choice of test system, and rational selection of substrate/interacting drug and their concentrations.
In Vitro Metabolism Studies
The metabolic profile of the investigational drug should be characterized from in vitro studies utilizing human liver microsomes, microsomes expressing recombinant enzymes, or freshly isolated or cryopreserved human hepatocytes. When metabolism constitutes ≥ 25% of the drug’s elimination (or when metabolism is unknown), in vivo studies using appropriate inhibitors/inducers are warranted, starting with strong inhibitors/inducers. If a positive interaction is seen, weaker agents should be investigated through a clinical study or physiologically based pharmacokinetic (PBPK) modeling.
Phase 1 metabolizing enzymes to be evaluated for the role of the investigational drug as substrate include the cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A. If the investigational drug is not a CYP substrate, other phase 1 enzymes such as monoamine oxidase (MAO), flavin monooxygenase (FMO), xanthine oxidase (XO), and alcohol/aldehyde dehydrogenase (ADH) should be explored. Phase 2 metabolizing enzymes involved in conjugation reactions have historically attracted less attention in DDI evaluations, largely due to imperfect tools and a lower incidence of adverse DDIs; however, if glucuronidation represents ≥25% of total metabolism, in vitro studies with recombinant UGTs (UDP glucuronosyl transferases) are recommended in the guidance and a decision tree is provided to aid evaluation.
In vitro characterization of the investigational drug as an inhibitor of CYP enzymes is usually investigated using human liver microsomes or cDNA-expressed microsomes to determine the inhibition mechanisms (e.g., reversible or time-dependent) and potency (e.g., Ki). Characterization of the drug as an inducer is typically assessed by measuring mRNA changes in CYP1A2, CYP2B6, and CYP3A (at a minimum, along with positive controls) using hepatocytes from at least 3 human donors. Quantitative analysis of these in vitro data combined with clinical pharmacokinetic data can be performed using a variety of algorithms, basic models, mechanistic static models, and/or more comprehensive PBPK models to indicate whether in vivo drug-drug interaction studies are warranted. Basic models, although conservative, have been widely used because they are simple and practical; these models rely on estimation of an R value, a ratio of intrinsic clearance values of probe substrates in the presence and absence of the interacting drug. Mechanistic static models incorporate more detailed drug disposition data such as bioavailability or fractional metabolism. Cutoffs and calculation methods for basic and static models are provided in the guidance. PBPK models (e.g., those available in GastroPlusTM and Simcyp®) integrate a variety of parameters based on human physiology and the drugs of interest and can be continuously refined. In some cases, well-developed PBPK models can offer alternatives to dedicated clinical studies if the predicted AUC ratio is between 0.8-1.25. Sponsors should contact the FDA early in the process and be prepared to provide:
- Description of structural model
- Source and justifications for system and drug-dependent parameters
- Type of error models
- Model output
- Data analysis
- Adequate sensitivity analyses
Regardless of which prediction model is used, sponsors should provide details of model assumptions, physiological and biological plausibility, the origin of the parameters, and information on uncertainty and variability.
In Vitro Transporter Studies
The transporters P-gp (P-glycoprotein) and BCRP (Breast cancer resistance protein) are expressed in the GI tract, liver, and kidney, and can have a role in limiting oral bioavailability. All investigational drugs should be evaluated in vitro to determine whether they are a potential substrate of these transporters, with exceptions where a drug is highly permeable and highly soluble (BCS class 1) and a waiver can be granted. A bidirectional assay in Caco-2 cells or overexpressed cell lines is the preferred method of evaluation. Additional evaluations of investigational drugs as substrates of hepatic uptake transporters (OATP1B1/OATP1B3), kidney transporters (OAT1/3 and OCT2), or other transporters (e.g., MRP) should be performed based on knowledge of the clearance pathway and other drugs in the same therapeutic class. Similar evaluations of the investigational drug as a potential inhibitor of these transporters should be considered. Decision trees provided in the guidance outline where in vitro results for these assessments might indicate a need for further in vivo studies. The role of the investigational drug as an inducer of transporters may be explored using in vitro tools such as a nuclear receptor assay or the adenocarcinoma cell line LS 180, which expresses P-gp; however, due to a lack of validated in vitro methodology, the definitive determination of induction potential relies on in vivo studies.
Learn more about how in vitro studies inform drug interactions by connecting with one of our senior scientists.