Enzyme Induction

FDA- and EMA-recommended studies

Cytotoxicity and stability assays

CYP mRNA and enzyme induction

While investigating your drug's metabolic qualities, you will also want to include enzyme induction studies to highlight potential issues with achieving efficacious plasma concentrations of concomitantly administered medications when your drug induces or enhances an enzyme expression. Enzyme induction can result in increased metabolic clearance or toxicity that is caused by increased systemic exposure of active metabolites.

Induction of CYP enzymes (typically CYP1A2, CYP2B6 and CYP3A4) is measured in vitro following exposure to test article in monolayer cultures of human hepatocytes.

Initial experiments should investigate the potential to induce CYP1A2, CYP2B6, and CYP3A4 enzymes. If induction of CYP3A4 enzymes is observed, the sponsor should also evaluate the induction potential of CYP2C enzymes (CYP2C8, CYP2C9, CYP2C19). The effects are then compared with those elicited by positive control inducers of the CYP enzymes under investigation. In addition, ex vivo animal livers (typically mouse, rat, dog, or monkey) can be processed to subcellular fractions and used to assess test article mediated effects on drug metabolizing enzymes following in vivo administration during safety assessment studies.

Regulatory considerations for enzyme induction studies

These studies are recommended by both FDA and EMA drug-drug interaction (DDI) guidelines to evaluate cytochrome P450 induction for CYP1A2, CYP2B6,CYP2C8, CYP2C9, CYP2C19, and CYP3A4 before going to first-in-human trials.

Induction data is used in determining the requirement and scope of clinical DDI studies.

Method

Test system: Individual human donor hepatocytes, cryopreserved
CYP enzymes evaluated: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A4/5.
Test Article Concentrations: Selected to bracket predicted clinical exposures (0.1 – 10X C_max), unless limited by solubility. Select 5 concentrations for cytotoxicity, 7 for mRNA induction,and 3 for enzyme activity induction
Incubation time: 72 hours, with refreshed media every 24 hours.

Cytotoxicity

In preliminary assays, hepatocytes from one donor are cultured in the presence of test article (five concentrations) or positive control tamoxifen (50 µM) in triplicate wells for 72 hours in a humidified incubator at 37°C in 5% CO2, with exchanges of medium ± test article or controls occurring every 24 hours. After 72 hours, viability of hepatocytes will be evaluated using the MTS assay. Results will be used to determine non-toxic test article concentrations for the induction assays.

Stability

In conjunction with the cytotoxicity assay, medium samples are collected to assess concentrations of test article over time.

CYP mRNA Induction

Hepatocytes from three donors are incubated in the presence or absence of test article (seven concentrations) or control inducers (see below) in triplicate wells for 72 hours in a humidified incubator at 37°C in 5% CO2, with exchange of medium ± test article or controls occurring every 24 hours. After 72 hours, mRNA is extracted and gene expression is determined by real-time quantitative PCR. The level of each CYP mRNA is normalized to that of an endogenous control gene (e.g., GAPDH). Fold induction of CYP gene expression is then calculated using the 2-ΔΔCT method. Test article-dependent increases in CYP mRNA levels ≥2-fold relative to the vehicle control, or a response ≥20% of the response of positive controls, may be interpreted as positive results.

CYP Enzyme Induction

Hepatocytes from three donors are grown in the presence or absence of test article (three concentrations) or control inducers (as above) in triplicate wells for 72 hours in a humidified incubator at 37°C in 5% CO2, with exchanges of medium ± test article or controls occurring every 24 hours. After 72 hours, hepatocytes are incubated in fresh medium containing appropriate CYP substrates (see below) for 2 hours, after which reactions are terminated and samples collected for analysis of metabolites (see below) using a liquid chromatography-mass spectrometry (LC-MS) analytical method. Test article-dependent increases in CYP enzyme activity ≥2-fold relative to the vehicle control, or a response ≥20% of the response of positive controls, may be interpreted as positive results.

			Enzyme Activity Assays
CYP Enzyme	Inducer	Non-inducer	Substrate	Analyte
CYP1A2	Omeprazole	Flumazenil	Phenacetin	Acetaminophen
CYP2B6	Phenobarbital	Flumazenil	Bupropion	Hydroxybupropion
CYP3A4/5	Rifampicin	Flumazenil	Midazolam	1’-Hydroxymidazolam

Deliverables

These assays will provide mRNA and enzyme activity levels in comparison with known inducers of CYP enzymes. The EC₅₀ and E_max will be provided if applicable.

If positive results are observed, pharmacometrics may allow additional guidance when assessing the need for a clinical trial.

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