When a drug induces the enzymes that metabolize other drugs, co-administered medicines can be cleared faster, reach lower exposures and lose efficacy. The drug can also induce its own metabolism (auto-induction), complicating dose selection. Assessing induction potential early, and correctly, is therefore a standard part of every drug discovery and development program.
The ICH M12 guideline has harmonized how regulators expect this assessment to be done. Below, we walk through the mechanisms, study design, endpoints and interpretation of in vitro CYP induction studies, and what to do when a risk is flagged.
The main mechanism behind induction of enzymatic activity is the increased transcription of the genes encoding the metabolic enzyme, leading to increased enzymatic protein abundance and ultimately increased enzymatic activity. This is typically caused by the activation of nuclear receptors regulating the transcription of the genes in question. Of particular interest in the drug discovery and development context are the nuclear receptors responsible for regulating CYP enzymes, namely pregnane X receptor (PXR, CYP3A) and constitutive androstane receptor (CAR, CYP2B6) as well as the common helix-loop-helix transcription factor aryl hydrocarbon receptor (AhR, CYP1A).
The ICH M12 guideline recommends that the induction potential of a new drug should always be investigated for the three major inducible enzymes CYP1A2, CYP2B6 and CYP3A4 in primary human hepatocytes, using at least 3 individual donors to account for interindividual variation. If induction of CYP3A4 is observed, the potential of the drug to induce CYP2C8, CYP2C9 and CYP2C19 should be further investigated. Like CYP3A4, the CYP2C enzyme family is also induced via the PXR pathway, even if generally to a lesser extent compared to CYP3A4. Consequently, a negative induction result obtained with a sensitive CYP3A4 substrate in a clinical study can be used as a basis to rule out the induction of CYP2C enzymes (given that the drug or its major metabolites are not CYP3A4 inhibitors).
Induction of the target enzymes can be monitored using several endpoints. Target enzyme mRNA level as measured by qRT-PCR is usually used as the primary endpoint, as also recommended by the ICH M12 guideline. Enzyme activity can also be measured using an enzyme-specific probe substrate but using it as an only readout is not recommended, as this can lead to false negative findings in cases where the drug also simultaneously acts as an inhibitor of the same target enzyme. For CYP2C19, mRNA response to inducers is often limited in primary human hepatocyte cultures, which is why enzyme activity should be used as the primary endpoint when assessing CYP2C19 induction potential.
Besides CYP enzymes, other enzyme families are also inducible. Since over a half of the most prescribed drugs exhibit UGT-mediated metabolism, the induction of UGT enzymes has recently gained more interest. However, currently the in vitro methodology for studying UGT induction is still evolving and not yet well established, which is why routine investigation of UGT induction potential is not required by the authorities. Data obtained from CYP induction studies can be used as a surrogate for UGT induction, as many UGT enzymes are regulated through the same nuclear receptors as CYPs. However, when of special interest, the UGT induction potential of a drug can also be studied in vitro, and this will be discussed in more detail separately.
Attention to major metabolites is also warranted when assessing the overall DDI liability of a new drug. However, in the context of enzyme induction, the in vitro evaluation of the parent drug can also reflect the induction potential of metabolites, because metabolites can be generated during the incubation when using hepatocytes. This is why a CYP induction study for major metabolites is not strictly required in the ICH M12 guideline. However, if dealing with a pro-drug or an extra-hepatically formed metabolite, a dedicated in vitro evaluation of the metabolite is recommended.
As in enzyme inhibition studies, CYP induction risk should also always be assessed using unbound incubation concentrations instead of nominal concentrations. The importance of using unbound drug concentrations is also emphasized in the ICH M12 Guideline. The unbound fraction in incubation systems (fu,inc) can be determined experimentally using methods such as rapid equilibrium dialysis (RED) or ultrafiltration or estimated computationally using predictive models based on physicochemical properties such as lipophilicity, charge, and protein binding affinity.
If a drug increases the mRNA expression of a CYP enzyme in a concentration-dependent manner, and the fold-change of mRNA expression is ≥ 2-fold at 50 times the expected unbound maximal in vivo plasma concentration at least in one of the tested donors, the risk for a clinically relevant drug-drug interaction cannot be ruled out. Notably, unlike previous agency-specific guidelines, the new ICH M12 guideline does not require an assessment of intestinal CYP3A induction risk.
If a CYP induction risk is flagged, more quantitative approaches can then be implemented to assess the risk in more detail. Kinetic induction parameters Emax and EC50,u (maximum fold induction and the unbound concentration resulting in half maximal induction, respectively), calculated from data spanning a wide test concentration range, can be used as inputs in mechanistic models to predict the probability of an in vivo drug-drug interaction. In vivo DDI risk can also be assessed using correlation methods such as RIS approach, which compares the induction effect of the investigational drug to that of established in vivo inducers by establishing a calibration curve using a set of inducers and their known in vivo effects. Especially for CYP3A4 there are good amount of low, moderate and high inducers characterized, and the correlation method may provide true additional value. For example, if the basic method cannot rule out CYP3A4 induction risk for the investigational drug, RIS approach is a potential follow-up in vitro method for quantitative assessment and for de-risking the interaction.
If you would like to learn more about how to incorporate CYP induction assessment into your drug development program, our experts are happy to help. Get in touch with us to discuss your specific needs and identify the most appropriate strategy for your compound!
