Cytochrome P450 Induction

Understand the potential drug-drug interaction liabilities of your compounds by using our cytochrome P450 (CYP) induction assay.

Cytochrome P450 induction is one of Cyprotex's in vitro experimental ADME services. Cyprotex delivers consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Determining potential induction of cytochrome P450 (CYP450) enzymes:

• Induction of cytochrome P450 enzymes is associated with an increased prevalence of clinical drug-drug interactions.
• Cyprotex’s cytochrome P450 induction assay identifies the potential of test compounds to induce CYP1A2, CYP2B6 or CYP3A4 in cultured human hepatocytes by evaluating mRNA levels and/or catalytic activity. Assays are designed to meet FDA¹ and EMA² guidelines.
• Test drug concentrations should be based on the expected human plasma drug concentrations and dose. Solubility, cytotoxicity and plasma protein binding should also be taken into consideration.
• Cyprotex’s cytochrome P450 induction assay delivers fold-induction data normalized to vehicle control which can be compared to positive control responses. If appropriate, data is fit using non-linear regression analysis to four-parameter sigmoidal equation to produce E_max and EC₅₀ values.
• The clinical consequences of induction may be therapeutic failure caused by a decreased systemic exposure of the drug itself or a co-administered therapy, or toxicity as a result of increased bioactivation.

Supplementary Assays

• Preliminary visual aqueous solubility assessment
• Preliminary cytotoxicity assessment using MTT in primary human hepatocytes or HepaRG^TM
• Measurement of parent drug on final day of dosing
• Relative induction score (RIS) analysis

Example data from Cyprotex’s CYP induction shows the increase in mRNA expression plotted against concentration for positive control compounds omeprazole and rifampicin for induction of CYP1A2 and CYP3A4 respectively in a single donor.

Cytochrome P450s (CYPs) are a family of enzymes which play a major role in the metabolism of drugs. Induction of CYPs is associated with an increased prevalence of clinical drug-drug interactions (DDIs). If a CYP enzyme is induced by a compound it may increase the metabolism of a concurrent therapy, or itself (autoinduction) leading to a reduction in plasma levels and a potential decrease in drug efficacy. Induction of CYP enzymes can also lead to toxicity by increasing reactive metabolite formation. Therefore, prior knowledge of potential interactions with co-administered therapy is needed to guide development of a drug and to identify possible DDIs or the need for clinical DDI studies.

There are two main mechanisms by which induction of cytochrome P450 enzymes may occur:

1. Nuclear receptor-mediated induction. The most common mechanism of CYP enzyme induction is transcriptional gene activation. Nuclear receptors, such as the aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR), mediate drug-induced changes in the expression of phase I and phase II enzymes and transporters. Induction of CYP1A2, CYP2B6 and CYP3A4 gene expression can serve as sensitive representative endpoints for activation of AhR, CAR and PXR respectively³.
2. Stabilization of the mRNA or enzyme. For example, troleandomycin induces rat CYP3A by decreasing the rate of CYP3A protein degradation with no increase in the rate of protein synthesis⁴.

Variability in the expression and function of CYP enzymes has been well described in the literature⁵ with donor variability a reflection of the inter-individual differences of CYP expression observed in the population. Regulatory bodies^1,2 recommend the use of preparations from at least 3 different donors, in order to account for this inter-donor variability. If the result from at least one donor’s hepatocytes exceeds the predefined threshold, the sponsor should consider the drug an inducer in vitro and conduct a follow-up evaluation¹.

CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 are all included in the CYP induction assay. It is recommended to evaluate CYP1A2, CYP2B6 and CYP3A4 initially as they are induced via different nuclear receptors. A positive in vitro result for CYP3A4 would indicate the need to perform follow up induction studies for CYP2C8, CYP2C9 and CYP2C19 as these isoforms are also induced via activation of the pregnane X receptor (PXR).

Industry standard and regulator recommended in vitro inducers^1,2,7 are assessed alongside the test compound(s) as positive controls; omeprazole for CYP1A2, phenobarbital for CYP2B6 and rifampicin for CYP2C8, CYP2C9, CYP2C19 and CYP3A4. A clinical non-inducer (flumazenil) is also included as a negative control.

Analysis of mRNA expression relative to a vehicle control is a robust and sensitive method of analysis to detect enzyme induction at mRNA level. Enzyme activity levels using a specific probe substrate may also be used to quantify CYP induction; however, the challenge of this approach is the possible masking of a positive induction result when concurrent inhibition occurs. However, if induction due to protein stabilization is suspected, it is recommended induction is also measured at activity level².

It is recommended that drug concentrations selected for CYP induction studies reflect either observed or expected clinical concentrations¹. To cover recommendations by both EMA and FDA regulatory bodies to account for hepatic exposure, it is recommended that the concentration is at least 50 times unbound C_max. If fraction unbound is less than 1% then a fraction unbound of 1% is used for the C_max unbound calculations.

In the case of CYP3A4, intestinal concentration should also be considered. In order to meet regulatory requirements, it is suggested that concentrations cover 0.1 times the dose/250 mL^1,2.

The concentrations tested may be limited by solubility or cell toxicity concerns. In this instance, the maximum possible concentration without causing detrimental effects to the cells should be tested².

To aid with extrapolation of in vitro CYP induction results to in vivo scenarios, it is important to understand the actual concentration of drug achieved in the experiment¹. Measuring the concentration of actual drug in the system provides an understanding of whether nominal concentrations have been achieved.

To achieve this in the Cyprotex CYP induction assay, the concentration of parent drug can be measured in the final 24 hrs of the experiment at several time points.

The in vitro induction analysis calculates fold induction relative to vehicle control. Basic methods of assessing in vitro induction analysis apply a cut-off to fold induction observed i.e. concentration-dependent increase of mRNA expression with fold changed ≥2-fold relative to vehicle control or increase >20% of positive control, at expected hepatic concentrations of drug. This provides a conservative potential risk of clinical DDI due to induction.

If a positive result of ≥2-fold or increase >20% of positive control is observed in at least one donor, the compound should be considered an in vitro inducer and in vivo follow up studies may be required¹.

Where appropriate and if a sufficient dose response relationship is observed, nonlinear regression analysis to a four-parameter sigmoidal equation is implemented to produce E_max (maximum fold induction) and EC₅₀ values (concentration of test compound which produces a fold induction of 50% of the calculated E_max). These parameters can be applied to basic models including R3 determination and/or correlation analysis and assessed in conjunction with predicted in vivo exposure data as described in the regulatory guidelines^1,2 to predict DDI risk and to determine whether follow-up clinical studies are required.

For correlation analysis, please see our relative induction score (RIS) page and FAQ.

If no apparent plateau is observed over the concentration range tested, the E_max will be constrained to the maximum observed E_max, and EC₅₀ will be determined accordingly. This avoids extrapolating beyond the measured data and is in accordance with literature. Equally, additional curve-fitting criteria in the case of atypical or bell-shaped curves are applied and data points may be excluded⁶. A decrease in induction at higher concentrations can usually be explained as a result of solubility or cytotoxicity issues.

CYP2C induction studies are only required if a positive CYP3A4 induction response has been identified, due to the co-regulatory pathways observed with these isoforms. Basic methods to determine induction risk as described previously, such as ≥2-fold induction with a concentration dependent increase observed, or increase >20% of positive control observed in at least one donor can also be utilized for these isoforms. However, it has been well documented in the literature that the inductive response of CYP2C isoforms can be variable from donor to donor, and in some cases the dynamic range of the induction response can be small⁷.

_{1) US Food and Drug Administration (2020) Final guidance for industry: In Vitro Drug Interaction Studies – Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions Guidance for Industry (available to view here).
2) EMA (2012) Guideline on the Investigation of Drug Interactions
3) Hewitt NJ et al., (2007) Induction of hepatic cytochrome P450 enzymes: methods, mechanisms, recommendations, and in vitro-in vivo correlations. Xenobiotica 37(10-11); 1196-1224
4) Watkins PB et al., (1986) Macrolide antibiotics inhibit the degradation of the glucocorticoid-responsive cytochrome P-450p in rat hepatocytes in vivo and in primary monolayer culture. J Biol Chem 261(14); 6264-6271
5) Kenny et al., (2018) Considerations from the IQ Induction Working Group in Response to Drug-Drug Interaction Guidances from Regulatory Agencies: Focus on CYP3A4 mRNA in vitro response thresholds, variability, and clinical relevance. DMD 46:1285-1303
6) Wong SG. et al., (2021) Considerations from the Innovation and Quality Induction Working Group in Response to Drug-Drug Interaction Guidance from Regulatory Agencies: Guidelines on Model Fitting and Recommendations on Time Course for In Vitro Cytochrome P450 Induction Studies Including Impact on Drug Interaction Risk Assessment., Drug Metab. Dispos., 49:94-110
7) Hariparsad et al., (2017) Considerations from the IQ Induction Working Group in Response to DrugDrug Interaction Guidances from Regulatory Agencies: Focus on Down-regulation, CYP2C Induction and CYP2B6 Positive Control. DMD 45; 1049–1059}