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ADME PK

Cytochrome P450 induction assay

Understand the potential drug-drug interaction liabilities of your compounds by using our cytochrome P450 (CYP450) induction assay.

Cytochrome P450 induction is one of Cyprotex's in vitro experimental ADME services. Cyprotex deliver 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 Emax 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.
Cultured hepatocytes (cryopreserved or fresh) are the preferred in vitro system for induction (and down-regulation) in vitro studies.

2EMA (2012) Guideline on the investigation of drug interactions

Protocol

Cytochrome P450 induction assay protocol

Test System Cryopreserved or fresh human hepatocytes (3 donors recommended)
HepaRG cells are available on request
Test Article Concentration 1, 3 or 6 concentrations (dependent upon unbound Cmax, dose, solubility and cytotoxicity) plus vehicle control, in triplicate
CYP Isoforms CYP1A2, CYP2B6 and CYP3A4
For CYP2C, UGT or transporter studies, please contact directly for information
Negative Control Non-inducer
Positive Control Omeprazole (CYP1A2)
Phenobarbital (CYP2B6)
Rifampicin (CYP3A4)
Test Article Requirements Dependent on top concentration (recommend 0.1% DMSO in incubation)
Exposure Period 72 hr (Media changed every 24 hrs)
Probe Substrates for Catalytic Activity Phenacetin (CYP1A2)
Bupropion (CYP2B6)
Midazolam (CYP3A4)
Analysis Method LC-MS/MS quantification of acetaminophen (CYP1A2), hydroxybupropion (CYP2B6) and 1-hydroxymidazolam (CYP3A4)
qRT-PCR for relative mRNA expression levels (CYP1A2, CYP2B6 and CYP3A4).
Data Delivery Report detailing methodology, donor demographics, mRNA levels, fold induction relative to vehicle control, concentration of metabolite of probe substrate, Emax, EC50 and F2 (concentration which leads to a 2-fold increase above Emin) if appropriate.

Supplementary Assays
Preliminary aqueous solubility assessment

Cytotoxicity assessment in primary human hepatocytes or HepaRG (e.g. MTT)

Assessment of non-specific binding

Measurement of parent drug on final day of dosing

Data

Data from Cyprotex's Cytochrome P450 Induction assay

Increase in mRNA expression plotted against concentration for positive control compounds omeprazole, phenobarbital and rifampicin for induction of CYP1A2, CYP2B6 and CYP3A4 respectively in a single donor.

 
CYP induction by omeprazole
Figure 1
Induction of CYP1A2 mRNA levels by omeprazole in cryopreserved human hepatocytes.
CYP induction by phenobarbital
Figure 2
Induction of CYP2B6 mRNA levels by phenobarbital in cryopreserved human hepatocytes.
CYP induction by rifampicin
Figure 3
Induction of CYP3A4 mRNA levels by rifampicin in cryopreserved human hepatocytes.

Q&A

Why should I assess the cytochrome P450 induction potential of my compound?

Cytochromes P450 are a family of enzymes which play a major role in the metabolism of drugs. If a cytochrome P450 enzyme is induced by a compound it may both increase the metabolism of itself (autoinduction), or a concurrent therapy, and so reduce plasma levels resulting in a decrease in efficacy. Induction of cytochrome P450 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 co-medication restrictions. The regulatory authorities2 recommend preferably evaluating CYP induction prior to Phase II clinical trials.

By what mechanisms can cytochrome P450 induction occur?

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

1) 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 synthesis3.

2) Nuclear receptor-mediated induction. The most common mechanism of cytochrome P450 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 respectively4.

Is inter-assay variation observed in the induction studies?

If the donor has been prescribed enzyme-inducing drugs prior to the resection, or is a smoker, then it is possible that background levels of cytochrome P450 may be elevated. The level of induction may be less dramatic in these circumstances. This is one of the reasons why it is recommended to test at least 3 different donors1. Typically, the response of the test compound is compared to the inductive effect of the positive control compounds in each donor to account for the variability across donors.

What controls are included in the cytochrome P450 induction assay?

Three positive control compounds are assessed alongside the test compounds. These control compounds are known cytochrome P450 inducers (i.e., rifampicin for CYP3A4, phenobarbital for CYP2B6 and omeprazole for CYP1A2). A compound which is a known non-inducer can also be assessed.

Should I assess both activity and mRNA expression?

The specific activity of the induced CYPs determine the relative change in drug metabolism activity at the protein level. However, in some cases both induction and inhibition may occur, masking the level of induction when catalytic activity only is measured. For this reason, the regulatory authorities now recommend assessing mRNA expression analysis1,2, unless induction due to protein stabilization is suspected, when activity should also be assessed2.

How do I interpret the data from the cytochrome P450 induction assay?

The fold change and the probability that a statistically significant difference exists between the wells exposed to test compound and the vehicle control wells is calculated and compared to positive control responses to allow for inter-donor comparison. Where appropriate and if a sufficient dose response relationship is observed, nonlinear regression analysis to a four-parameter sigmoidal equation is implemented to produce Emax (maximum fold induction), EC50 values (concentration of test compound which produces a fold induction of 50% of the calculated Emax) and F2 (concentration which leads to a 2-fold increase above Emin). These parameters can be applied to basic models stated in the regulatory guidelines1,2 to estimate DDI risk and to determine whether follow-up clinical studies are required.

References

1 FDA (2012) Draft Guidance for Industry: Drug Interaction Studies - Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations
2 EMA (2012) Guideline on the Investigation of Drug Interactions
3 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
4 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

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