Cytochrome P450 induction assay

Cytochrome P450 induction assay protocol

Test System Fresh human hepatocytes Test Compound Concentration 0.1 μM, 1 μM, 10 μM (alternative concentrations available on request) CYP Isoforms CYP1A2, CYP2B6 and CYP3A4 Number of Replicates 3 Negative Control Vehicle (typically 0.1 % DMSO) Positive Control Omeprazole (CYP1A2) Phenobarbital (CYP2B6) Dexamethasone and rifampicin (CYP3A4) Compound Requirements 100 μL of 10 mM solution Exposure Period 72 hr (media changed every 24 hrs) Probe Substrates Ethoxyresorufin (CYP1A2) Bupropion (CYP2B6) Midazolam (CYP3A4) Analysis method Fluorescent quantification of resorufin (CYP1A2); LC-MS/MS quantification of hydroxybupropion (CYP2B6) and 1-hydroxymidazolam (CYP3A4); RT-PCR of mRNA expression (CYP1A2, CYP2B6 and CYP3A4). Data Delivery Report detailing methodology, donor demographics, concentration of metabolite of probe substrate (activity), mRNA levels, fold induction above vehicle control, probability (p value) of induction and % of positive control induction.

According to a recent PhRMA perspective (Chu et al., 2009)1, the most commonly used and recommended experimental protocol for assessing enzyme induction in regulatory submissions involves the use of primary hepatocytes and evaluates catalytic activity and mRNA levels for 3 cytochrome P450 isoforms, CYP1A2, CYP2B6 and CYP3A4.

Data from the Cloe Select Cytochrome P450 Induction assay

Induction of CYP1A2 by omeprazole, CYP2B6 by phenobarbital and CYP3A4 by rifampicin was investigated using cells from three individual human donors. All donors responded to the inducers, both in terms of catalytic activity and mRNA assessment.

Figure 1

Induction of CYP1A2 catalytic activity and mRNA levels by omeprazole (50 μM) in human hepatocytes (Data represent mean fold induction of CYP1A2 ± standard deviation for a single donor, n=3 replicates)

 

Figure 2

Induction of CYP2B6 catalytic activity and mRNA levels by phenobarbital (500 μM) in human hepatocytes (Data represent mean fold induction of CYP2B6 ± standard deviation for a single donor, n=3 replicates)

 

Figure 3

Induction of CYP3A4 catalytic activity and mRNA levels by rifampicin (10 μM) in human hepatocytes (Data represent mean fold induction of CYP3A4 ± standard deviation for a single donor, n=3 replicates)

 

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Questions and answers on cytochrome P450 induction

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 either, 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. According to a recent PhRMA perspective, the most commonly used and recommended experimental protocol for assessing enzyme induction in regulatory submissions involves the use of primary hepatocytes and evaluates catalytic activity and mRNA levels for 3 cytochrome P450 enzyme isoforms, CYP1A, CYP2B6 and CYP3A4¹.

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 synthesis².
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 CYP1A, CYP2B6 and CYP3A4 gene expression can serve as sensitive representative endpoints for activation of AhR, CAR and PXR³.

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?

Four positive control compounds are screened alongside the test compounds. These control compounds are known cytochrome P450 inducers (i.e., dexamethasone and rifampicin for CYP3A4, phenobarbital for CYP2B6 and omeprazole for CYP1A).

Why should I assess both activity and mRNA expression?

The most important endpoint is the specific activity of the induced CYPs because this will ultimately determine the relative change in drug metabolism activity. However, in some cases both induction and inhibition may occur, impacting on the activity measured. Assays which include CYP mRNA expression analysis will provide mechanistic information. Due to the frequency of cytochrome P450 enzyme inhibition, conducting both activity and mRNA expression studies is recommended⁴.

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

A statistically significant increase in the formation of the isoform specific metabolite in the presence of the test compound suggests the compound is inducing the cytochrome P450 isoform. Typically, the response of the test compound is compared to the inductive effect of the positive control. For the test compound, an induction of at least 40% of the positive control induction level would indicate a positive inductive effect⁵. The plasma levels observed in vivo must be considered when interpreting the in vitro result.

What does it mean if the results of activity and mRNA expression do not appear to correlate?

Increases in CYP mRNA expression or enzyme activity at low drug concentrations with decreases in CYP mRNA expression or enzyme activity at higher drug concentrations is typical of compounds that exhibit poor solubility in culture media or cytotoxicity. Such an outcome across multiple isoforms is suggestive of cytotoxicity rather than potential enzyme inhibition. Increases in CYP mRNA expression with little or no increase in enzyme activity may occur when the test compound is both a P450 inducer and mechanism based inhibitor of the same enzyme. The mRNA expression is unaffected by the inhibition, however, the enzyme activities measurements may be confounded by concomitant inhibition and induction. When a positive induction response is seen in only one of several donor hepatocyte preparations, the result can not be ignored and may require further repeat hepatocyte studies or additional mechanistic studies to understand the spurious positive result¹.

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