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CYP induction assays – which way to go?

5 September 2018

In drug discovery and development, there are often several options available for addressing research questions, which may vary considerably in laboriousness, costs and the level of data they produce. Therefore, it is important to choose the right study set up at the right time to obtain correct and relevant answers for the project. As an example of different study approaches, let’s have a look at alternatives for studying CYP enzyme induction in vitro.

The induction of CYP enzymes can increase the elimination of co-administered drugs and lead to sub-therapeutic drug concentrations in the body. Therefore, the drug regulatory authorities require CYP induction evaluation for all NCEs prior entering clinical studies. Due to the mechanisms behind the CYP induction, it can be investigated in vitro in different ways.

Typically, CYP induction is caused by increased gene expression which leads to increased enzyme synthesis. This is regulated by nuclear receptors, mainly PXR (pregnane X receptor), AhR (aryl hydrocarbon receptor) and CAR (constitutive androstane receptor), and their activation leads to increased gene expression. Therefore, measuring nuclear receptor activation can be utilised to predict CYP enzyme induction indirectly. In nuclear receptor activation assays, the endpoint can be measured as increase in luciferase activity, when cells transfected with a luciferase reporter gene are used. PXR regulates the expression of most important CYP enzymes responsible for drug metabolism, mainly the CYP3A family, and hence, the most common studies for screening purposes are based on measuring PXR activation. In addition, investigating AhR and CAR receptor activation may provide valuable information about the induction of CYP1A and CYP2B family, respectively. The nuclear receptor approach is very useful in early drug development for screening possible CYP induction properties.

Perhaps one step more sophisticated system to evaluate CYP enzyme induction uses hepatic cell-lines, such as HepaRG, which are easily available and express multiple CYP enzymes providing information of CYP enzymes regulated by different pathways. HepaRG is a widely used cell-line for studying CYP enzyme induction providing a robust and reproducible method, without the effect of donor variability, as is the case also with other immortal cell lines. Importantly, the HepaRG has high CYP activity and responds consistently to generally accepted inducers, which are used as positive controls. The most sensitive endpoint to measure CYP induction is to analyse mRNA levels with real-time qPCR. In addition, the enzyme activity might be worthwhile to study to gain additional information, as the compound may act as an inducer and inhibitor of the same enzyme. Furthermore, in some cases protein stabilisation may be the reason for increased enzymatic activity. Compared to the nuclear receptor activation approach, mRNA level and enzyme activity measurement studies provide more comprehensive information and more accurate predictions can be done based on the results.

The most ideal experimental environment to study CYP induction uses primary human hepatocytes, as they present the analogous metabolic capacity of human liver. Pooled, cryopreserved human hepatocytes are easily available and a good option to evaluate CYP induction. From the regulatory point of view, single donor human hepatocytes at least from three different donors are required to assess enzyme induction using mRNA levels and/or enzyme activity as the endpoint. Although, the regulatory compliant CYP induction studies give the most comprehensive results, previously mentioned options are more cost-efficient for screening purposes and for providing sufficient information for internal decision-making also later in development.

When designing and choosing the study set up, it is important to consider various aspects as different approaches affect also the gained information. In some cases, a very robust, fast and cost-efficient experiment may be a good option, whereas in other cases the whole regulatory requirements should be addressed. In addition, it might be worthwhile to consider, whether additional, e.g. cytotoxicity studies, could be performed simultaneously. If feeling unsure which assay to perform, many CROs are able to assist in the study design to find the best solutions to meet the customers’ needs.

Written by Miia Kovalainen

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