May 2019

Induction Junction, What’s Your Function?

Author:  Madison Knapp, Rebecca Campbell, Dr. Joanna Barbara
Posted:  13 May 2019

Why do induction studies?

Induction potential is an important piece of the drug-drug interaction (DDI) component of an IND submission. Simply put, we are evaluating the upregulation of drug metabolizing enzymes (or induction), cytochrome P450 (CYP) enzymes in this case. If a drug is an inducer (or perpetrator) of a CYP enzyme that is responsible for the metabolism of another drug (or victim), then administration of the perpetrator and victim concomitantly can mean faster clearance of the victim drug-- or of the perpetrator itself in the case of autoinduction. This can lead to decreased therapeutic effects due to a lower concentration of the victim drug or toxicity attributed to increased metabolite formation.
 
Common examples of victim drugs with a narrow therapeutic index include oral contraceptives and the blood thinner warfarin. Knowing the induction potential of your drug candidate is necessary in understanding safety parameters and can inform labeling and dosing considerations in the clinic. SEKISUI XenoTech offers induction studies using primary human hepatocytes to evaluate induction potential in accordance with FDA and EMA guidelines.
 

What receptor pathways are involved in preclinical definitive studies?

In our standard induction studies, we evaluate induction of CYP1A2 (regulated by the AhR pathway), CYP2B6 (regulated by CAR), and CYP3A4 (regulated by PXR). PXR is a receptor pathway commonly involved in expression of several drug metabolizing enzymes and transporters; induction of CYP 3A4 may create the need to explore induction of other proteins. Therefore, we offer follow-up or concurrent analysis of the CYP2C enzymes when there is induction of CYP3A4.
 
Enzyme Receptor Acronym
CYP1A2 Aryl hydrocarbon receptor AhR
CYP2B6 Constitutive androstane receptor CAR
CYP3A4 etc. Pregnane X receptor PXR
 

What is used to measure induction?

CYP induction is often a result of the activation of gene transcription, most commonly by nuclear receptor activation, so the measurement of mRNA expression is the agency-recommended method of evaluating induction (which reduces any potential effects from enzyme inhibition masking induction). You can evaluate induction potential using either mRNA or enzyme activity endpoints by comparing the response of the test article to the response of a vehicle control (i.e., fold change). The FDA recommends mRNA, whereas the EMA requires it. We choose to measure mRNA rather than activity in our standard study design for CYPs 1A2, 3A4, and 2B6 so clients can submit to either or both authorities, and because mRNA is cost-saving for the customer compared to activity measurements. However, where appropriate we can measure activity instead, as would be the case for CYP2C induction.
 

What is involved in a standard study?

Two supporting assessments are performed to determine the concentrations evaluated during the experiment: the first assessment determines the test article solubility in an acceptable solvent for the test system and determines solubility in test system (i.e., cell culture media) with minimal organic solvent.
 
The second assessment is used to evaluate potential cytotoxicity associated with the test article. Cell health is monitored throughout the study to ensure cytotoxicity is not a factor; if it is, cell function is compromised and we cannot accurately detect induction. In addition to the qualitative cell morphology photomicrograph comparison, we test for LDH (lactate dehydrogenase) release to evaluate cell health and to determine the concentration at which cell membrane disruption starts to occur. Cytotoxicity can be evaluated prior to the induction evaluation or concurrently.
 
All human hepatocytes used are pre-characterized for induction, and if appropriate we have the option to use lots characterized for RIS.
 
In the main experiment, we treat cryopreserved hepatocytes from three individual donors cultured in a collagen-sandwich configuration, as required by FDA and EMA, by applying test article (7 concentrations) and controls once daily for three consecutive days in cell culture media. Samples are then collected for mRNA harvest or enzyme activity assays are performed.The mRNA samples are then analyzed by qRT-PCR (real time quantitative reverse transcription-polymerase change reaction). Activity assays involve LC-MS/MS. Relative quantitation compares the expression of the test article-treated samples to the vehicle control-treated samples (i.e., fold change over vehicle control), samples are analyzed for mRNA change (or enzyme activity).
 
Concurrent to the main assay, spent media analysis is performed to measure drug concentration in media over the course of the last day of incubation through 24 hours post the final treatment.
 

Why do spent media analysis?

Spent media analysis is necessary because it is required by FDA and EMA; the test system might not take up as much test article as predicted, or you might find that you need to dose more than once per day (although this is rare). Standard dosing is once daily for three days, and while it is rare to see results necessitate dose adjustment, it is possible as the drug may be immediately cleared precluding accurate induction assessment. In spent media analysis, “spent” (used) incubation media is collected and analyzed using LC-MS/MS to measure drug concentration during and following incubation.
 

Why use cryopreserved hepatocytes rather than fresh or immortalized cell lines?

Cryopreserved hepatocytes are the most appropriate choice for convenience and quality in induction assays. Because we use hepatocyte lots that are processed from donor tissue in-house, we can select the most appropriate available lots. Our cryopreserved lots are pre-characterized, so we know they are able to achieve required standards. Immortalized cell lines are not ideal for use in induction assays because most are known to show poor expression of different key enzymes. Additionally, while immortalized cell lines are acceptable test systems for FDA submission, the EMA does not consider them to be appropriate primary test systems, but rather complementary.
 

Is using fewer TA concentrations a good way to save money?

No. Most of the cost is associated with setting up the cultures and incubation. In the case of a positive response, having fewer than seven TA concentrations to compare can make it difficult to generate a useful sigmoidal induction curve. In the long run it will be more expensive to go back and repeat to get data points to establish concentration-dependence and determine induction parameters.
 

Does your standard study meet both EMA and FDA’s recently revised standards for submission?

Yes. In its most recent guidance update, the FDA made standard some study components previously appropriate for some clients but not all. Requirements by the FDA now closely mirror those by the EMA. Although there are a few key differences, our studies are designed to meet the most stringent criteria.
 

How are TA concentrations selected?

Both guidance documents suggest consideration of intestinal concentration (dose/250mL ) if the drug will be orally administered. In addition, potential plasma exposures are considered. FDA suggests treating up to one order of magnitude (10x) over Cmax steady state, unbound, whereas the EMA suggests 50x Cmax steady state, unbound. Both recommendations are rooted in conservative safety factors, so in our evaluations we use 50x above in order to cover both sets of guidance requirements.
 

What constitutes a positive response?

Fold-change in mRNA expression or enzyme activity over a vehicle control is the primary endpoint. If mRNA or activity fold-change is more than 2-fold compared to the vehicle control and more than 20% of fold-change observed compared to the positive control in any of the three donors, this qualifies as a positive result according to both agencies. Concentration-dependence is also a consideration.
 

What positive controls are used?

Known inducers for each of the three nuclear receptors (AhR, CAR and PXR) are used in standard studies as positive controls against which we can compare a test article’s potential. While the FDA and EMA agree on what compounds ought to be used to evaluate AhR and PXR (omeprazole and rifampin respectively), there is some discrepancy in what is best for the CAR. Upon our team’s review of current relevant literature, we have decided to use phenobarbital over CITCO to evaluate CYP2B6 induction. The reason for this is not just because most of our clients are submitting to the FDA, but also that it’s more consistent and accounts for ‘crosstalk’ between CAR and PXR which does occur in vivo. CITCO has been demonstrated to be more selective for CAR but the fold change is highly variable compared to phenobarbital, negatively impacting dependability of results. It is important to remember that guidance documents published by agencies describe recommendations, not stringent requirements. While we can use CITCO if requested by the client, the EMA does accept studies conducted using phenobarbital as the positive control to evaluate CYP2B6 induction.
 

Is a negative control necessary?

FDA does require it so it is included in our study design, although some experts argue it is no more useful than the vehicle control.
 

What happens if there is a positive induction response?

Knowing it’s a positive isn’t very helpful unless you know what to do with that information-- consistent with the conservative nature of FDA guidance, any positive response observed in even just one donor warrants further investigation. If a positive response was observed in the CYP3A4, then CYP2C data must be gathered to further elucidate PXR interaction. The follow-up may be further in vitro evaluation or clinical evaluations.
 
As follow up to any concentration-dependent positive response, we use the dose and fold change values to calculate the induction parameters of EC50 (the concentration associated with half-maximal induction) and Emax (maximal fold induction in vitro). We determine these values by plotting experimental data (fold change) using sigmoidal curve plotting.
 

How is the 2C response evaluated?

If CYP3A4 induction is observed, it must be followed up with analysis of the CYP2C subfamily of CYP enzymes (CYP2C8, CYP2C9, and CYP2C19) because they are known to be induced through the same pathway (PXR). If you know from screening data that there will be CYP3A4 induction, we recommend including CYP2C analysis in the study from the beginning to save time and money. CYP2C induction is measured by enzyme activity rather than mRNA because mRNA fold change is highly variable for all CYP2C enzymes and often the the positive control response is less than 2-fold. Though we only use hepatocyte cultures characterized for CYP2C induction, even activity fold change may still be less than 2-fold. Even so, activity has shown to be the more reliable endpoint for definitive studies.
 

What do I do with EC50 and Emax values in the event of a positive response?

Once EC50 and Emax values are established, follow-up actions are proceeded stepwise from simple to complex: basic modeling, correlation methods, static mechanistic modeling, and dynamic mechanistic model (PBPK). We routinely perform correlation analysis, basic modeling, and static mechanistic modeling to explore relevant ratios. While the 2017 draft guidance for in vitro DDI provides a framework for such studies, there is a separate more prescriptive document published called Physiologically-Based Pharmacokinetic Analyses – Format and Content, which can be used in planning follow-up PBPK modeling.
 

This digest reflects major concepts from a recent webinar delivered by Dr. Joanna Barbara, Vice President of Scientific Operations at SEKISUI XenoTech, titled “In Vitro Induction Studies: Elements of Design and Important Considerations in Data Analysis.” Still have questions or want to get started on an induction study with us? SEKISUI XenoTech has spent 25 years building a reputation for expertise and dependable, high-quality data tailored to industry needs. Talk to an expert today! 

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About the Authors
Dr. Joanna Barbara obtained her Ph.D. in Analytical Chemistry from the University of Florida. She joined SEKISUI XenoTech in 2007, has authored or coauthored numerous scientific posters and papers, and has represented SEKISUI XenoTech as an invited speaker at various analytical and drug metabolism conferences. Joanna has extensive experience in DMPKregulatory compliance, and process and project management. As Vice President of Scientific Operations, she is responsible for the development, design, operation, and improvement of SEKISUI XenoTech's Scientific Division.

Madison Knapp received her BS from the University of Missouri – Columbia and became SEKISUI XenoTech’s Scientific Communications Coordinator in 2019 after serving in similar positions at CropLife America, Bond Life Sciences Center and the CAFNR Office of Communications.

Rebecca Campbell joined SEKISUI XenoTech in 2001. Prior to joining Sekisui XenoTech, she obtained her B.S. in Biology from the University of Central Missouri and studied toxicology and pharmaceutical sciences at the University of Missouri-Kansas City School of Pharmacy. Rebecca has extensive experience in in vitro CYP induction studies, regulatory compliance, and project management. As a Principal Scientist in Scientific Operations, she is responsible for the conduct and oversight of the nonclinical, drug interaction-related contract studies in the Program Oversight Department of SEKISUI XenoTech's Scientific Division.

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