In Vitro Enzyme Induction

Assay SelectionTest SystemAutomation and AnalysisControls

Studies of in vitro enzyme induction are conducted in primary cultures of human hepatocytes. These studies provide information on clinically significant issues, such as:

•     Pharmacokinetic tolerance
•     Drug-drug interactions
•     Enhanced biotransformation
•     Enhanced bioactivation
•     Increased elimination (auto-induction)
•     Explanation of in vivo animal tumor formation

XenoTech excels in enzyme induction studies in human hepatocytes (the FDA's "gold standard"), which are conducted in accordance with the FDA's recommendation.  Induction can be evaluated based on changes in gene expression (qRT-PCR analysis of mRNA levels) and/or changes in enzyme activity determined either in the hepatocytes (in situ) or in microsomes prepared from the cultured hepatocytes.  Whatever your choice, you can be certain that our strict selection criteria for the cultures used in our induction studies ensure reliable results.

Enzyme Induction Studies During Drug Development

XenoTech's "gold standard" approach involves treating cultured hepatocytes from three human livers for three consecutive days with three or more concentrations of drug candidate (up to 10 x plasma Cmaxss, if possible) and appropriate positive controls (omeprazole, phenobarbital and rifampin).  Cell morphology and LDH release are used to assess cell toxicity.  Changes in the expression of genes encoding CYP enzymes (CYP1A2, CYP2B6 and CYP3A4, at a minimum), UGT enzymes and transporters (like P-glycoprotein) are based on measurement of mRNA levels by qRT-PCR.  Changes in enzyme activity are determined in microsomes isolated from the hepatocytes or in situ, as summarized below.

Enzymatic activities measured in microsomes: Microsomes isolated from cultured hepatocytes can be used to measure multiple CYP and UGT enzymes, and they can also be analyzed by western immunoblotting to support mechanistic studies (for example, to identify cases where induction of an enzyme, such as CYP3A4, is masked by irreversible inhibition, or to establish whether a decrease in enzyme activity is due to enzyme inhibition or suppression).  Analysis of mRNA levels can provide similar mechanistic information.  With this approach, microsomes prepared directly from human livers can be included as additional controls. A distinct advantage of using microsomes to measure enzymatic activities is that the initial analysis can be confined to CYP1A2, CYP2B6 and CYP3A4 and, if requested by the FDA, the analysis can be expanded to include other enzymes (such as the CYP2C enzymes) without the need to prepare three new cultures of human hepatocytes.  Additional enzymes can readily be measured at a later date using the stored microsomes from the original study cultures.

Enzymatic activities measured in hepatocytes in situ: Treatment-related changes in CYP activity can be assessed in situ. This method allows activity data to be delivered quickly and at a lower cost than the gold standard approach. We recommend supplementing activity endpoints with mRNA expression and cell toxicity assessment to aid in the interpretation of enzymatic activity results.

Enzyme Induction Screen with qRT-PCR arrays

Assay Selection

CYP1A2        Phenacetin O-dealkylation
CYP2A6        Coumarin 7-hydroxylation
CYP2B6        Bupropion hydroxylation
CYP2B6        Efavirenz 8-hydroxylation
CYP2C8        Amodiaquine N-dealkylation
CYP2C8        Paclitaxel 6α-hydroxylation
CYP2C9        Diclofenac 4´-hydroxylation
CYP2C19      S-Mephenytoin 4´-hydroxylation
CYP2D6*      Dextromethorphan O-demethylation
CYP2E1        Chlorzoxazone 6-hydroxylation
CYP3A4/5     Testosterone 6β-hydroxylation
CYP3A4/5     Nifedipine oxidation
CYP3A4/5     Midazolam 1´-hydroxylation
CYP4A11      Lauric acid 12-hydroxylation

UGT1A1        17β-Estradiol 3-glucuronidation
UGT1A4        Trifluoperazine glucuronidation
UGT1A6        1-Naphthol glucuronidation
UGT1A9        Propofol glucuronidation
UGT2B7        Morphine 3-glucuronidation

At a minimum, an assessment of enzyme induction in human hepatocytes should include CYP1A2, CYP2B6 and CYP3A4, as recommended by the FDA.

*The FDA recognizes CYP2D6 as a non-inducible enzyme. It can be included as part of an enzyme induction study upon request.

Test System

XenoTech offers an assessment of enzyme induction in the following in vitro test systems:

• Freshly plated cultures of human hepatocytes (with enzyme analysis in microsomes or in situ)
• Cryopreserved attaching human hepatocytes (with enzyme analysis in microsomes or in situ)
• Immortalized human hepatocytes, such as Fa2N-4 cells (with in situ enzyme analysis)

Automation and Analysis

•     Tecan liquid-handling system
•     Validated LC/MS/MS methods
•     High-throughput Shimadzu autosamplers
•     Deuterated internal standards

Controls

Appropriate vehicle and positive controls (prototypical inducers) are included all enzyme induction studies to permit an assessment of the Percent Effectiveness of any enzyme induction observed with the drug candidate.  The FDA requires a clinical assessment of enzyme induction when a drug candidate, at pharmacologically-relevant concentrations, is 40% or more effective as the appropriate positive control: omeprazole for CYP1A2, phenobarbital for CYP2B6 and rifampin for CYP3A4, CYP2C8, CYP2C9 and CYP2C19.