title: The use of a high-throughput luminescent method to assess CYP3A enzyme induction in cultured rat hepatocytes.
abstract: Assessment of a new chemical entity for cytochrome P450 (CYP) enzyme induction at an early stage in discovery is crucial to prevent potential drug-drug interactions. CYP3A, the most abundant CYP isoform in the liver, metabolizes approximately 50% of drugs currently on the market and is also a highly inducible enzyme. The use of both rat and human hepatocyte culture for the prediction of in vivo CYP3A induction has become refined and validated and is considered a standard in vitro model. The current evaluation of CYP3A enzyme induction involves the use of substrates requiring subsequent analysis of metabolites by high-performance liquid chromatography/mass spectrometry, which adds considerable time and cost. In the present study, we describe the use of a novel luminogenic substrate, luciferin-6'-pentafluoro-benzyl ether (PFBE), which allows for a fast and selective measurement of CYP3A enzyme induction in cultured rat hepatocytes. The extent of induction was evaluated using cells treated for 3 d with the prototypical inducers, dexamethasone, phenobarbital, and pregnenolone 16 alpha-carbonitrile (PCN). Enzyme activity was measured in the treated cells either by the depentafluorobenzylation of luciferin-PFBE or the testosterone 6-beta-hydroxylation. Using both methods, dexamethasone and PCN-treated cells exhibited strong CYP3A activity, whereas phenobarbital treatment resulted in a weak response. The fold induction varied between both methods, but this variability can be controlled by normalizing data from each treatment to a positive control. The results indicate that luciferin-PFBE is an attractive alternative to the use of conventional substrate, testosterone, providing a sensitive, robust, and rapid method compatible with the multiwell plate format for the assessment of CYP3A induction.
authors.affiliation: Institute for Diabetes Discovery, 23 Business Park Drive, Branford, CT 06405, USA. Martha.garcia@idd-diabetes.com
authors.forename: Martha C
authors.initials: MC
authors.lastname: Garcia
authors.forename: Dongping
authors.initials: D
authors.lastname: Ma
authors.forename: A Thomas
authors.initials: AT
authors.lastname: Dicioccio
authors.forename: James
authors.initials: J
authors.lastname: Cali
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis*; D004790:Enzyme Induction; D049454:Firefly Luciferin / Q000737:chemistry / Q000378:metabolism; D022781:Hepatocytes / Q000201:enzymology*; D008163:Luminescent Measurements / Q000379:methods*; D051381:Rats; D015203:Reproducibility of Results; D013379:Substrate Specificity
chemical_list: D049454:Firefly Luciferin; D051544:Cytochrome P-450 CYP3A