Abstract
Rifampicin is a strong inducer of cytochrome P450 (CYP3A4) and P-glycoprotein (P-gp/ABCB1), leading to profound drug–drug interactions. In contrast, the chemically related rifabutin does not show such pronounced induction properties in vivo. The aim of our study was to conduct a comprehensive analysis of the different induction potentials of rifampicin and rifabutin in primary human hepatocytes and to analyze the mechanism of potential differences. Therefore, we evaluated CYP3A4/ABCB1 mRNA expression (polymerase chain reaction), CYP3A4/P-gp protein expression (immunoaffinity–liquid chromatography–mass spectrometry, IA-LC-MS/MS), CYP3A4 activity (testosterone hydroxylation), and considered intracellular drug uptake after treatment with increasing rifamycin concentrations (0.01–10 µM). Furthermore, rifamycin effects on the protein levels of CYP2C8, CYP2C9, and CYP2C19 were analyzed (IA-LC-MS/MS). Mechanistic analysis included the evaluation of possible suicide CYP3A4 inhibition (IC50 shift assay) and drug impact on translational efficiency (cell-free luminescence assays). Rifabutin accumulated 6- to 15-fold higher in hepatocytes than rifampicin, but induced CYP3A4 mRNA comparably to rifampicin (e. g. rifampicin 61-fold vs. rifabutin 44-fold, 72 h). While rifampicin for example enhanced protein (10 µM: 21-fold) and activity levels considerably (53-fold), rifabutin only slightly increased CYP3A4 protein expression (10 µM: 3.3-fold) or activity (11-fold) compared to rifampicin after 72 h. Both rifamycins similarly influenced expression of other eliminating proteins. A potential CYP3A4 suicide inhibition by a specific rifabutin metabolite or disruption of ribosome function were excluded experimentally. In conclusion, the lack of protein enhancement, could explain rifabutin’s weaker induction-related drug–drug interaction risk in vivo.
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Acknowledgements
We thank Vanessa Scherer, Michaela Rolf, and Stephanie Liehner for the excellent technical assistance. We also thank Dr. Thomas Ebner, Dr. Eva Ludwig-Schwellinger, and Prof. Dr. Gerd Mikus for their valuable input regarding experimental approaches in our studies. We acknowledge the support of the non-profit foundation Human Tissue & Cell Research, which holds human tissue on trust, making it broadly available for research on an ethical and legal basis.
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Julie Nilles receives an allowance from the Boehringer Ingelheim Pharma GmbH & Co. KG. The company, however, had no role in the study design, the collection, analysis or interpretation of the data; and in the decision to submit the article for publication.
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Experiments, data collection, and analyses were performed by JN and SR. The first draft of the manuscript was written by JN, SR, and DT. JW and WE.H guided experimental approaches and commented on previous versions of the manuscript, which led to considerable improvements of the first draft. All authors read and approved the final manuscript.
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Nilles, J., Theile, D., Weiss, J. et al. Lack of CYP3A4 protein induction despite mRNA induction in primary hepatocytes exposed to rifabutin as a possible explanation for its low interaction risk in vivo. Arch Toxicol (2024). https://doi.org/10.1007/s00204-024-03763-w
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DOI: https://doi.org/10.1007/s00204-024-03763-w