# In Silico Comparison of Rifampicin and 25-desacetyl Rifampicin-Induced PXR-Mediated CYP450 Transcriptional Response in 3D Primary Human Hepatocytes

## TL;DR

This study compares how two forms of rifampicin affect liver enzyme activity in human cells using mathematical models and gene analysis.

## Contribution

The study introduces a novel in silico approach to compare PXR-mediated CYP450 transcriptional responses between rifampicin and its metabolite.

## Key findings

- 25-DRIF activates PXR at a rate 20 times lower than RIF.
- CYP3A4 transcription rate constants were higher in 25-DRIF-treated cells compared to RIF-treated cells.
- CYP2C9 transcription rate constants were similar in RIF- and 25-DRIF-treated cells.

## Abstract

The pregnane X receptor (PXR) regulates the expression of cytochrome P450 (CYP) enzymes and plays a crucial role in the metabolism of various drugs. Rifampicin (RIF) is a PXR ligand that forms the primary metabolite, 25-desacetyl rifampicin (25-DRIF), which retains the antimicrobial activity of the original drug. In this study, we quantified PXR activation and its associated effects on CYP3A4, CYP2C9, and CYP2B6 enzymes in response to 25-DRIF treatment by combining mathematical modeling with long-term mRNA expression analysis of these enzymes in 3D primary human hepatocyte (3D PHH) spheroids. Our estimates suggest that 25-DRIF activates PXR at a rate 20 times lower than RIF. The PXR-dependent rate constant for CYP3A4 transcription was estimated to be higher in 3D PHHs treated with 25-DRIF than in those treated with RIF and also higher than that for CYP2B6 transcription in 3D PHHs treated with 25-DRIF. The rate constants driving PXR-dependent transcription of CYP2C9 were comparable in RIF- and 25-DRIF-treated 3D PHHs. These results demonstrate the ligand-specific nature of PXR activation and suggest that the transcription of PXR-controlled CYP enzymes is ligand- and CYP-specific in 3D PHHs. Finally, we showed that the half-maximal effective concentration (\documentclass[12pt]{minimal}
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\begin{document}$$\hbox {EC}_{50}$$\end{document}EC50) evaluated from our mathematical predictions was time-dependent, which was further validated by CYP3A4 gene reporter assays that measured RIF-induced PXR activity.

The online version contains supplementary material available at 10.1007/s11538-026-01619-1.