The differences in drug disposition gene induction by rifampicin and rifabutin are unlikely due to different effects on important pregnane X receptor (NR1I2) splice variants

Abstract

Rifampicin and rifabutin can activate the pregnane X receptor (PXR, NR1I2), thereby inducing pharmacokinetically important genes/proteins and reducing exposure to co-administered drugs. Because induction effects vary considerably between these antibiotics, differences could be due to unequal rifamycin-induced activation or tissue expression of the three major NR1I2 splice variants, PXR.1 (NM_003889), PXR.2 (NM_022002), and PXR.3 (NM_033013). Consequently, PXR activation (PXR reporter gene assays) and mRNA expression levels of total NR1I2, PXR.1, PXR.2, and PXR.3 were investigated by polymerase chain reaction in colon and liver samples from eleven surgical patients, in LS180 cells, and primary human hepatocytes. Compared to the colon, total NR1I2 mRNA expression was higher in the liver. Both tissues showed similar expression levels of PXR.1 and PXR.3, respectively. PXR.2 was not quantifiable in the colon samples. Rifampicin and rifabutin similarly enhanced PXR.1 and PXR.2 activity when transfected into LS180 cells, while PXR.3 could not be activated. In LS180 cells, rifampicin (10 μM) reduced total NR1I2 and PXR.3 expression 2-fold after 24 h, while rifabutin (10 μM) increased total NR1I2, PXR.1, PXR.2, and PXR.3 mRNA by approx. 50% after 96-h exposure. In primary human hepatocytes, rifampicin (10 μM) suppressed total NR1I2, PXR.1, and PXR.3 after 48-h exposure, and rifabutin (10 μM) had no significant impact on total NR1I2 or any of the splice variants studied. In conclusion, both antibiotics activated the studied PXR splice variants similarly but modified their expression differently. While rifampicin can suppress mRNA of PXR forms, rifabutin rather increases their expression levels.

Keywords: Expression; Pregnane X receptor; Primary human hepatocytes; Rifabutin; Rifampicin; Splice variant.

Fig. 1

Structure of the three NR1I2 mRNA transcripts (PXR.1, PXR.2, PXR.3) and binding sites of the respective primer pairs. Exons 2 through 9 are represented as gray rectangles, the alternative exon 1 (1a or 1b) is shown in white. The region of exon 5 that is only expressed in PXR.1 and PXR.2 is highlighted in black. The primer pair for total NR1I2 (NR1I2-for; NR1I2-rev; gray arrows) binds to exon 8/9 (forward primer) and 9 (reverse primer). Primer pair combinations only for NR1I2 splice variants bind to regions being selectively present in the respective transcript variant. PXR.1-for recognizes PXR.1 and PXR.3; PXR.1-rev recognizes PXR.1 and PXR.2; PXR2-for recognizes only PXR.2; PXR.2-rev recognizes PXR.1-3; PXR.3-for recognizes only PXR.3; PXR.3-rev recognizes PXR1-3. The combination of PXR.1-for/rev, PXR.2-for/rev, and PXR.3-for/rev is thus selective for only one splice variant

Fig. 2

Relative PXR reporter activity in LS180 cells after selective overexpression of NR1I2 splice variants (PXR.1, PXR.2, or PXR.3) and subsequent 72-h exposure to rifampicin (open circles) or rifabutin (closed circles). Data shown is the mean ± S.D. of three independent biological replicates with triplicates for each concentration. Data were fitted according to an E_max model (4-parameter logistic equation; variable with slope)

Fig. 3

Impact of 10 μM rifampicin (white bars) or rifabutin (gray bars) for 24 h, 96 h, or 144 h on mRNA expression of total NR1I2 or its splice variants in LS180 cells, normalized to solvent control (black bars). Data shown is the mean ± S.D. of four independent biological replicates. Statistical significance was evaluated by non-parametric Kruskal-Wallis test and Dunn’s test, correcting for multiple comparisons

Fig. 4

Relative mRNA expression of total NR1I2 (A) or its splice variants (B) in primary human hepatocytes after treatment with rifampicin or rifabutin (both 10 μM) for 48 h. Data points shown are the mean ± S.D. of hepatocyte samples (pooled from three independent experiments), evaluated by two technical replicates (PCR runs). Statistical significance was evaluated by non-parametric Kruskal-Wallis test and Dunn’s test (correcting for multiple comparisons), comparing all solvent control hepatocyte samples with all rifampicin-treated or rifabutin-treated hepatocyte samples

Fig. 5

Relative mRNA expression of total NR1I2 (A) or its splice variants (B) in matched samples of healthy liver and colon. Data points shown are the mean relative expression of each sample, evaluated by two technical replicates (PCR runs). Statistical significance was evaluated by non-parametric Wilcoxon matched-pairs signed-rank test. Samples with unreliable housekeeping gene expression or outliers identified by ROUT testing (Q-level at 1%) were excluded from the analysis