Effects of commonly used excipients on the expression of CYP3A4 in colon and liver cells

Abstract

Purpose: The objective of this investigation was to assess whether common pharmaceutical excipients regulate the expression of drug-metabolizing enzymes in human colon and liver cells.

Methods: Nineteen commonly used excipients were evaluated using a panel of experiments including cell-based human PXR activation assays, real-time RT-PCR assays for CYP3A4 mRNA expression, and immunoblot analysis of CYP3A4 protein expression in immortalized human liver cells (HepG2 and Fa2N4), human primary hepatocytes, and the intestinal LS174T cell models.

Results: No excipient activated human PXR or practically induced CYP3A4. However, three excipients (polysorbate 80, pregelatinized starch, and hydroxypropyl methylcellulose) tended to decrease mRNA and protein expression across experimental models.

Conclusion: This study represents the first investigation of the potential role of excipients in the expression of drug-metabolizing enzymes. Findings imply that some excipients may hold potential for excipient-drug interactions by repression of CYP3A4 expression.

Figure 1

Activation of human PXR in cell-based promoter reporter assays. HepG2 cells were cotransfected with PXR expression vector and CYP2B6-PBREM/XREM reporter construct as described in “Materials and Methods”. Twenty-four hours after transfection, cells were treated with excipients at concentrations indicated in Table 1, RIF (10 μM), DMSO (0.1%), or PBS (none) for 24 hrs. After harvest, firefly luciferase activities were measured using luciferase reagent normalized to protein concentration. Data represent mean ± SD (n = 3) expressed as fold over vehicle control. The dot line indicates 30% of RIF activation.

Figure 2

Excipients affect CYP3A4 expression in immortalized hepatocytes Fa2N4. Cultured Fa2N4 were treated with excipients at concentrations indicated in Table 1, positive control (RIF 10 μM) or PBS (none) and DMSO (0.1%) as respective solvent controls. (A) After treatment for 24 hrs, total RNA was isolated and realtime RT-PCR was used to detect CYP3A4 mRNA expression as outlined in “Materials and Methods”. (B) After treatment for 72 hrs, cell homogenates (20 μg) from each group were subjected to immunoblot analysis of CYP3A4 protein expression.

Figure 2

Excipients affect CYP3A4 expression in immortalized hepatocytes Fa2N4. Cultured Fa2N4 were treated with excipients at concentrations indicated in Table 1, positive control (RIF 10 μM) or PBS (none) and DMSO (0.1%) as respective solvent controls. (A) After treatment for 24 hrs, total RNA was isolated and realtime RT-PCR was used to detect CYP3A4 mRNA expression as outlined in “Materials and Methods”. (B) After treatment for 72 hrs, cell homogenates (20 μg) from each group were subjected to immunoblot analysis of CYP3A4 protein expression.

Figure 3

Excipients affect CYP3A4 expression in human primary hepatocytes. Human primary hepatocytes were prepared from two different donors (HL#14 and HL#17) and treated with RIF (10 μM), DMSO (0.1%), PBS (none) or selected excipients at concentrations indicated in Table 1 for 24 hrs mRNA (A) and 72 hrs protein (B) analysis, respectively. (A) Total RNA was isolated, reverse transcribed, then subjected to realtime RT-PCR analysis of CYP3A4 mRNA expression. (B) Whole cell homogenates (20 μg) from each group were subjected to immunoblot analysis of CYP3A4 protein expression as described in “Materials and Methods”.

Figure 4

Excipients affect CYP3A4 and MDR1 mRNA expression in LS174T cells. Human colon carcinoma LS174T cells were treated for 24 hrs with RIF (10 μM), DMSO (0.1%), PBS (none) or excipients at concentrations indicated in Table 1. As indicated in “Material and Methods”, total RNA was isolated from each group, reverse transcribed, and subjected to realtime RT-PCR analysis of CYP3A4 (A) and MDR1 (B).