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Inhibitory effects of phytochemicals on metabolic capabilities of CYP2D6(*)1 and CYP2D6(*)10 using cell-based models in vitro.

Qu Q, Qu J, Han L, Zhan M, Wu LX, Zhang YW, Zhang W, Zhou HH - Acta Pharmacol. Sin. (2014)

Bottom Line: Herbal products have been widely used, and the safety of herb-drug interactions has aroused intensive concerns.This study aimed to investigate the effects of phytochemicals on the catalytic activities of human CYP2D6(*)1 and CYP2D6(*)10 in vitro.HepG2-CYP2D6(*)1 and HepG2-CYP2D6(*)10 cell lines were successfully constructed.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Xiangya Hospital, Central South University, Changsha 410078, China [2] Xiangya Hospital, Central South University, Changsha 410008, China.

ABSTRACT

Aim: Herbal products have been widely used, and the safety of herb-drug interactions has aroused intensive concerns. This study aimed to investigate the effects of phytochemicals on the catalytic activities of human CYP2D6(*)1 and CYP2D6(*)10 in vitro.

Methods: HepG2 cells were stably transfected with CYP2D6(*)1 and CYP2D6(*)10 expression vectors. The metabolic kinetics of the enzymes was studied using HPLC and fluorimetry.

Results: HepG2-CYP2D6(*)1 and HepG2-CYP2D6(*)10 cell lines were successfully constructed. Among the 63 phytochemicals screened, 6 compounds, including coptisine sulfate, bilobalide, schizandrin B, luteolin, schizandrin A and puerarin, at 100 μmol/L inhibited CYP2D6(*)1- and CYP2D6(*)10-mediated O-demethylation of a coumarin compound AMMC by more than 50%. Furthermore, the inhibition by these compounds was dose-dependent. Eadie-Hofstee plots demonstrated that these compounds competitively inhibited CYP2D6(*)1 and CYP2D6(*)10. However, their Ki values for CYP2D6(*)1 and CYP2D6(*)10 were very close, suggesting that genotype-dependent herb-drug inhibition was similar between the two variants.

Conclusion: Six phytochemicals inhibit CYP2D6(*)1 and CYP2D6(*)10-mediated catalytic activities in a dose-dependent manner in vitro. Thus herbal products containing these phytochemicals may inhibit the in vivo metabolism of co-administered drugs whose primary route of elimination is CYP2D6.

Show MeSH
(A) The sequencing results of the C188T and G4268C mutations. (B) mRNA levels of CYP2D6 in HepG2-CYP2D6*1, HepG2-CYP2D6*10, and HepG2-pcDNA3.1. Total RNA was isolated and gene expression was assessed by quantitative real-time PCR. (C) Protein levels of CYP2D6 in HepG2-CYP2D6*1, HepG2-CYP2D6*10, and HepG2-pcDNA3.1(−). Total protein was extracted and detected by Western blotting. The results represent the mean±SD for three independent experiments.
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fig1: (A) The sequencing results of the C188T and G4268C mutations. (B) mRNA levels of CYP2D6 in HepG2-CYP2D6*1, HepG2-CYP2D6*10, and HepG2-pcDNA3.1. Total RNA was isolated and gene expression was assessed by quantitative real-time PCR. (C) Protein levels of CYP2D6 in HepG2-CYP2D6*1, HepG2-CYP2D6*10, and HepG2-pcDNA3.1(−). Total protein was extracted and detected by Western blotting. The results represent the mean±SD for three independent experiments.

Mentions: To construct the CYP2D6 expression vector, human CYP2D6*1 cDNA (wild type) was inserted into pcDNA3.1(−) and verified by sequencing. The mutations of 188T and 4268C were introduced into the wild type cDNA by site-directed mutagenesis and verified by sequencing (Figure 1A). The HepG2 cells were transfected with the control vector (pcDNA3.1), the wild-type vector (CYP2D6*1) or the mutation vector (CYP2D6*10). As shown in Figure 1B and 1C, the mRNA and protein levels of CYP2D6*1 and *10 in their respective transfected cells were remarkably higher than that in the control cells transfected with pcDNA3.1. We selected those transfected cells which did not show a significant difference between the expression of CYP2D6*1 and *10.


Inhibitory effects of phytochemicals on metabolic capabilities of CYP2D6(*)1 and CYP2D6(*)10 using cell-based models in vitro.

Qu Q, Qu J, Han L, Zhan M, Wu LX, Zhang YW, Zhang W, Zhou HH - Acta Pharmacol. Sin. (2014)

(A) The sequencing results of the C188T and G4268C mutations. (B) mRNA levels of CYP2D6 in HepG2-CYP2D6*1, HepG2-CYP2D6*10, and HepG2-pcDNA3.1. Total RNA was isolated and gene expression was assessed by quantitative real-time PCR. (C) Protein levels of CYP2D6 in HepG2-CYP2D6*1, HepG2-CYP2D6*10, and HepG2-pcDNA3.1(−). Total protein was extracted and detected by Western blotting. The results represent the mean±SD for three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4814035&req=5

fig1: (A) The sequencing results of the C188T and G4268C mutations. (B) mRNA levels of CYP2D6 in HepG2-CYP2D6*1, HepG2-CYP2D6*10, and HepG2-pcDNA3.1. Total RNA was isolated and gene expression was assessed by quantitative real-time PCR. (C) Protein levels of CYP2D6 in HepG2-CYP2D6*1, HepG2-CYP2D6*10, and HepG2-pcDNA3.1(−). Total protein was extracted and detected by Western blotting. The results represent the mean±SD for three independent experiments.
Mentions: To construct the CYP2D6 expression vector, human CYP2D6*1 cDNA (wild type) was inserted into pcDNA3.1(−) and verified by sequencing. The mutations of 188T and 4268C were introduced into the wild type cDNA by site-directed mutagenesis and verified by sequencing (Figure 1A). The HepG2 cells were transfected with the control vector (pcDNA3.1), the wild-type vector (CYP2D6*1) or the mutation vector (CYP2D6*10). As shown in Figure 1B and 1C, the mRNA and protein levels of CYP2D6*1 and *10 in their respective transfected cells were remarkably higher than that in the control cells transfected with pcDNA3.1. We selected those transfected cells which did not show a significant difference between the expression of CYP2D6*1 and *10.

Bottom Line: Herbal products have been widely used, and the safety of herb-drug interactions has aroused intensive concerns.This study aimed to investigate the effects of phytochemicals on the catalytic activities of human CYP2D6(*)1 and CYP2D6(*)10 in vitro.HepG2-CYP2D6(*)1 and HepG2-CYP2D6(*)10 cell lines were successfully constructed.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Xiangya Hospital, Central South University, Changsha 410078, China [2] Xiangya Hospital, Central South University, Changsha 410008, China.

ABSTRACT

Aim: Herbal products have been widely used, and the safety of herb-drug interactions has aroused intensive concerns. This study aimed to investigate the effects of phytochemicals on the catalytic activities of human CYP2D6(*)1 and CYP2D6(*)10 in vitro.

Methods: HepG2 cells were stably transfected with CYP2D6(*)1 and CYP2D6(*)10 expression vectors. The metabolic kinetics of the enzymes was studied using HPLC and fluorimetry.

Results: HepG2-CYP2D6(*)1 and HepG2-CYP2D6(*)10 cell lines were successfully constructed. Among the 63 phytochemicals screened, 6 compounds, including coptisine sulfate, bilobalide, schizandrin B, luteolin, schizandrin A and puerarin, at 100 μmol/L inhibited CYP2D6(*)1- and CYP2D6(*)10-mediated O-demethylation of a coumarin compound AMMC by more than 50%. Furthermore, the inhibition by these compounds was dose-dependent. Eadie-Hofstee plots demonstrated that these compounds competitively inhibited CYP2D6(*)1 and CYP2D6(*)10. However, their Ki values for CYP2D6(*)1 and CYP2D6(*)10 were very close, suggesting that genotype-dependent herb-drug inhibition was similar between the two variants.

Conclusion: Six phytochemicals inhibit CYP2D6(*)1 and CYP2D6(*)10-mediated catalytic activities in a dose-dependent manner in vitro. Thus herbal products containing these phytochemicals may inhibit the in vivo metabolism of co-administered drugs whose primary route of elimination is CYP2D6.

Show MeSH