• YAKHAK HOEJI
• /
• v.59 no.2
• /
• pp.49-54
• /
• 2015

In the present study we evaluated comparative herb-drug interaction potential of red ginseng total powder, ginsenoside Rg1, and Rb1 by inhibition of CYP isoforms including CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 using pooled human liver microsomes (HLMs). As measured by liquid chromatography-electrospray ionization tandem mass spectrometry, red ginseng total powder inhibited significantly activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and testosterone 6-beta hydroxylation by CYP3A4, but the $IC_{50}$ values were higher than $556{\mu}g/ml$. Activities of CYP2B6, CYP2C9, CYP2D6 and CYP3A4 were inhibited by ginsenoside Rb1. Also, activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and testosterone 6-beta hydroxylation by CYP3A4 were inhibited by ginsenoside Rg1. The $IC_{50}$ values of ginsenoside Rb1 and Rg1 were higher than $200{\mu}g/ml$. Based on $IC_{50}$ values against CYP isoforms, ginsenosides-drug interactions by CYP inhibition may be very low in clinical situations.

• Toxicological Research
• /
• v.13 no.1_2
• /
• pp.117-125
• /
• 1997

In order to assess the possibility whether CYP2D is involved in caffeine metabolism, we have purified and characterized the rat liver microsomal cytochrome P4502D1 (CYP2D1), equivalent to CYP2D6 in human liver, and have utilized the reconstituted CYP2D1 in the metabolism of 4 primary caffeine (1, 3, 7-trimethylxanthine) metabolites such as paraxanthine (1, 7-dimethylxanthine), 1, 3, 7-trimethylurate, theophylline (1, 3-dimethylxanthine) and theobromine (3, 7-dimethylxanthine). Rat liver CYP 2D1 has been purified to a specific content of 8.98 nmole/mg protein (13.4fold purification, 1.5% yield) using $\omega$-aminooctylagarose, hydroxlapatite, and DE52 columns in a sequential manner. As judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified CYP2D1 was apparently homogeneous. Molecular weight of the purified CYP2D1 was found to be 51, 000 Da. Catalytic activity of the purified and then reconstituted CYP2D1 was confirmed by using bufuralol, a known subsFate of CYP2D1. The reconstituted CYP2D1 was found to produce to 1-hydroxylbufuralol at a rate of 1.43$\pm$0.13 nmol/min/nmol P450. The kinetic analysis of bufuralol hydroxylation indicated that Km and Vmax values were 7.32$\mu M$ and 1.64 nmol/min/nmol P450, respectively. The reconstituted CYP2D1 could catalyze the 7-demethylation of PX to 1-methylxanthine at a rate of 12.5 pmol/min/pmol, and also the 7- and 3- demethylations of 1, 3, 7-trimethylurate to 1, 3-dimethylurate and 1, 7-dimethylurate at 6.5 and 12.8 pmol/min/pmol CYP2D1, respectively. The reconstituted CYP2D1 could also 3-demethylate theophylline to 1-methylxanthine at 5 pmol/min/pmol and hydroxylate the theophylline to 1, 3-dimethylurate at 21.8 pmol/min/pmol CYP2D1. The reconstituted CYP2D1, however, did not metabolize TB at all (detection limits were 0.03 pmol/min/pmol). This study indicated that CYP2D1 is involved in 3-and 7-demethylations of paraxanthine and theophylline and suggested that CYP2D6 (equivalent to CYP2D1 in rat liver) present in human liver may be involved in the secondary metabolism of the primary metabolites of caffeine.

• Biomolecules & Therapeutics
• /
• v.16 no.4
• /
• pp.336-342
• /
• 2008

The resveratrol analogue piceatannol (3,5,3',4'-tetrahydroxy-trans-stilbene) is a polyphenol present in grapes and wine and reported to have anti-carcinogenic activities. To investigate the mechanism of anticarcinogenic activities of piceatannol, the effects on CYP 1 enzymes were determined in Escherichia coli membranes coexpressing recombinant human CYP1A1, CYP1A2 or CYP1B1 with human NADPH-P450 reductase. Piceatannol showed a strong inhibition of CYP1A1 and CYP1B1 in a concentration-dependent manner, and $IC_{50}$ of human CYP1A1 and CYP1B1 was 5.8 ${\mu}M$ and 16.6 ${\mu}M$, respectively. However, piceatannol did not inhibit CYP1A2 activity in the concentration of up to 100 ${\mu}M$. Piceatannol exhibited 3-fold selectivity for CYP1B1 over CYP1A1. The mode of inhibition of piceatannol was non-competitive for CYP1A1 and CYP1B1. The result that piceatannol did not inhibit CYP1B1-mediated $\alpha$-naphthoflavone ($\alpha$-NF) metabolism suggests piceatannol may act as a non-competitive inhibitor as well. In human prostate carcinoma PC-3 cells, piceatannol induces apoptosis and prevents Aktmediated signal pathway. Taken together, abilities of piceatannol to induce apoptotic cell death as well as CYP1 enzyme inhibition make this compound a useful tool for cancer chemoprevention.

• Archives of Pharmacal Research
• /
• v.20 no.5
• /
• pp.404-409
• /
• 1997

Trout CYP1A-CAT expression construct was generated by cloning -3.5 Kb $5^I$ flanking DNA of trout liver CYP1A gene in front of CAT gene at pCAT-basic vector. Hepa 1 cells, which are known to contain a functional arylhydrbcarbon $receptor^I$ were transfected with trout CYP1A-CAT using lipofectin. 3-Methylcholanthrene (1 nM) was added into hepa 1 cells in culture in order to examine if $5^I$ flanking DNA of trout CYP1A gene could interact with mouse transactivating factors to bring about transcription of the chloramphenicol acetyltransferase(CAT) reporter gene. The level of CAT protein was measured by CAT ELISA and the level of CAT mRNA was determined by RTPCR. The treatment of 1 nM 3-methylcholanthrene resulted in two fold increases in CAT protein as well as CAT mRNA compared to untreated control hepa 1 cells. These data indicate that arylhydrocarbon receptors of mouse hepa 1 cells are functional to activate exogenously transfected trout CYP1A-CAT construct in terms of both transcription and translation of CAT. We also examined the effect of 3-methylcholanthrene on endogenous cyplal activity in hepa 1 cell. 3-Methylcholanthrene (1 nM) treatment to hepa 1 cells trahsfected with trout CYP1A-CAT construct stimulated the level of cyp1a1 mRNA by two folds and the activity of ethoxyresorufin-O-deethylase by two fold compared to that of control cells. In this study we reported that trout CYP1A-CAT reporter gene expression construct could be expressed by 3-methylcholanthrene treatment in mouse hepa 1 cells. Thus trout CYP1A-CAT could serve as a good model to study the mechanism of regulation of CYP1A1 gene expression.

• Toxicological Research
• /
• v.14 no.4
• /
• pp.587-594
• /
• 1998

In order to understand the mechanism if the regulation of CYP 1A1 gene expression and ethoxyresorufin deethylase (EROD) activity in ex vivo system, we have studied the action of TCDD and 3MC in the isolated perfused female rat liver. CYP1A1 mRNA level and EROD activity were measured in rat liver that was isolated and perfused with various chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3MC), 17$\beta$-estradiol (E$_2$), morin. TCDD or 3MC alone perfusion into female rat liver resulted in increase of CYP 1A1 mRNA level and the magnitude of stimulation was six times higher with TCDD treatment than 3MC treatment. However E$_2$ perfusion into female rat liver showed inhibition of CYP 1A1 mRNA level. When 10$^{-8}$ M E$_2$ was administered concomitantly with either 10$^{-9}$ M TCDD or 10$^{-9}$ M 3MC, stimulated CYP 1A1 mRNA by either TCDD or 3MC was inhibited. Morin was examined for its effects on CYP 1A1 mRNA level and result was similar to that was observed with estrogen. EROD activity was also stimulated with either TCDD or 3MC perfusion, and the magnitude of EROD stiumlation was smaller than that of CYP 1A1 mRNA stimulation in response to TCDD or 3MC perfusion. Unlike CYP1A1 mRNA level, stimulation of EROD activity was greater with 3MC than TCDD. Concomitant perfusion either E$_2$ or morin with TCDD or 3MC inhibited 3MC perfusion or TCDD perfusion stimulated EROD activity. These data suggested that TCDD and 3MC might act diffrently in terms of regulation of CYP 1A1 gene expression in rat liver.

• Toxicological Research
• /
• v.16 no.2
• /
• pp.95-100
• /
• 2000

In previous study, both constitutive expression and 3-methylcholanthrene (3MC)-mediated elevation of CYP1A2 mRNA were demonstrated in human hepatoma HepG2 cells by reverse transcription-polymerase chain reaction (RT-PCR), suggesting that HepG2 cells would be appropriate for the study of human CYP1A2 regulation(Chung and Bresnick, 1994). Further studies were conducted to determine the basis of this induction phenomenon that is observed in HepG2 cells. Since CYP1A1 gene, another polycyclic hydrocarbon(PH)-inducible gene, is regulated by PHs through their interactions via receptors with cis-elements, the 5'-flanking region of human CYP 1A2 gene was analyzed to search such responsive elements. The promoter activity of various lengths of CYP1A2 gene sequence (-3203/+58bp) was measured in transiently-transfected HepG2 cells by fusion constructs containing the CAT, hGH or luciferase genes as a reporter. This region of the CYP1A2 gene, although containing a XRE, was only weakly responsive (less than 2 fold induction) to 10 nM of TCDD or 1 $\mu$M 3 MC treatment. This small enhancement of promoter activity is inconsistent with the previous observation, i.e., 12 to 14 fold-enhanced CYP1A2 mRNA from 1 $\mu$M 3 MC treated HepG2 cells, suggesting that additional mechanisms would exist for PH-mediated induction of CYP1A2 in these cells.

• Journal of Life Science
• /
• v.26 no.1
• /
• pp.42-49
• /
• 2016

The retinoic acid (RA) plays an important role in the growth and development of many cells, and bioactive RA concentration is regulated by several enzymes, including CYP26A1. The expression of the CYP26A1 gene is regulated by RA, and the CYP26A1 gene is one of the candidates for RA-responsive genes. Although CYP26A1 genes are cloned from several animals, cloning of the CYP26A1 gene from cows has not been reported yet. The promoter region of CYP26A1 from cows was cloned by PCR and analyzed by sequence alignment with human and mouse CYP26A1. The RA-responsive element (RARE), DR-5 (ttggg), was located in this region and was perfectly conserved. The promoter region of bovine CYP26A1, which contains DR-5, was ligated to the luciferase reporter gene on transient transfection assays. The expression of CYP26A1-Luc promoter was activated by ATRA treatment in lung-derived mtCC cells. Co-transfection with RAR-α or -β with ATRA significantly activates the expression of CYP26A1-Luc promoter; however, it was less effective with either RAR-γ or RXR-γ. In addition, the endogenous gene expressions measured by Q-RT-PCR in mtCC cells were not significantly affected by ATRA treatment for 2 days; however, the expression of the endogenous CYP26A1 gene was diminished sharply at day 3 with ATRA treatment. In conclusion, the promoter region of bovine CYP26A1 contains conserved DR-5 RARE, which functions as a binding site for RAR-α or -β, and it is involved in the regulation of CYP26A1 gene expression and the control of RA signaling in mtCC cells.

• Archives of Pharmacal Research
• /
• v.27 no.3
• /
• pp.319-323
• /
• 2004

Cytochrome P450 (CYP) 3A4 is of great interest because of its important roles in the oxidation of numerous drugs and xenobiotics. HDJ-1, a molecular chaperone in human, is known to assist the correct folding of unfolded proteins. To achieve a high yield of recombinant human CYP3A4 in Escherichia coli, the CYP3A4 encoding gene was co-expressed with the chaperone HDJ-1, under the control of an inducible tac promoter in a bicistronic format. The levels of expression of the CYP3A4 in the bicistronic construct reached up to 715 nmol $(liter culture)^{-1}$ within 16 h at $37^{\circ}C$, which was about a 3.3-fold increase compared to that of the CYP3A4 alone without the HDJ-1. By co-expression with HDJ-1, the catalytic activity of CYP3A4 was also increased by -15-fold. The amount of activity increase was similar to that of the CYP production at the whole cell level. The present over-expression system may be useful for the rapid production of large amounts of active CYP3A4 in E. coli.

• YAKHAK HOEJI
• /
• v.57 no.3
• /
• pp.194-198
• /
• 2013

In the present study we evaluated drug-drug interaction potential of ambroxol and cetirizine mediated by inhibition of CYP isoforms including CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 using pooled human liver microsomes (HLMs). As measured by liquid chromatography-electrospray ionization tandem mass spectrometry, cetirizine and ambroxol inhibited significantly CYP2E1 but the maximal inhibition was approximately 36% at 10 ${\mu}M$ cetirizine and 28% at 3 ${\mu}M$ ambroxol. In addition, CYP2D6 activity was decreased to approximately 83% of control activity in pooled HLM incubated with 3 ${\mu}M$ ambroxol. Activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 were not significantly inhibited by cetirizine and ambroxol. Considering their maximal plasma concentration in human ($C_{max}$ of cetirizine is approximately 0.67 ${\mu}M$ and $C_{max}$ of ambroxol is 0.044 ${\mu}M$), these two drugs have very low possibility in drug-drug interaction by CYP inhibition in clinical situations.

• The Journal of Korean Medicine
• /
• v.35 no.2
• /
• pp.47-59
• /
• 2014

Objectives: Most drug interactions are attributed to the inhibition or induction of the activity of cytochrome P450s (CYP450). Although the regulation of CYP450s by drugs has been widely reported, there have been few studies on influence of traditional herbal formulas on the drug-metabolizing enzymes. Because herbal formulas have been used traditionally to treat various diseases and because herb-drug interactions are crucial factors determining therapeutic efficacies, a systematic evaluation of the effects of herbal formulas is important. Methods: The effects of Galgeun-tang (GGT, gegen tang), Gumiganghwal-tang (GMGHT, jiuweiqianghuo tang), Insampaedok-san (ISPDS, renshenbaidu powder), Samsoeum (SSE, shensu drink), Socheongryong-tang (SCRT, xiaoqinglong-tang) and Sosiho-tang (SSHT, xiaochaihu tang) that are traditional herbal formulas used to treat common cold, on drug-metabolizing enzymes were evaluated through an in vitro CYP3A4, CYP2D6, CYP2C19 and CYP2E1 inhibition assay to assess its interaction potential with synthetic drugs. The inhibitory effects of herbal formulas were characterized with $IC_{50}$ values. Results: These six herbal formulas inhibited the activities of CYP3A4, 2C19, 2D6 and 2E1, in a concentration-dependent manner. Among the six herbal formulas, GGT critically inhibited CYP2C19, CYP2D6 and CYP2E1. GMGHT also inhibited CYP2D6 and CYP2E1 to a greater extent than the other CYP450 isozymes. Additionally, SSE and SSHT may change the effects of medicines that depend primarily on the CYP2C19 and CYP2E1 pathways. On the other hand, ISPDS and SCRT were not inhibited CYP3A4, CYP2C19, CYP2D6 and CYP2E1-mediated metabolism. Conclusions: These findings provide useful information regarding the safety and effectiveness of herbal formulas.