- Email: [email protected]
Inhibitory effect of medicinal plant-derived carboxylic acids on the human transporters hOAT1, hOAT3, hOATP1B1, and hOATP2B1
Chinese Journal of Natural Medicines 2014, 12(2): 01310138
Chinese Journal of Natural Medicines
Inhibitory effect of medicinal plant-derived carboxylic acids on the human transporters hOAT1, hOAT3, hOATP1B1, and hOATP2B1 ZHANG Zhi-Yu1, 2, SI Duan-Yun2, YI Xiu-Lin2*, LIU Chang-Xiao2* 1
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China;
2
State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin
300193, China Available online 20 Feb. 2014
[ABSTRACT] A significant number of organic carboxylic acids have been shown to influence the absorption and distribution of drugs mediated by organic anion transporters (OATs). In this study, uptake experiments were performed to assess the inhibitory effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1, hOAT3, hOATP1B1, and hOATP2B1. After a drug-drug interaction (DDI) investigation, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were found and validated to inhibit hOAT1 in a competitive manner, and deoxycholic acid was found to be an inhibitor of all four transporters. The apparent 50% inhibitory concentrations of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were estimated to be 133.87, 3.69, 90.03 and 6.03 μmolL1 for hOAT1, respectively. The apparent 50% inhibitory concentrations of deoxycholic acid were estimated to be 9.57 μmolL1 for hOAT3, 70.54 μmolL1 for hOATP1B1, and 168.27 μmolL1 for hOATP2B1. Because cinnamic acid, ferulic acid, and cynarin are ingredients of food or food additives, the present study suggests there are new food-drug interactions to be disclosed. In addition, deoxycholic acid may be used as a probe for studying the correlation of OATs and OATPs. [KEY WORDS] hOAT1; hOAT3; hOATP1B1; hOATP2B1; Transport; Food-drug interactions
[CLC Number] R969.1
[Document code] A
[Article ID] 2095-6975 (2014)02-0131-08
Introduction Organic anion transporters (OATs) expressed in renal proximal tubule and organic anion transporting polypeptides (OATPs) expressed in hepatocytes regulate blood concentrations of various drugs [1]. In particular, the human organic anion transporters hOAT1 and hOAT3, which are mainly expressed in the basolateral membrane of the renal proximal
[Received on] 10-Nov.-2012 [Research funding] This project was supported by the National 973 Project Foundation of China (Nos. 2010CB735602, 2012CB72400-2) and the National Significant New Drug Creation Project Foundation of China (Nos. 2011ZX09102-009-002, 2012ZX09304-002). [ Corresponding author] YI Xiu-Lin: Prof., LIU Chang-Xiao: Prof., Tel: 86-22-84845243. E-mail: [email protected], liuchangxiao@ 163.com These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
tubules, are thought to play the major roles in the tubular uptake of various drugs from blood [2]. The human organic anion transporting polypeptides (hOATPs), hOATP1B1 and hOATP2B1, are mainly expressed in the basolateral membrane of hepatocytes, and play key roles for drug absorption and distribution in the liver [3]. Accordingly, renal organic anion transporters and hepatic organic anion transporting polypeptides can result in drug-drug interactions when their substrates and inhibitors are co-administered to patients. For instance, probenecid, which is a typical inhibitor of hOAT1 and hOAT3, could elevate the blood level of cephalosporin; cyclosporine A, which is a typical inhibitor of both hOATP1B1 and hOATP2B1, could elevate the blood level of bilirubin [4-5]. Drug-drug interactions mediated by these drug transporters should be investigated as an important aspect for improving clinical medication safety. Cinnamic acid and its derivative ferulic acid are the principal components of several plant medicines. Previous studies have shown that cinnamic acid and ferulic acid have hepatoprotective, anti-oxidant, and anti-diabetic activities [6-8]. Oleanolic
– 131 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
acid, which is also abundant in plant tissues, has several pharmacological properties, such as hepatoprotective, anti- inflammatory, anti-oxidant, and anticancer activities [9]. Deoxycholic acid, which is a hydrophobic acid, could be used as antifungal agent [10]. Cynarin, occurring in artichokes, is a derivative of caffeoylquinic acid, which has a variety of bioactivities, including antioxidant, antibacterial, anticancer, and antihistamic [11-12]. From a literature review, studies regarding the inhibitory effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1, hOAT3, hOATP1B1 and hOATP2B1 have not been carried out. The structures of cinnamic acid (A), ferulic acid (B),
Fig. 1
oleanolic acid (C), deoxycholic acid (D), and cynarin (E) are shown in Fig. 1. Each of them contains a carboxyl group. The purpose of the present study is to investigate the inhibitory effect of these organic anion compounds on hOAT1, hOAT3, hOATP1B1, and hOATP2B1 by performing uptake experiments using S2 cell lines transfected with hOAT1 and hOAT3 genes, and HEK293 cell lines transfected with hOATP1B1 and hOATP2B1 genes. Here it is reported that cinnamic acid, ferulic acid, deoxycholic acid, and cynarin are competitive inhibitors of hOAT1, and that deoxycholic acid is also an inhibitor of hOAT3, hOATP1B1, and hOATP2B1.
Chemical structures of cinnamic acid (A), ferulic acid (B), oleanolic acid (C), deoxycholic acid (D) and cynarin (E)
Materials and Methods Materials [3H]Estrone sulfate (1.687 TBq/mmol) and [14C]p-aminohippurate (2.039 GBq/mmol) were purchased from Perkin Elmer (Boston, MA, USA). DMEM culture solution, DPBS and fetal bovine serum (FBS) were bought from Gibco (Carlsbad, CA, USA). Cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid and cynarin were bought from National Institutes for Food and Drug Control (Beijing, China). Unlabeled estrone sulfate and p-aminohippurate were purchased from Sigma-Aldrich (St. Louis, MO, USA). OAT1-, OAT3-transfected S2 cells and OATP1B1-, OATP2B1transfected HEK293 cells were provided as a gift by Fuji Biomedix Co., Ltd (Yamanashi, Japan). All other chemical reagents used were of the highest purity available. In vitro models preparation HEK293 cell lines were incubated in a CO 2 incubator (Thermo Scientific, Waltham, MA, USA) at 37 °C and 5% CO 2 . S2 cell lines were incubated in a CO 2 incubator at 33 °C and 5% CO 2 . The culture solution was DMEM with added 10% FBS. After passage at least twice, the cell growth status and transporter protein expression were stable. Then HEK293 and S2 cell lines were seeded in poly-D-lysine coated, 24-well plates (Biocoat, BD. San Jose,
CA, USA) and non-poly-D-lysine coated plates, respectively. The initial cell concentrations were 1.5 × 105 cells/mL and 2 × 105 cells/mL, respectively. After incubating for 40 h, the 24-well plates were covered with monolayer cells. Then gene expression validation was performed through RT-PCR (Fig. 2). Uptake experiment After preparation, the cells were washed with uptake DPBS buffer and then incubated in DPBS buffer at 37 °C for 10 min. The uptake reaction was initiated by adding each radiolabeled substrate to incubating cells at a series of indicated concentrations at 37 °C. At the completion of this assay, which lasted 1 min for hOATP1B1 and hOATP2B1, and 2 min for hOAT1 and hOAT3, uptake was stopped by three washes with ice-cold DPBS buffer. The cells were then dissolved in 0.1 molL1 NaOH solution. After extraction, each sample was added to scintillation solution (3 mL) and quantified with a Tri-Carb 2910TR Liquid Scintillation Analyzer (PerkinElmer, Boston, MA, USA). Protein quantification was investigated with BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) to standardize the experimental results. An uptake experiment was also performed with MOCK cells (which have no transporter protein expression) for subtracting the influence of other factors, except for transporter proteins.
– 132 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
Fig. 2 Gene expression validation of hOAT1, hOAT3, hOATP1B1 and hOATP2B1 According to the GenBank numbers of hOAT1 (AF097490.1), hOAT3 (AF097491.1), hOATP1B1 (NM006446.4), and hOATP2B1 (BC041095.1), specific primers were designed by Primer Premier 5.0. Through RT-PCR, specific gene expression of these four genes was validated. S2 MOCK and HEK 293 MOCK, which had no transporter gene expressed, were used as the negative control. Each pair of primers was used to amplify the cDNA of hOAT1, hOAT3, hOATP1B1, hOATP2B1, S2 MOCK, and HEK293 MOCK cell lines, respectively. PCR products were confirmed by gene sequencing
To examine if an inhibitory effect exists, the uptake of 5 μmolL1 p-aminohippurate mediated by hOAT1, and 50 n molL1 estrone sulfate mediated by hOAT3, hOATP1B1, and hOATP2B1 was performed, as outlined above in the absence or presence of 600 μmolL1 cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin. Experiments were performed as three replicates. To test the inhibition kinetics, the uptake of 5 μmolL1 p-aminohippurate mediated by hOAT1 and 50 nmolL1 estrone sulfate mediated by hOAT3, hOATP1B1, and hOATP2B1 was performed in the absence or presence of inhibitors at the indicated concentrations. Experiments were performed as three replicates. To investigate the inhibition mode of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin on hOAT1, uptake of p-aminohippurate was performed at a range of indicated concentrations in the absence or presence of inhibitors at the concentration of each IC 50 value, respectively. Experiments were performed as three replicates. Statistical analysis Data are presented as means ± SEM, Data were analyzed by the unpaired t-test using GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA, USA). Differences in
the mean values were considered to be significant when P < 0.05. Nonlinear regression analysis of the data was also performed using GraphPad Prism 3.0 software.
Results Concentration-dependent uptake of p-aminohippurate by hOAT1 and of estrone sulfate by hOAT3, hOATP1B1, and hOATP2B1 Fig. 3A shows the concentration-dependent uptake of [14C]p-aminohippurate, a prototypical substrate of hOAT1, in S2 cells expressing hOAT1. Least-squares nonlinear regression analysis of the data gave an apparent Km value of (43.84 ± 3.90) μmolL1. Fig. 3B shows the concentration-dependent uptake of [3H]estrone sulfate, a prototypical substrate of hOAT3, in S2 cells expressing hOAT3. The apparent Km value was (79.71 ± 12.59) μmolL1. Fig. 3C shows the concentration-dependent uptake of [3H]estrone sulfate, a prototypical substrate of hOATP1B1, in HEK293 cells expressing hOATP1B1. The apparent Km value was (6.76 ± 1.54) μmolL1. Fig. 3D shows the concentration-dependent uptake of [3H]estrone sulfate, a prototypical substrate of hOATP2B1, in HEK293 cells expressing hOATP2B1. The apparent Km value was (25.09 ± 4.68) μmolL1.
– 133 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
Fig. 3 Concentration-dependent uptake of p-aminohippurate by hOAT1, and of estrone sulfate by hOAT3, hOATP1B1 and hOATP2B1. (A) Uptake of [14C] p-aminohippurate by hOAT1 measured over a range of p-aminohippurate concentrations. Non-linear regression analysis generated an apparent Km of (43.84 ± 3.90) μ " 1. (B) Uptake of [3H]estrone sulfate by hOAT3 measured over a range of estrone sulfate concentrations. Non-linear regression analysis generated an apparent Km of (79.71 ± 12.59) μ " 1. (C) Uptake of [3H]estrone sulfate by hOATP1B1 measured over a range of estrone sulfate concentrations. Non-linear regression analysis generated an apparent Km of (6.76 ± 1.54) μ " 1. (D) Uptake of [3H]estrone sulfate by hOATP2B1 measured over a range of estrone sulfate concentrations. Non-linear regression analysis generated an apparent Km of (25.09 ± 4.68) μ " 1. mean ± SEM, n = 3
Effects of five carboxylic acids on the uptake of p-aminohippurate by hOAT1 and the uptake of estrone sulfate by hOAT3, hOATP1B1, and hOATP2B1 The inhibitory effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1, hOAT3, hOATP1B1, and hOATP2B1. As shown in Figure 4A, in the presence of 600 ³ 1 cinnamic acid, ferulic acid, deoxycholic acid, and cynarin, the uptake of p-aminohippurate mediated by hOAT1 was inhibited (P < 0.001). As shown in Fig. 4B-D, in the presence of 600 ³ 1 deoxycholic acid, the uptake of estrone sulfate mediated by hOAT3, hOATP1B1, and hOATP2B1 was inhibited (P < 0.001). Although cinnamic acid, ferulic acid, and cynarin could inhibit hOAT3 (Fig. 4B), and oleanolic acid and cynarin could inhibit hOATP2B1 (Fig. 4D), estrone sulfate uptake was still over 25% of control in the presence of 600 ³ 1 inhibitors. This demonstrated that their IC 50 values probably would be quite large. Because a large IC 50 value means a low possibility of adverse drug reaction (ADR) rooted from DDI or daily food intake, their further investigation is not discussed here. Concentration-dependence of the inhibitory effects of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin on hOAT1 To estimate the IC 50 values of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin for hOAT1, the inhibitory effects of these four carboxylic acids were investigated over a range of concentrations. As shown in Fig. 5A-D, cinnamic
acid, ferulic acid, deoxycholic acid, and cynarin displayed concentration-dependent inhibition of p-aminohippurate uptake mediated by hOAT1 with apparent IC 50 values of (133.87 ± 24.33), (3.69 ± 0.32), (90.03 ± 7.14) and (6.03 ± 0.44) μ 1, respectively (n = 3). The potency rank of these four carboxylic acids was ferulic acid > cynarin > deoxycholic acid > cinnamic acid. Concentration dependence of the inhibitory effect of deoxycholic acid on hOAT3, hOATP1B1, and hOATP2B1 To estimate the IC 50 values of deoxycholic acid for hOAT3, hOATP1B1, and hOATP2B1, the dose dependence of the inhibitory effect was investigated. As shown in Figure 6A-C, deoxycholic acid inhibited the transport of estrone sulfate by hOAT3, hOATP1B1, and hOATP2B1. The apparent IC 50 values were (9.57 ± 1.35) μ 1 for hOAT3, (70.54 ± 2.84) μ 1 for hOATP1B1, and (168.27 ± 9.80) μ 1 for hOATP2B1 (n = 3). Mode of inhibition of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin on hOAT1 To elucidate the inhibition mode of these four acids on hOAT1, the uptake of p-aminohippurate was performed at a range of indicated concentrations in the absence or presence of inhibitors, and Eadie-Hofstee plot analysis was performed. As shown in Fig. 7A-D, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin significantly affected the Km value of p-aminohippurate uptake mediated by hOAT1. The
– 134 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
Km value was increased from (37.06 ± 2.23) to (163.0 ± 6.12) μmolL1 (P < 0.000 1), (95.65 ± 8.28) μmolL1 (P = 0.002 4), (72.22 ± 2.61) μmolL1 (P = 0.000 5), and (129.8 ± 14.5) μmolL1 (P = 0.003 2), respectively. However, V max variation was not significant for ferulic acid, deoxycholic acid, and cynarin which changed from (359.1 ± 14.41) to (426.1 ± 29.36) μmolL1 (P = 0.109 9), (345.3 ± 9.08) μmolL1 (P = 0.463 2) and (465.3 ± 43.42) μmolL1 (P = 0.081), respectively. Although the V max variation was significant for cinnamic acid, which changed from (359.1 ± 14.41) to (608.1 ± 19.16) μmolL1 (P = 0.000 5), the Km variation was found to be more significant than V max because the fold change of Km is larger than that of V max . According to these results, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin should inhibit hOAT1 in a competitive manner, which means that they are probably substrates of hOAT1.
Discussion and Conclusions
Fig. 4 The effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1-mediated p-aminohippurate and hOAT3-, hOATP1B1-, hOATP2B1mediated estrone sulfate uptake (A) Uptake of 5 μmol"L 1 [14C]p-aminohippurate by hOAT1 measured in the presence or absence of 600 μmol"L 1 of selected acids. (B) Uptake of 50 nmol"L 1 [3H]estrone sulfate by hOAT3 measured in the presence or absence of 600 μmol"L 1 of selected acids. (C) Uptake of 50 nmol"L 1 [3H]estrone sulfate by hOATP1B1 measured in the presence or absence of 600 μmol"L 1 of selected acids. (D) Uptake of 50 nmol"L 1 [3H] estrone sulfate by hOATP2B1 measured in the presence or absence of 600 μmol"L 1 of selected acids. The data are expressed as mean ± SEM, n = 3. *P < 0.05; **P < 0.01; *** P < 0.001
The transporters hOAT1 and hOAT3 are important for drug absorption and distribution in the kidneys, and the transporters hOATP1B1 and hOATP2B1 are important in the liver. Therefore, identification of their inhibitors is quite useful for improvement in clinical medication safety. Because the determination of drug-drug interactions is one of the main directions for new drug development which concerns the FDA, the inhibitory effects of a variety of drugs towards these transporters have been widely investigated during the last decade. A number of carboxylic acids have been found to be substrates or inhibitors of these transporters. It was considered that there should be more carboxylic acids which have a high or low affinity with these transporters. Consequently, the present study was designed to investigate the inhibitory effects of several carboxylic acids on hOAT1, hOAT3, hOATP1B1, and hOATP2B1. To examine if the inhibitory effect exists, the uptake of p-aminohippurate was assessed using hOAT1-transfected S2 cell lines, and the uptake of estrone sulfate was also investigated using hOAT3-transfected S2 cell lines, and hOATP1B1and hOATP2B1-transfected HEK293 cell lines in the absence or presence of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin, respectively. Of the acids tested, cinnamic acid, ferulic acid, and cynarin had inhibitory effects on hOAT1-mediated p-aminohippurate uptake and hOAT3-mediated estrone sulfate uptake. In addition, oleanolic acid and cynarin could inhibit hOATP2B1, and deoxycholic acid could inhibit all four transporters. Concentration-dependence of the inhibitory effects of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin on hOAT1 was investigated, and the IC 50 values were determined. Eadie-Hofstee plot analysis suggests that they inhibit hOAT1 in a competitive manner. Although ferulic acid is a derivative of cinnamic acid and they have the same core structure, the difference between their IC 50 values is quite apparent (one is over 100 μmolL1 and the other is less than 5 μmolL1).
– 135 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
Fig. 5 Kinetics of the inhibition of hOAT1-mediated [14C] p-aminohippurate uptake by cinnamic acid, ferulic acid, deoxycholic acid, and cynarin. (A) cinnamic acid, (B) ferulic acid, (C) deoxycholic acid, and (D) cynarin were tested for their impact on the uptake of 5 μmol"L 1 [14C] p-aminohippurate by hOAT1. The extent of inhibition is expressed as the percentage of p-aminohippurate uptake in the absence of inhibitors (control). Non-linear regression analysis of the data gave apparent IC 50 values of (133.87 ± 24.33), (3.69 ± 0.32), (90.03 ± 7.14) and (6.03 ± 0.44) μmol"L 1, respectively. Each point is the mean ± SEM (n = 3)
Fig. 6 Kinetics of inhibition of hOAT3-, hOATP1B1-, hOATP2B1-mediated [3H]estrone sulfate uptake by deoxycholic acid. Deoxycholic acid was tested for its impact on the uptake of 50 nmol"L 1 [3H]estrone sulfate by hOAT3 (A), hOATP1B1 (B), and hOATP2B1 (C). Non-linear regression analysis of the data gave apparent IC 50 values of (9.57 ± 1.35), (70.54 ± 2.84) and (168.27 ± 9.80) μmol"L 1, respectively. Each point is the mean ± SEM (n = 3)
This result demonstrates that the additional –OH and –OCH 3 functional groups profoundly influence the inhibitory effect. Interestingly, it was found that deoxycholic acid was an inhibitor for hOAT1, hOAT3, hOATP1B1, and hOATP2B1. Therefore, the concentration-dependence of the inhibitory effect of deoxycholic acid on hOAT3, hOATP1B1, and hOATP2B1 was investigated and the IC 50 values determined. Because the inhibitory effect of deoxycholic acid on hOAT3 is much stronger than for the other transporters, it was concluded that deoxycholic acid is a specific inhibitor for hOAT3, but is not specific for hOAT1, hOATP1B1, and hOATP2B1. It was reported that chenodeoxycholic and ursodeoxycholic acids are substrates of the hepatic uptake transporters hOATP1B1 and hOATP1B3 [13-15]. Deoxycholic acid is one of the bile acids and is also the analogue of chenodeoxycholic and ursodeoxycholic acids, so it probably is also a substrate of hOATP1B1 and hOATP2B1. These results established that deoxycholic acid could inhibit hOATP1B1 and hOATP2B1. In addition, it has an inhibitory effect on hOAT1 and hOAT3, which is really interesting. To date, few inhibitors can inhibit
organic anion transporters and organic anion transporting polypeptides, simultaneously. The reason is probably that they prefer different reactive groups in most cases. However, sometimes a special case exists, for example, deoxycholic acid possibly contains the functional attributes that can be identified by both hOATs and hOATPs. Food-drug interactions mediated by transporters have attracted the attention of researchers during the past ten years. Several papers have reported that the ingredients of food or vegetables have inhibitory effects on drug transporters [16-18]. Cinnamic acid could be used as a food additive and spice, and it is also an ingredient of the widely-used cinnamon and of cinnamon tea which is very famous in China. Ferulic acid is one of the choices for a food preservative. Cynarin is one of the ingredients of artichoke, which is a very popular vegetable in Europe, the Americas, and China. In this paper, it was found that these three acids have inhibitory effects on hOAT1 and hOAT3. In addition, their inhibitory ability on hOAT1 was determined and the results showed that the IC 50 values of ferulic acid and cynarin were less than 10 μmolL1. There
– 136 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
Fig. 7 Concentration dependent uptake of [14C]p-aminohippurate by hOAT1 in the presence or absence of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin. (A) Uptake of [14C]p-aminohippurate at various concentrations by hOAT1 in the absence (triangle) or presence (square) of 134 μmol"L 1 cinnamic acid. (B) Uptake of [14C]p-aminohippurate at various concentrations by hOAT1 in the absence (triangle) or presence (square) of 3.7 μmol"L 1 ferulic acid. (C) Uptake of [14C]p-aminohippurate at various concentrations by hOAT1 in the absence (triangle) or presence (square) of 90 μmol"L 1 deoxycholic acid. (D) Uptake of [14C]p-aminohippurate at various concentrations by hOAT1 in the absence (triangle) or presence (square) of 6 μmol"L 1 cynarin. Inset: Eadie-Hofstee plots of data; V, uptake rate (pmol/mg protein/min); S, concentration of [14C] p-aminohippurate (μmol"L 1). Each point is the mean ± SEM (n = 3) 3
fore, attention should be paid to the daily intake of these compounds during the process of drug treatment. In conclusion, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were validated as competitive inhibitors of hOAT1. Meanwhile, the present study suggests some new food-drug interactions. Furthermore, deoxycholic acid was found to be an inhibitor for hOAT1, hOAT3, hOATP1B1, and hOATP2B1, which probably could be used for revealing the correlation between hOATs and hOATPs.
References [1]
[2]
[3]
[4]
Giacomini KM, Huang SM, Tweedie DJ, et al. Membrane transporters in drug development [J]. Nat Rev Drug Discov, 2010, 9 (3): 215-236. El-Sheikh AAK, Masereeuw R, Russel FGM. Mechanisms of renal anionic drug transport [J]. Eur J Pharmacol, 2008, 585 (2-3): 245-255. Bohan A, Boyer JL. Mechanisms of hepatic transport of drugs: implications for cholestatic drug reactions [J]. Semin Liver Dis, 2002, 22 (2): 123-136. Campbell SD, de Morais SM, Xu JJ. Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia [J]. Chem Biol Interact, 2004, 150 (2): 179-187.
– 137 –
[5]
Shitara Y, Sato H, Sugiyama Y. Evaluation of drug-drug interaction in the hepatobiliary and renal transport of drugs [J]. Annu Rev Pharmacol Toxicol, 2005, 45: 689-723. [6] Lee EJ, Kim SR, Kim J, et al. Hepatoprotective phenylpropanoids from Scrophularia buergeriana roots against CCl 4-induced toxicity: action mechanism and structureactivity relationship [J]. Planta Med, 2002, 68 (5): 407-411. [7] Liu IM, Hsu FL, Chen CF, et al. Antihyperglycemic action of isoferulic acid in streptozotocin-induced diabetic rats [J]. Br J Pharmacol, 2000, 129 (4): 631-636. [8] Natella F, Nardini M, Di Felice M, et al. Benzoic and cinnamic acid derivatives as antioxidants: structure-activity relation [J]. J Agric Food Chem, 1999, 47 (4): 1453-1459. [9] Pollier J, Goossens A. Oleanolic acid [J]. Phytochemistry, 2012, 77: 10-15. [10] Marples BA, Stretton RJ. Antifungal utility of bile acids: US4681876 A [P]. Google Patents, 1987. [11] Horman I, Badoud R, Ammann W. Food-related applications of one- and two-dimensional high-resolution proton-NMR: structure and conformation of cynarin [J]. J Agric Food Chem, 1984, 32 (3): 538-540. [12] Miyamae Y, Kurisu M, Han J, et al. Structure-activity relationship of caffeoylquinic acids on the accelerating activity on ATP production [J]. Chem Pharm Bull, 2011, 59 (4): 502- 507.
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138 [13] König J, Klatt S, Dilger K, et al. Characterization of ursodeoxycholic and norursodeoxycholic acid as substrates of the hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1 and NTCP [J]. Basic Clin Pharmacol Toxicol, 2012, 111 (2): 81-86. [14] Maeda K, Kambara M, Tian Y, et al. Uptake of ursodeoxycholate and its conjugates by human hepatocytes: role of Na+-taurocholate cotransporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1 (OATP-C), and OATP1B3 (OATP8) [J]. Mol Pharm, 2006, 3 (1): 70-77. [15] Yamaguchi H, Okada M, Akitaya S, et al. Transport of fluorescent chenodeoxycholic acid via the human organic
anion transporters OATP1B1 and OATP1B3 [J]. J Lipid Res, 2006, 47 (6): 1196-1202. [16] Bailey DG. Fruit juice inhibition of uptake transport: a new type of food–drug interaction [J]. Br J Clin Pharmacol, 2010, 70 (5): 645-655. [17] Harris RZ, Jang GR, Tsunoda S. Dietary effects on drug metabolism and transport [J]. Clin Pharmacokinet, 2003, 42 (13): 1071-1088. [18] Uwai Y, Ozeki Y, Isaka T, et al. Inhibitory effect of caffeic acid on human organic anion transporters hOAT1 and hOAT3: A novel candidate for food–drug interaction [J]. Drug Metab Pharmacokinet, 2011, 26 (5): 486-493.
Cite this article as: ZHANG Zhi-Yu, SI Duan-Yun, YI Xiu-Lin, LIU Chang-Xiao. Inhibitory effect of medicinal plant-derived carboxylic acids on the human transporters hOAT1, hOAT3, hOATP1B1, and hOATP2B1 [J]. Chinese Journal of Natural Medicines, 2014, 12(2): 131-138
– 138 –
Chinese Journal of Natural Medicines
Inhibitory effect of medicinal plant-derived carboxylic acids on the human transporters hOAT1, hOAT3, hOATP1B1, and hOATP2B1 ZHANG Zhi-Yu1, 2, SI Duan-Yun2, YI Xiu-Lin2*, LIU Chang-Xiao2* 1
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China;
2
State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin
300193, China Available online 20 Feb. 2014
[ABSTRACT] A significant number of organic carboxylic acids have been shown to influence the absorption and distribution of drugs mediated by organic anion transporters (OATs). In this study, uptake experiments were performed to assess the inhibitory effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1, hOAT3, hOATP1B1, and hOATP2B1. After a drug-drug interaction (DDI) investigation, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were found and validated to inhibit hOAT1 in a competitive manner, and deoxycholic acid was found to be an inhibitor of all four transporters. The apparent 50% inhibitory concentrations of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were estimated to be 133.87, 3.69, 90.03 and 6.03 μmolL1 for hOAT1, respectively. The apparent 50% inhibitory concentrations of deoxycholic acid were estimated to be 9.57 μmolL1 for hOAT3, 70.54 μmolL1 for hOATP1B1, and 168.27 μmolL1 for hOATP2B1. Because cinnamic acid, ferulic acid, and cynarin are ingredients of food or food additives, the present study suggests there are new food-drug interactions to be disclosed. In addition, deoxycholic acid may be used as a probe for studying the correlation of OATs and OATPs. [KEY WORDS] hOAT1; hOAT3; hOATP1B1; hOATP2B1; Transport; Food-drug interactions
[CLC Number] R969.1
[Document code] A
[Article ID] 2095-6975 (2014)02-0131-08
Introduction Organic anion transporters (OATs) expressed in renal proximal tubule and organic anion transporting polypeptides (OATPs) expressed in hepatocytes regulate blood concentrations of various drugs [1]. In particular, the human organic anion transporters hOAT1 and hOAT3, which are mainly expressed in the basolateral membrane of the renal proximal
[Received on] 10-Nov.-2012 [Research funding] This project was supported by the National 973 Project Foundation of China (Nos. 2010CB735602, 2012CB72400-2) and the National Significant New Drug Creation Project Foundation of China (Nos. 2011ZX09102-009-002, 2012ZX09304-002). [ Corresponding author] YI Xiu-Lin: Prof., LIU Chang-Xiao: Prof., Tel: 86-22-84845243. E-mail: [email protected], liuchangxiao@ 163.com These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
tubules, are thought to play the major roles in the tubular uptake of various drugs from blood [2]. The human organic anion transporting polypeptides (hOATPs), hOATP1B1 and hOATP2B1, are mainly expressed in the basolateral membrane of hepatocytes, and play key roles for drug absorption and distribution in the liver [3]. Accordingly, renal organic anion transporters and hepatic organic anion transporting polypeptides can result in drug-drug interactions when their substrates and inhibitors are co-administered to patients. For instance, probenecid, which is a typical inhibitor of hOAT1 and hOAT3, could elevate the blood level of cephalosporin; cyclosporine A, which is a typical inhibitor of both hOATP1B1 and hOATP2B1, could elevate the blood level of bilirubin [4-5]. Drug-drug interactions mediated by these drug transporters should be investigated as an important aspect for improving clinical medication safety. Cinnamic acid and its derivative ferulic acid are the principal components of several plant medicines. Previous studies have shown that cinnamic acid and ferulic acid have hepatoprotective, anti-oxidant, and anti-diabetic activities [6-8]. Oleanolic
– 131 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
acid, which is also abundant in plant tissues, has several pharmacological properties, such as hepatoprotective, anti- inflammatory, anti-oxidant, and anticancer activities [9]. Deoxycholic acid, which is a hydrophobic acid, could be used as antifungal agent [10]. Cynarin, occurring in artichokes, is a derivative of caffeoylquinic acid, which has a variety of bioactivities, including antioxidant, antibacterial, anticancer, and antihistamic [11-12]. From a literature review, studies regarding the inhibitory effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1, hOAT3, hOATP1B1 and hOATP2B1 have not been carried out. The structures of cinnamic acid (A), ferulic acid (B),
Fig. 1
oleanolic acid (C), deoxycholic acid (D), and cynarin (E) are shown in Fig. 1. Each of them contains a carboxyl group. The purpose of the present study is to investigate the inhibitory effect of these organic anion compounds on hOAT1, hOAT3, hOATP1B1, and hOATP2B1 by performing uptake experiments using S2 cell lines transfected with hOAT1 and hOAT3 genes, and HEK293 cell lines transfected with hOATP1B1 and hOATP2B1 genes. Here it is reported that cinnamic acid, ferulic acid, deoxycholic acid, and cynarin are competitive inhibitors of hOAT1, and that deoxycholic acid is also an inhibitor of hOAT3, hOATP1B1, and hOATP2B1.
Chemical structures of cinnamic acid (A), ferulic acid (B), oleanolic acid (C), deoxycholic acid (D) and cynarin (E)
Materials and Methods Materials [3H]Estrone sulfate (1.687 TBq/mmol) and [14C]p-aminohippurate (2.039 GBq/mmol) were purchased from Perkin Elmer (Boston, MA, USA). DMEM culture solution, DPBS and fetal bovine serum (FBS) were bought from Gibco (Carlsbad, CA, USA). Cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid and cynarin were bought from National Institutes for Food and Drug Control (Beijing, China). Unlabeled estrone sulfate and p-aminohippurate were purchased from Sigma-Aldrich (St. Louis, MO, USA). OAT1-, OAT3-transfected S2 cells and OATP1B1-, OATP2B1transfected HEK293 cells were provided as a gift by Fuji Biomedix Co., Ltd (Yamanashi, Japan). All other chemical reagents used were of the highest purity available. In vitro models preparation HEK293 cell lines were incubated in a CO 2 incubator (Thermo Scientific, Waltham, MA, USA) at 37 °C and 5% CO 2 . S2 cell lines were incubated in a CO 2 incubator at 33 °C and 5% CO 2 . The culture solution was DMEM with added 10% FBS. After passage at least twice, the cell growth status and transporter protein expression were stable. Then HEK293 and S2 cell lines were seeded in poly-D-lysine coated, 24-well plates (Biocoat, BD. San Jose,
CA, USA) and non-poly-D-lysine coated plates, respectively. The initial cell concentrations were 1.5 × 105 cells/mL and 2 × 105 cells/mL, respectively. After incubating for 40 h, the 24-well plates were covered with monolayer cells. Then gene expression validation was performed through RT-PCR (Fig. 2). Uptake experiment After preparation, the cells were washed with uptake DPBS buffer and then incubated in DPBS buffer at 37 °C for 10 min. The uptake reaction was initiated by adding each radiolabeled substrate to incubating cells at a series of indicated concentrations at 37 °C. At the completion of this assay, which lasted 1 min for hOATP1B1 and hOATP2B1, and 2 min for hOAT1 and hOAT3, uptake was stopped by three washes with ice-cold DPBS buffer. The cells were then dissolved in 0.1 molL1 NaOH solution. After extraction, each sample was added to scintillation solution (3 mL) and quantified with a Tri-Carb 2910TR Liquid Scintillation Analyzer (PerkinElmer, Boston, MA, USA). Protein quantification was investigated with BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) to standardize the experimental results. An uptake experiment was also performed with MOCK cells (which have no transporter protein expression) for subtracting the influence of other factors, except for transporter proteins.
– 132 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
Fig. 2 Gene expression validation of hOAT1, hOAT3, hOATP1B1 and hOATP2B1 According to the GenBank numbers of hOAT1 (AF097490.1), hOAT3 (AF097491.1), hOATP1B1 (NM006446.4), and hOATP2B1 (BC041095.1), specific primers were designed by Primer Premier 5.0. Through RT-PCR, specific gene expression of these four genes was validated. S2 MOCK and HEK 293 MOCK, which had no transporter gene expressed, were used as the negative control. Each pair of primers was used to amplify the cDNA of hOAT1, hOAT3, hOATP1B1, hOATP2B1, S2 MOCK, and HEK293 MOCK cell lines, respectively. PCR products were confirmed by gene sequencing
To examine if an inhibitory effect exists, the uptake of 5 μmolL1 p-aminohippurate mediated by hOAT1, and 50 n molL1 estrone sulfate mediated by hOAT3, hOATP1B1, and hOATP2B1 was performed, as outlined above in the absence or presence of 600 μmolL1 cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin. Experiments were performed as three replicates. To test the inhibition kinetics, the uptake of 5 μmolL1 p-aminohippurate mediated by hOAT1 and 50 nmolL1 estrone sulfate mediated by hOAT3, hOATP1B1, and hOATP2B1 was performed in the absence or presence of inhibitors at the indicated concentrations. Experiments were performed as three replicates. To investigate the inhibition mode of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin on hOAT1, uptake of p-aminohippurate was performed at a range of indicated concentrations in the absence or presence of inhibitors at the concentration of each IC 50 value, respectively. Experiments were performed as three replicates. Statistical analysis Data are presented as means ± SEM, Data were analyzed by the unpaired t-test using GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA, USA). Differences in
the mean values were considered to be significant when P < 0.05. Nonlinear regression analysis of the data was also performed using GraphPad Prism 3.0 software.
Results Concentration-dependent uptake of p-aminohippurate by hOAT1 and of estrone sulfate by hOAT3, hOATP1B1, and hOATP2B1 Fig. 3A shows the concentration-dependent uptake of [14C]p-aminohippurate, a prototypical substrate of hOAT1, in S2 cells expressing hOAT1. Least-squares nonlinear regression analysis of the data gave an apparent Km value of (43.84 ± 3.90) μmolL1. Fig. 3B shows the concentration-dependent uptake of [3H]estrone sulfate, a prototypical substrate of hOAT3, in S2 cells expressing hOAT3. The apparent Km value was (79.71 ± 12.59) μmolL1. Fig. 3C shows the concentration-dependent uptake of [3H]estrone sulfate, a prototypical substrate of hOATP1B1, in HEK293 cells expressing hOATP1B1. The apparent Km value was (6.76 ± 1.54) μmolL1. Fig. 3D shows the concentration-dependent uptake of [3H]estrone sulfate, a prototypical substrate of hOATP2B1, in HEK293 cells expressing hOATP2B1. The apparent Km value was (25.09 ± 4.68) μmolL1.
– 133 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
Fig. 3 Concentration-dependent uptake of p-aminohippurate by hOAT1, and of estrone sulfate by hOAT3, hOATP1B1 and hOATP2B1. (A) Uptake of [14C] p-aminohippurate by hOAT1 measured over a range of p-aminohippurate concentrations. Non-linear regression analysis generated an apparent Km of (43.84 ± 3.90) μ " 1. (B) Uptake of [3H]estrone sulfate by hOAT3 measured over a range of estrone sulfate concentrations. Non-linear regression analysis generated an apparent Km of (79.71 ± 12.59) μ " 1. (C) Uptake of [3H]estrone sulfate by hOATP1B1 measured over a range of estrone sulfate concentrations. Non-linear regression analysis generated an apparent Km of (6.76 ± 1.54) μ " 1. (D) Uptake of [3H]estrone sulfate by hOATP2B1 measured over a range of estrone sulfate concentrations. Non-linear regression analysis generated an apparent Km of (25.09 ± 4.68) μ " 1. mean ± SEM, n = 3
Effects of five carboxylic acids on the uptake of p-aminohippurate by hOAT1 and the uptake of estrone sulfate by hOAT3, hOATP1B1, and hOATP2B1 The inhibitory effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1, hOAT3, hOATP1B1, and hOATP2B1. As shown in Figure 4A, in the presence of 600 ³ 1 cinnamic acid, ferulic acid, deoxycholic acid, and cynarin, the uptake of p-aminohippurate mediated by hOAT1 was inhibited (P < 0.001). As shown in Fig. 4B-D, in the presence of 600 ³ 1 deoxycholic acid, the uptake of estrone sulfate mediated by hOAT3, hOATP1B1, and hOATP2B1 was inhibited (P < 0.001). Although cinnamic acid, ferulic acid, and cynarin could inhibit hOAT3 (Fig. 4B), and oleanolic acid and cynarin could inhibit hOATP2B1 (Fig. 4D), estrone sulfate uptake was still over 25% of control in the presence of 600 ³ 1 inhibitors. This demonstrated that their IC 50 values probably would be quite large. Because a large IC 50 value means a low possibility of adverse drug reaction (ADR) rooted from DDI or daily food intake, their further investigation is not discussed here. Concentration-dependence of the inhibitory effects of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin on hOAT1 To estimate the IC 50 values of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin for hOAT1, the inhibitory effects of these four carboxylic acids were investigated over a range of concentrations. As shown in Fig. 5A-D, cinnamic
acid, ferulic acid, deoxycholic acid, and cynarin displayed concentration-dependent inhibition of p-aminohippurate uptake mediated by hOAT1 with apparent IC 50 values of (133.87 ± 24.33), (3.69 ± 0.32), (90.03 ± 7.14) and (6.03 ± 0.44) μ 1, respectively (n = 3). The potency rank of these four carboxylic acids was ferulic acid > cynarin > deoxycholic acid > cinnamic acid. Concentration dependence of the inhibitory effect of deoxycholic acid on hOAT3, hOATP1B1, and hOATP2B1 To estimate the IC 50 values of deoxycholic acid for hOAT3, hOATP1B1, and hOATP2B1, the dose dependence of the inhibitory effect was investigated. As shown in Figure 6A-C, deoxycholic acid inhibited the transport of estrone sulfate by hOAT3, hOATP1B1, and hOATP2B1. The apparent IC 50 values were (9.57 ± 1.35) μ 1 for hOAT3, (70.54 ± 2.84) μ 1 for hOATP1B1, and (168.27 ± 9.80) μ 1 for hOATP2B1 (n = 3). Mode of inhibition of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin on hOAT1 To elucidate the inhibition mode of these four acids on hOAT1, the uptake of p-aminohippurate was performed at a range of indicated concentrations in the absence or presence of inhibitors, and Eadie-Hofstee plot analysis was performed. As shown in Fig. 7A-D, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin significantly affected the Km value of p-aminohippurate uptake mediated by hOAT1. The
– 134 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
Km value was increased from (37.06 ± 2.23) to (163.0 ± 6.12) μmolL1 (P < 0.000 1), (95.65 ± 8.28) μmolL1 (P = 0.002 4), (72.22 ± 2.61) μmolL1 (P = 0.000 5), and (129.8 ± 14.5) μmolL1 (P = 0.003 2), respectively. However, V max variation was not significant for ferulic acid, deoxycholic acid, and cynarin which changed from (359.1 ± 14.41) to (426.1 ± 29.36) μmolL1 (P = 0.109 9), (345.3 ± 9.08) μmolL1 (P = 0.463 2) and (465.3 ± 43.42) μmolL1 (P = 0.081), respectively. Although the V max variation was significant for cinnamic acid, which changed from (359.1 ± 14.41) to (608.1 ± 19.16) μmolL1 (P = 0.000 5), the Km variation was found to be more significant than V max because the fold change of Km is larger than that of V max . According to these results, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin should inhibit hOAT1 in a competitive manner, which means that they are probably substrates of hOAT1.
Discussion and Conclusions
Fig. 4 The effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1-mediated p-aminohippurate and hOAT3-, hOATP1B1-, hOATP2B1mediated estrone sulfate uptake (A) Uptake of 5 μmol"L 1 [14C]p-aminohippurate by hOAT1 measured in the presence or absence of 600 μmol"L 1 of selected acids. (B) Uptake of 50 nmol"L 1 [3H]estrone sulfate by hOAT3 measured in the presence or absence of 600 μmol"L 1 of selected acids. (C) Uptake of 50 nmol"L 1 [3H]estrone sulfate by hOATP1B1 measured in the presence or absence of 600 μmol"L 1 of selected acids. (D) Uptake of 50 nmol"L 1 [3H] estrone sulfate by hOATP2B1 measured in the presence or absence of 600 μmol"L 1 of selected acids. The data are expressed as mean ± SEM, n = 3. *P < 0.05; **P < 0.01; *** P < 0.001
The transporters hOAT1 and hOAT3 are important for drug absorption and distribution in the kidneys, and the transporters hOATP1B1 and hOATP2B1 are important in the liver. Therefore, identification of their inhibitors is quite useful for improvement in clinical medication safety. Because the determination of drug-drug interactions is one of the main directions for new drug development which concerns the FDA, the inhibitory effects of a variety of drugs towards these transporters have been widely investigated during the last decade. A number of carboxylic acids have been found to be substrates or inhibitors of these transporters. It was considered that there should be more carboxylic acids which have a high or low affinity with these transporters. Consequently, the present study was designed to investigate the inhibitory effects of several carboxylic acids on hOAT1, hOAT3, hOATP1B1, and hOATP2B1. To examine if the inhibitory effect exists, the uptake of p-aminohippurate was assessed using hOAT1-transfected S2 cell lines, and the uptake of estrone sulfate was also investigated using hOAT3-transfected S2 cell lines, and hOATP1B1and hOATP2B1-transfected HEK293 cell lines in the absence or presence of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin, respectively. Of the acids tested, cinnamic acid, ferulic acid, and cynarin had inhibitory effects on hOAT1-mediated p-aminohippurate uptake and hOAT3-mediated estrone sulfate uptake. In addition, oleanolic acid and cynarin could inhibit hOATP2B1, and deoxycholic acid could inhibit all four transporters. Concentration-dependence of the inhibitory effects of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin on hOAT1 was investigated, and the IC 50 values were determined. Eadie-Hofstee plot analysis suggests that they inhibit hOAT1 in a competitive manner. Although ferulic acid is a derivative of cinnamic acid and they have the same core structure, the difference between their IC 50 values is quite apparent (one is over 100 μmolL1 and the other is less than 5 μmolL1).
– 135 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
Fig. 5 Kinetics of the inhibition of hOAT1-mediated [14C] p-aminohippurate uptake by cinnamic acid, ferulic acid, deoxycholic acid, and cynarin. (A) cinnamic acid, (B) ferulic acid, (C) deoxycholic acid, and (D) cynarin were tested for their impact on the uptake of 5 μmol"L 1 [14C] p-aminohippurate by hOAT1. The extent of inhibition is expressed as the percentage of p-aminohippurate uptake in the absence of inhibitors (control). Non-linear regression analysis of the data gave apparent IC 50 values of (133.87 ± 24.33), (3.69 ± 0.32), (90.03 ± 7.14) and (6.03 ± 0.44) μmol"L 1, respectively. Each point is the mean ± SEM (n = 3)
Fig. 6 Kinetics of inhibition of hOAT3-, hOATP1B1-, hOATP2B1-mediated [3H]estrone sulfate uptake by deoxycholic acid. Deoxycholic acid was tested for its impact on the uptake of 50 nmol"L 1 [3H]estrone sulfate by hOAT3 (A), hOATP1B1 (B), and hOATP2B1 (C). Non-linear regression analysis of the data gave apparent IC 50 values of (9.57 ± 1.35), (70.54 ± 2.84) and (168.27 ± 9.80) μmol"L 1, respectively. Each point is the mean ± SEM (n = 3)
This result demonstrates that the additional –OH and –OCH 3 functional groups profoundly influence the inhibitory effect. Interestingly, it was found that deoxycholic acid was an inhibitor for hOAT1, hOAT3, hOATP1B1, and hOATP2B1. Therefore, the concentration-dependence of the inhibitory effect of deoxycholic acid on hOAT3, hOATP1B1, and hOATP2B1 was investigated and the IC 50 values determined. Because the inhibitory effect of deoxycholic acid on hOAT3 is much stronger than for the other transporters, it was concluded that deoxycholic acid is a specific inhibitor for hOAT3, but is not specific for hOAT1, hOATP1B1, and hOATP2B1. It was reported that chenodeoxycholic and ursodeoxycholic acids are substrates of the hepatic uptake transporters hOATP1B1 and hOATP1B3 [13-15]. Deoxycholic acid is one of the bile acids and is also the analogue of chenodeoxycholic and ursodeoxycholic acids, so it probably is also a substrate of hOATP1B1 and hOATP2B1. These results established that deoxycholic acid could inhibit hOATP1B1 and hOATP2B1. In addition, it has an inhibitory effect on hOAT1 and hOAT3, which is really interesting. To date, few inhibitors can inhibit
organic anion transporters and organic anion transporting polypeptides, simultaneously. The reason is probably that they prefer different reactive groups in most cases. However, sometimes a special case exists, for example, deoxycholic acid possibly contains the functional attributes that can be identified by both hOATs and hOATPs. Food-drug interactions mediated by transporters have attracted the attention of researchers during the past ten years. Several papers have reported that the ingredients of food or vegetables have inhibitory effects on drug transporters [16-18]. Cinnamic acid could be used as a food additive and spice, and it is also an ingredient of the widely-used cinnamon and of cinnamon tea which is very famous in China. Ferulic acid is one of the choices for a food preservative. Cynarin is one of the ingredients of artichoke, which is a very popular vegetable in Europe, the Americas, and China. In this paper, it was found that these three acids have inhibitory effects on hOAT1 and hOAT3. In addition, their inhibitory ability on hOAT1 was determined and the results showed that the IC 50 values of ferulic acid and cynarin were less than 10 μmolL1. There
– 136 –
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138
Fig. 7 Concentration dependent uptake of [14C]p-aminohippurate by hOAT1 in the presence or absence of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin. (A) Uptake of [14C]p-aminohippurate at various concentrations by hOAT1 in the absence (triangle) or presence (square) of 134 μmol"L 1 cinnamic acid. (B) Uptake of [14C]p-aminohippurate at various concentrations by hOAT1 in the absence (triangle) or presence (square) of 3.7 μmol"L 1 ferulic acid. (C) Uptake of [14C]p-aminohippurate at various concentrations by hOAT1 in the absence (triangle) or presence (square) of 90 μmol"L 1 deoxycholic acid. (D) Uptake of [14C]p-aminohippurate at various concentrations by hOAT1 in the absence (triangle) or presence (square) of 6 μmol"L 1 cynarin. Inset: Eadie-Hofstee plots of data; V, uptake rate (pmol/mg protein/min); S, concentration of [14C] p-aminohippurate (μmol"L 1). Each point is the mean ± SEM (n = 3) 3
fore, attention should be paid to the daily intake of these compounds during the process of drug treatment. In conclusion, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were validated as competitive inhibitors of hOAT1. Meanwhile, the present study suggests some new food-drug interactions. Furthermore, deoxycholic acid was found to be an inhibitor for hOAT1, hOAT3, hOATP1B1, and hOATP2B1, which probably could be used for revealing the correlation between hOATs and hOATPs.
References [1]
[2]
[3]
[4]
Giacomini KM, Huang SM, Tweedie DJ, et al. Membrane transporters in drug development [J]. Nat Rev Drug Discov, 2010, 9 (3): 215-236. El-Sheikh AAK, Masereeuw R, Russel FGM. Mechanisms of renal anionic drug transport [J]. Eur J Pharmacol, 2008, 585 (2-3): 245-255. Bohan A, Boyer JL. Mechanisms of hepatic transport of drugs: implications for cholestatic drug reactions [J]. Semin Liver Dis, 2002, 22 (2): 123-136. Campbell SD, de Morais SM, Xu JJ. Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia [J]. Chem Biol Interact, 2004, 150 (2): 179-187.
– 137 –
[5]
Shitara Y, Sato H, Sugiyama Y. Evaluation of drug-drug interaction in the hepatobiliary and renal transport of drugs [J]. Annu Rev Pharmacol Toxicol, 2005, 45: 689-723. [6] Lee EJ, Kim SR, Kim J, et al. Hepatoprotective phenylpropanoids from Scrophularia buergeriana roots against CCl 4-induced toxicity: action mechanism and structureactivity relationship [J]. Planta Med, 2002, 68 (5): 407-411. [7] Liu IM, Hsu FL, Chen CF, et al. Antihyperglycemic action of isoferulic acid in streptozotocin-induced diabetic rats [J]. Br J Pharmacol, 2000, 129 (4): 631-636. [8] Natella F, Nardini M, Di Felice M, et al. Benzoic and cinnamic acid derivatives as antioxidants: structure-activity relation [J]. J Agric Food Chem, 1999, 47 (4): 1453-1459. [9] Pollier J, Goossens A. Oleanolic acid [J]. Phytochemistry, 2012, 77: 10-15. [10] Marples BA, Stretton RJ. Antifungal utility of bile acids: US4681876 A [P]. Google Patents, 1987. [11] Horman I, Badoud R, Ammann W. Food-related applications of one- and two-dimensional high-resolution proton-NMR: structure and conformation of cynarin [J]. J Agric Food Chem, 1984, 32 (3): 538-540. [12] Miyamae Y, Kurisu M, Han J, et al. Structure-activity relationship of caffeoylquinic acids on the accelerating activity on ATP production [J]. Chem Pharm Bull, 2011, 59 (4): 502- 507.
ZHANG Zhi-Yu, et al. / Chin J Nat Med, 2014, 12(2): 131138 [13] König J, Klatt S, Dilger K, et al. Characterization of ursodeoxycholic and norursodeoxycholic acid as substrates of the hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1 and NTCP [J]. Basic Clin Pharmacol Toxicol, 2012, 111 (2): 81-86. [14] Maeda K, Kambara M, Tian Y, et al. Uptake of ursodeoxycholate and its conjugates by human hepatocytes: role of Na+-taurocholate cotransporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1 (OATP-C), and OATP1B3 (OATP8) [J]. Mol Pharm, 2006, 3 (1): 70-77. [15] Yamaguchi H, Okada M, Akitaya S, et al. Transport of fluorescent chenodeoxycholic acid via the human organic
anion transporters OATP1B1 and OATP1B3 [J]. J Lipid Res, 2006, 47 (6): 1196-1202. [16] Bailey DG. Fruit juice inhibition of uptake transport: a new type of food–drug interaction [J]. Br J Clin Pharmacol, 2010, 70 (5): 645-655. [17] Harris RZ, Jang GR, Tsunoda S. Dietary effects on drug metabolism and transport [J]. Clin Pharmacokinet, 2003, 42 (13): 1071-1088. [18] Uwai Y, Ozeki Y, Isaka T, et al. Inhibitory effect of caffeic acid on human organic anion transporters hOAT1 and hOAT3: A novel candidate for food–drug interaction [J]. Drug Metab Pharmacokinet, 2011, 26 (5): 486-493.
Cite this article as: ZHANG Zhi-Yu, SI Duan-Yun, YI Xiu-Lin, LIU Chang-Xiao. Inhibitory effect of medicinal plant-derived carboxylic acids on the human transporters hOAT1, hOAT3, hOATP1B1, and hOATP2B1 [J]. Chinese Journal of Natural Medicines, 2014, 12(2): 131-138
– 138 –