Effects of Diosmetin on Nine Cytochrome P450 Isoforms, Ugts

Toxicology and Applied Pharmacology 334 (2017) 1–7

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Toxicology and Applied Pharmacology

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Eﬀects of diosmetin on nine cytochrome P450 isoforms, UGTs and three MARK drug transporters in vitro

Jun-Jun Chena,1, Jing-Xian Zhanga,1, Xiang-Qi Zhanga, Mei-Juan Qib, Mei-Zhi Shia, Jiao Yanga, ⁎ Ke-Zhi Zhangc,d, Cheng Guoa, Yong-Long Hana, a Department of Pharmacy, Shanghai Jiao Tong University Aﬃliated Sixth People's Hospital, 600 Yi Shan Road, Shanghai 200233, China b College of Food Science and Technology, Shanghai Ocean University, 999 Hucheng Huan Road, Shanghai 201306, China c State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China d KG Pharma Limited, Floor 2, Building 3, No. 5-13 Wuhua Road, Chancheng, Foshan 528000, China

ARTICLE INFO ABSTRACT

Keywords: Diosmetin (3′, 5, 7-trihydroxy-4′-methoxyflavone), a natural flavonoid from traditional Chinese herbs, has been Diosmetin used in various medicinal products because of its anticancer, antimicrobial, antioxidant, estrogenic and anti- Cytochrome P450 inflammatory activity. However, flavonoids could affect the metabolic enzymes and cause drug-drug interactions UDP-glucuronyltransferases (DDI), reducing the efficacy of co-administered drugs and potentially resulting in serious adverse reactions. To Hepatic uptake transporters evaluate its potential to interact with co-administered drugs, the IC value of phase I cytochrome P450 enzymes Drug-drug interactions 50 (CYPs), phase II UDP-glucuronyltransferases (UGTs) and hepatic uptake transporters (organic cation transporters (OCTs), organic anion transporter polypeptides (OATPs) and Na+-taurocholate cotransporting polypeptides (NTCPs)) were examined in vitro by LC-MS/MS. Diosmetin showed strong inhibition of CYP1A2 in a concentration-dependent manner. The intensity of the inhibitory effect was followed by CYP2C8, CYP2C9, CYP2C19 and CYP2E1. For CYP2A6, CYP2B6, CYP2D6 and CYP3A4, diosmetin was found to have no significant inhibitory effects, and the induction effect on CYPs was not significant. For UGTs, diosmetin had a minimal inhibitory effect. In addition, the inhibitory effects of diosmetin on OATP and OCT1 were weak, and it had little effect on NTCP. This finding indicated that drug-drug interactions induced by diosmetin may occur through co-administration of drugs metabolized by CYP1A2.

1. Introduction superfamilies include CYP1, CYP2 and CYP3. CYP3A4 is most abun- dantly expressed in the liver and accounts for approximately 34% of Biotransformation, the metabolism of endogenous and exogenous drug metabolism, which is followed by CYP2D6 at 19%, CYP2C8 and xenobiotics in the body catalyzed through a variety of metabolic en- CYP2C9 at 16%, and CYP2C19 and CYP1A2 at 8% (Lewis, 2003; Zanger zymes, is the main mechanism by which the body maintains home- et al., 2008). Inhibition or induction of any isoforms of CYPs has been ostasis. The liver is the main organ of biotransformation through related recognized as the pivotal cause of metabolic DDI. The metabolites enzymes, such as cytochrome P450 enzymes (CYPs), UDP-glucuronyl- formed by Phase I drug-metabolizing enzymes are further metabolized transferase (UGTs), hepatic transporter and others, which are presented by phase II drug-metabolizing enzymes, such as UGTs, sulfo- in liver microsomes, cytosol and mitochondria. The inhibition of these transferases, and glutathione S-transferases. Among these enzymes, biotransformation-related proteins was the most common mechanism UGTs contribute the most to drug metabolism (Jancova et al., 2010). underlying drug-drug interactions (DDI) when patients were on mul- Evidence has shown that UGTs participate in 35% of the drug meta- tiple drug therapy. This is one of the major causes of drug withdrawal bolism related to phase II enzymes. Increasing studies have demon- from the market. strated that the inhibition of UGTs may mediate DDI and cause serious It is generally accepted that phase I drug-metabolizing enzymes, adverse reactions. Chemical medicine, herb medicine and the active CYPs, metabolize nearly 90% of pharmaceutical agents. CYP ingredients could also inhibit the activity of UGTs (Kim et al., 2016; Li

Abbreviations: CYPs, Cytochrome P450 enzymes; DDI, drug-drug interactions; HDI, herb-drug interactions; HLMs, human liver microsomes; NTCP, Na+-taurocholate cotransporting polypeptide; OAT, organic anion transporters; OCT, organic cation transporters; OATP, organic anion transporter polypeptides; P-gp, p-glycoprotein; UGTs, UDP-glucuronyltransferases ⁎ Corresponding author. E-mail address: [email protected] (Y.-L. Han). 1 Jun-Jun Chen and Jing-Xian Zhang contributed equally to this work. http://dx.doi.org/10.1016/j.taap.2017.08.020 Received 1 May 2017; Received in revised form 23 August 2017; Accepted 30 August 2017 Available online 01 September 2017 0041-008X/ © 2017 Elsevier Inc. All rights reserved. J.-J. Chen et al. Toxicology and Applied Pharmacology 334 (2017) 1–7

Mephenytoin, dextrorphan, N-deacetylation amodiaquine, 4-hydro- xydiclofenac, 4-hydroxymephenytoin, 6-hydroxychlorzoxazone and N- 3-benzylnirvanol were purchased from Toronto Research Chemicals (Toronto, Canada). Quercetin was purchased from Aladdin (Los Angeles, CA, USA). Ketoconazole was purchased from Civi Chem & Applications (Shanghai, China). Ham's F12 medium, penicillin, streptomycin, and insulin-transferrin-selenous solution (ITS-G) was purchased from Gibco (Carlsbad, CA, USA).

2.2. Animals

Fig. 1. Chemical structure of diosmetin. Male Sprague-Dawley rats (200 g to 300 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). et al., 2012). Animal care and treatment were performed strictly in accordance with A previous study showed that metabolic enzymes are the main the Provision and General Recommendation of Chinese Experimental factor inducing DDI. However, with an in-depth study of transporters, Animals Administration Legislation with a 12/12 h light/dark cycle in transporter-mediated DDI has gradually attracted the attention of re- colony room maintained at constant temperature (22 ± 1 °C) with air ffl searchers. Drug transporters include uptake and e ux transporters conditioning. All animals had free access to sterilized food and water. (Konig et al., 2013). Exogenous xenobiotics are transported into the Experimental procedures were in compliance with the Guide for Care ff liver due to uptake transporters, which a ect the drug concentration in and Use of Laboratory Animals (NIH publication, revised 1996). the organ and are thus associated with drug metabolism and excretion (Tsuji, 2006). Uptake transporters consist of the following four iso- 2.3. Primary human hepatocyte culture forms: organic anion transporters (OATs), organic cation transporters + (OCTs), organic anion transporter polypeptides (OATPs) and Na - Human liver tissue in this study was obtained from surgical resec- taurocholate cotransporting polypeptides (NTCPs). The FDA has pro- tion of paracancerous tissue from hepatocellular carcinoma in patients vided recommendations to evaluate transporter-mediated drug inter- who signed informed consent. The tissue acquisition protocol was in actions for new drug applications (Keogh, 2012). Therefore, it is im- accordance with the requirements issued by the local ethical commis- portant to explore whether the drug is an inhibitor or substrate of the sion in China. Three batches of primary human hepatocytes (ZS006, transporter pathway. ZS011 and FY016) were cultured. No.ZS006 hepatocytes was isolated ′ ′ fl fl Diosmetin (3 , 5, 7-trihydroxy-4 -methoxy avone) (Fig. 1), a a- from the liver of a Chinese woman aged 45. The donors of No.ZS011 vone found in plants belonging to the genus Teucrium (Lamiaceae) and and FY016 liver were Chinese men with 53 and 36 years old respec- in Portuguese olive leaves, is reported to exhibit a variety of pharma- tively. cological activities, such as anticancer, antimicrobial, antioxidative, Primary human hepatocytes were prepared with the classic two-step fl ff estrogenic and anti-in ammatory e ects (Kadifkova Panovska et al., collagenase perfusion method (Godoy et al., 2013). In the first step, the fl ′ 2005; Meirinhos et al., 2005). The avone glycoside diosmin (3 ,5,7- liver pieces were perfused with EGTA-containing D-hank's solution to ′ fl trihydroxy-4 -methoxy-methoxy avone-7-ramnoglucoside) has clini- remove residual blood and warm up the tissue. The end of this perfu- cally been used for treating venous tone and microcirculation and sion step is indicated by a light-red color of the out-flowing perfusion protecting capillaries. Diosmetin is the hydrolyzed and observed com- solution. During step two of the perfusion process, cell–matrix contacts ponent of diosmin in the intestines after oral administration (Hnátek, are destroyed by the digestion of ECM proteins with collagenase IV. 2015). Diosmetin is a potent inhibitor of CYP1A1 and CYP1B1, which Following the digestion procedure, the cannulae have to be pulled out can induce DDI, especially with concomitant administration of drugs quickly. BSA-containing stop solution is immediately poured over the metabolized by the same enzyme (Androutsopoulos et al., 2009). tissue for enzyme inactivation to prevent the over-digestion of the liver However, to the best of our knowledge, only a few studies have eval- tissue. The digested liver tissue is gently tweezed and cut into two ff uated the e ect of diosmetin on CYPs, and even fewer have evaluated halves with a scalpel without cutting through the capsule. Then tweeze ff its e ects on UGTs or transporters. Due to the potential clinical appli- the tissue gently and release the cells from the tissue into the sur- cation of diosmetin and adverse herb-drug interactions (HDIs) observed rounding stop solution. The cell suspension was collected with a ser- in many patients receiving conventional pharmacotherapy combined ological pipette and passed through the prepared funnel with gauze into ff herbal medicines, this study investigated the possible e ect of dios- ice-cooled 50 mL tubes in order to eliminate tissue debris. The cells metin on CYPs, UGTs and drug transporters to provide evidence for were centrifugated at 100 g at 4 °C for 5 min and resuspended in Wil- avoiding metabolic DDI (Levy et al., 2017). liams' medium E (WME) and Ham's F-12 (1:1) medium supplemented with 5% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 6.25 μg/ 2. Materials and methods mL insulin, 6.25 μg/mL transferrin, 0.01 μM dexamethasone, and 2 mM L-glutamine for human hepatocyte culture. The cell number and via- 2.1. Chemicals and reagents bility was determined by the trypan blue exclusion assay. Hepatocyte viability should be higher than 80%. Cells were resuspended and seeded Diosmetin (purity > 98%, FY20331102) was obtained from in collagen-coated 48-well plates at a concentration of 1.4 × 105 cells/

Nantong Feiyu Biological Technology Co., Ltd. (Nantong, Jiangsu, well. After 4 h of incubation at 37 °C and 5% CO2 in a humidified at- China). Human liver microsomes (HLMs, Lot1210223) were purchased mosphere, hepatocytes were overlaid with fresh medium containing from XenoTech (Kansas, KS, USA). Dexamethasone, L-glutamine, matrigel (0.25 mg/mL). Williams' medium E (WME), phenacetin, coumarin, dextromethorphan, amodiaquine, chlorzoxazone, testosterone, 7-hydroxycoumarin, para- 2.4. Primary rat hepatocyte culture cetamol, hydroxyamfebutamone, 6β-hydroxytestosterone, 7-hydroxycoumarin-D-glucuronide, α-naphthoflavone, tranylcypromine, ticlo- Primary rat pepatocytes were prepared with the method of two-step pidine and quinidine were purchased from Sigma-Aldrich (St Louis, collagenase perfusion in situ liver tissue (Godoy et al., 2013). In brief, MO, USA). Amfebutamone, diclofenac and fluconazole were purchased rats were anesthetized with pentobarbital sodium (30 mg/kg) and ab- from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). S- dominal cavity were opened by scalpel. During the two steps of

2 J.-J. Chen et al. Toxicology and Applied Pharmacology 334 (2017) 1–7

Table 1 Speciﬁc substrates and positive inhibitors of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 in human liver microsomes.

Isoforms Substrates Final concentration (μM) Positive inhibitor Final concentration (μM)

CYP1A2 Phenacetin 15 α-naphthoﬂavone 0.1 CYP2B6 Amfebutamone 5 Ticlopidine 0.5 CYP2C8 Amodiaquine 10 Quercetin 2.5 CYP2C9 Diclofenac 10 Fluconazole 20 CYP2C19 S-mephenytoin 40 N-benzylnirvanol 0.2 CYP2D6 Dextromethorphan 10 Quinidine 0.2 CYP3A4 Testosterone 10 Ketoconazole 0.05 Fluconazole 20 CYP2E1 Chlorzoxazone 40 Tranylcypromine 10 CYP2A6 Coumarin 2.5 Tranylcypromine 0.5

perfusion, diﬀerent kinds of solutions were perfused into hepatic portal MS/MS. The formation rate of 7-hydroxycoumarin-D-glucuronide in the vein. The procedure have been described in the section of primary presence of diosmetin was compared with controls in which diosmetin human hepatocytes culture. was replaced by vehicle solvent.

2.5. CYP inhibition assay 2.8. Hepatic uptake transporters inhibition assay

The inhibitory effects of diosmetin on different CYPs in human liver Inhibition of diosmetin on hepatic uptake transporters NCTP, OATP microsomes (HLMs) were detected. The NADPH working solution and OCT1 was determined in human hepatocytes. NCTP, OATP and consisted of the following: 3.0 mM NADP, 6.0 mM G6P, 3 U/mL G6PDH OCT1 mediated uptake of taurocholic acid-d5 sodium salt, rosuvastatin and 7.2 mM MgCl2. Positive controls and specific substrates are listed and 1- methyl 4- phenyl pyridine ion, respectively, was examined. below (Table 1). 40 μL human liver microsomes (0.25 mg/mL) were Human hepatocytes were seeded in 48-well plates and incubated with pre-incubated with 10 μLofdifferent concentrations of diosmetin different concentrations of diosmetin (0.137, 0.412, 1.23, 3.7, 11.1, (0.412, 1.23, 3.7, 11.1, 33.3, and 100 μM) and 10 μL substrates at 37 °C 33.3, and 100 μM) for 15 min at 37 °C in 5% CO2. After pre-incubation, for 10 min. 40 μL NADPH working solution was added for another specific substrates were co-incubated for 1 min at 37 °C. Cells were then 20 min of incubation. The reactions were terminated by ice-cold acet- washed with ice-cold HBSS and maintained in HBSS for 10 min at 4 °C. onitrile containing 100 ng/mL tolbutamide (internal standards). The Cells were incubated with ice-cold substrate solution for another 1 min incubation supernatants were collected and then analyzed by liquid at 4 °C. After washing, cells were incubated with 100 ng/mL tolbuta- chromatography-tandem mass spectrometry (LC-MS/MS). mide for 30 min. 150 μL incubation supernatants with tolbutamide and 10 μL cell lysate were mixed and diluted with 150 μL ddH2O. The 2.6. CYP induction assay substrate concentration were analyzed by LC-MS/MS.

To assess the induction effect of diosmetin on CYP1A2, CYP2B6 and 2.9. LC-MS/MS analysis of probe substrates CYP3A4, human or rat hepatocytes were seeded in 48-well plates. Following attachment, cells were incubated with different concentra- Chromatographic separations were performed using a Phenomenex μ tions of diosmetin (0.5, 1.28, 3.2, 8, 20, and 50 μM) at 37 °C in 5% CO2. C18 (30 mm × 2 mm, 4 m) column. The column was maintained at Three days later, the mixture medium was replaced by fresh medium ambient temperature (25 °C). The flow rate was maintained at 400 μL/ and incubated for another 1 h to remove residual xenobiotics. Then, the min. The mobile phase consisted of a mixture of 0.1% formic acid medium was removed and the substrate solutions (phenacetin, amfe- (solvent A) and 0.1% formic acid in chromatographic pure acetonitrile butamone and testosterone respectively) in the final concentration of (solvent B). The HPLC gradient elution was performed as follows: (1) 100 μM were incubated with hepatocytes for 30 min. The incubation mobile phase B was set to 5% at 0 min, (2) a linear gradient was in- supernatants were collected and mixed with 100 ng/mL labetalol (increased to 40% B in 3.0 min, (3) a linear gradient was increased to 90% ternal standards). Then, CYP1A2, CYP2B6 and CYP3A4 metabolic B from 3.0 min to 4.0 min, (4) an isocratic elution was maintained at products were analyzed by LC-MS/MS. For the data calculation, a ve- 90% B from 4.0 min to 4.5 min, and (5) the solvent composition was hicle control is run and the signal for the positive controls and the test returned to 5% B in 0.1 min for re-equilibration for 0.5 min. The mass article are compared to that to determine a relative fold over control transitions for the metabolites of probe substrates were detected by LC- value. Positive control for CYP1A2, CYP2B6 and CYP3A4 was ome- MS/MS. prazole, phenobarbital and rifampicin respectively. Cells in culture medium with 0.1% DMSO (without diosmetin) were designed as ne- 2.10. Data analysis gative control. The formula is as follow: The relative induction ratio % = (The signal for diosmetin – The signal for negative control)/(The The peak area ratios of the metabolites and internal standards were signal for positive control – The signal for negative control) × 100. acquired using Analyst 1.5 software packages (Applied Biosystems). The peak area ratios were plotted as a percentage of the relevant ne- 2.7. UGTs inhibition assay gative control for each reaction, and the IC50 values were calculated using GraphPad Prism 5.0 (San Diego, CA, USA). The inhibition of diosmetin on UGT activity in vitro was performed as follows. Human hepatocytes were co-incubated with substrate 7- 3. Results hydroxycoumarin (100 μM) and different concentrations of diosmetin (0.137, 0.412, 1.23, 3.7, 11.1, 33.3, and 100 μM) at 37 °C in WME 3.1. Inhibitory effect of diosmetin on CYPs medium for 15 min. Then, acetonitrile was added to the incubation mixture to terminate the reaction. Afterward, 7-hydroxycoumarin-D- Diosmetin inhibited the metabolic reaction of phenacetin to acet- glucuronide, the metabolite of 7-hydroxycoumarin, was detected by LC- aminophen, which was catalyzed by CYP1A2 in a concentration-

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Fig. 2. Inhibitory eﬀect of increasing concentrations of diosmetin on CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 in human liver microsomes. Each data point indicates the average values calculated from triplicate incubations as the means ± SEM using GraphPad 5.0 software.

Table 2 Table 2.

IC50 value of diosmetin on nine CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in human liver microsomes. 3.2. Induction of diosmetin on CYPs

Isoform Positive inhibitor Inhibitory ratio Diosmetin IC50 (μM) According to the FDA guidance in drug interaction studies, the CYP1A2 α-naphthoflavone 52.3% (0.1 μM) 2.6 [95%CI:1.8–3.9] evaluation of CYP enzyme induction should begin with studies of μ CYP2A6 Tranylcypromine 72.9% (0.5 M) > 100 CYP1A2, CYP2B6, and CYP3A in vitro. Therefore, these three CYPs were CYP2B6 Ticlopidine 9.57% (0.5 μM) > 100 CYP2C8 Quercetin 5.19% (2.5 μM) 12.1 [95%CI:8.2–17.8] chosen for exploring the induction of diosmetin on CYPs in this study. CYP2C9 Fluconazole 54.9% (20 μM) 15.5 [95%CI:9.8–24.3] Rat hepatocytes and three batches of human hepatocytes were cultured. CYP2C19 N-benzylniranol 44.1% (0.2 μM) 19.9 [95%CI:18.5–43.6] The data on rat hepatocytes showed that diosmetin (concentration CYP2D6 Quinidine 69.2% (0.2 μM) > 100 ranged from 0.5 μMto50μM) had no apparent induction of CYP1A2, CYP2E1 Tranylcypromine 69.0% (10 μM) 20.5 [95%CI:18.5–29.2] μ CYP2B and CYP3A (Table 3a). Moreover, the results were consistent in CYP3A4 Ketoconazole 45.6% (0.05 M) > 100 ff Fluconazole 73.2% (20 μM) the three di erent batches of human hepatocytes, showed no apparent induction of CYP1A2, CYP2B6 and CYP3A4 (Table 3b). These data fi The IC50 values are reported as geometric mean with 95% con dent interval (CI). suggest that diosmetin does not induce these CYPs.

μ dependent manner with an IC50 value of 2.6 M in human liver mi- 3.3. Inhibitory effect of diosmetin on UGTs crosomes. For CYP2C8, CYP2C9, CYP2C19 and CYP2E1, diosmetin ff μ showed moderate inhibitory e ects with IC50 values of 12.1 M, UGTs are key phase II metabolizing enzymes that participate in the 15.5 μM, 19.9 μM and 20.5 μM, respectively (Fig. 2). Diosmetin was metabolic elimination of various endobiotics and xenobiotics (drugs, found to have not significant inhibitory effects on CYP2A6, CYP2B6, herbs, and pollutants). The inhibition of UGT activity significantly af- CYP2D6 and CYP3A4 within the concentration range of 0.412 μMto fects the plasma exposure of drugs and herbs as well as induces DDI. μ 100 M. The IC50 values of diosmetin for the various CYPs are given in Diosmetin with different concentrations (0.137, 0.412, 1.23, 3.7, 11.1,

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Table 3a Induction ratio of diﬀerent concentrations of diosmetin on primary rat hepatocytes.

Diosmetin concentration CYP2B CYP1A2 CYP3A (μM)

50 −26.8 −70.6 −64.6 20 −5.0 17.7 3.1 8 −0.5 9.1 8.0 3.2 −3.3 −13.0 1.2 1.28 −0.8 −4.0 −2.3 0.5 −1.8 −6.5 −2.0

33.3, and 100 μM) showed weak inhibitory effects on UGTs. Even the concentration of diosmetin up to 100 μM, the inhibition ratio is only 44.7% (Fig. 3 and Table 4). The IC value could not be calculated. To 50 Fig. 3. Inhibitory effects of increasing concentrations of diosmetin on UGTs in human evaluate the rationality of the method, ketoconazole was used as a liver mirosomes. Each data point indicates the average values calculated from triplicate positive control and showed an obvious inhibitory effect on UGTs. determinations as the means ± SEM using GraphPad 5.0 software.

3.4. Inhibitory effect of diosmetin on hepatic uptake transporters Table 4 Inhibition effect of diosmetin on UGTs. The ability of diosmetin to inhibit the uptake of probe substrates Isoforms IC50(μM) Inhibitory ratio of taurocholic acid-d5 sodium salt, rosuvastatin and 1- methyl 4- phenyl pyridine ion by NCTP, OATP and OCT1, respectively, was examined in Ketoconazole Diosmetin 100 μM diosmetin human hepatocytes. The functionality of the system was assessed by determining the uptake of the specific substrate in the presence of a UGTs 40.4 > 100 44.7% known NCTP inhibitor (Cyclosporin A), OATP inhibitor (rifamycin) and OCT1 inhibitor (Verapamil). The data showed that diosmetin inhibited CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP2E1 and the OATP-mediated uptake of rosuvastatin and OCT1-mediated uptake CYP2A6. The potential of drugs to inhibit CYPs is usually investigated in μ of 1- methyl 4- phenyl pyridine ion with IC50 values of 21.51 M and vitro using HLMs. HLMs appear to be the most applicable in vitro system μ ff 44.98 M respectively, which indicated moderate inhibitory e ects on since the various CYPs are present in proportion to their in vivo re- OATP and OCT1 (Table 5). The maximal inhibition ratio of NTCP- presentation (Youdim et al., 2008). Our results that diosmetin inhibited mediated uptake of taurocholic acid-d5 sodium salt was 46.19% at a CYP1A2 with an IC of 2.6 μM were consistent with previous reports μ 50 diosmetin concentration up to 100 M. The IC50 value could not be (Androutsopoulos et al., 2010; Doostdar et al., 2000). Otherwise, re- calculated. porter showed that single oral administration of Daflon 500 mg (450 mg diosmin and 50 mg hesperidin), diosmin was not detected in 4. Discussion human plasma. The maximum concentration of diosmetin (the metabolites of diosmin) was 1.32 ± 0.67 μM[Campanero et al., 2010]. This

Drug biotransformation consists a series of complex processes that concentration is a little lower than the IC50 in our study. While, taking fl include entrance into hepatocytes via passive diffusion and active up- the daily dosage of Da on (1000 mg) in consideration, the IC50 for take mediated by hepatic uptake transporters, metabolism through CYP1A2 is highly consistent. For CYP2C8 and CYP2C9 in the present μ phase I and phase II enzymes, and excretion to bile or back to the study, diosmetin showed moderate inhibition with an IC50 of 12.1 M systemic circulation by the efflux transporters (Bi et al., 2012). The and 15.5 μM, respectively. A published study reported a stronger effect induction of DDI with these processes is the major root of adverse re- of diosmetin on these two CYP450 isoforms (Quintieri et al., 2011). The actions in the clinical setting, representing significant challenges in reason for the discrepancy may be the different substrates and cells drug research and development. To avoid diosmetin-related DDI, we used in each experiment. Furthermore, considering the difference be- used LC-MS/MS to comprehensively assess the biotransformation re- tween rats and human, we explored the induction effect of diosmetin on lated enzymes and transporters. In this study, nine isoforms of CYPs CYPs in rat and human hepatocytes. Primary hepatocytes are currently belonging to phase I enzymes, UGTs as phase II enzymes and hepatic the most accepted laboratory cell line in this field, and they have been transporters (NTCP, OATP and OCT1) were investigated. approved by the FDA as an effective tool to predict the drug-induced Diosmetin has been previously demonstrated to have an inhibitory potential effect of CYPs (LeCluyse, 2001). The induction effect of effect on CYP1A1 and CYP1A2 activity (Androutsopoulos et al., 2009). diosmetin on CYP1A2, CYP2B6, and CYP3A in human was assessed Our study investigated the inhibitory potential of diosmetin on more according to the FDA guidance. Although, the expression quantity and CYPs, including CYP1, CYP2 and CYP3 superfamilies, such as CYP1A2, isoforms of CYP450 are different between human and rats. The two

Table 3b Induction ratio of diﬀerent concentrations of diosmetin on primary human hepatocytes.

Diosmetin (μM) Human hepatocytes (ZS006) Human hepatocytes (FY016) Human hepatocytes (ZS011)

2B6 1A2 3A4 2B6 1A2 3A4 2B6 1A2 3A4

50 −5.6 6.8 −25.7 −1.9 5.5 −5.1 −5.3 4.9 13.4 20 −4.7 0.7 −20.8 −2.9 1.3 −5.7 −5.3 6.2 −5.4 8 −0.8 −0.8 −17.5 −1.6 0 −4.4 −5.3 1.4 −4.2 3.2 1.8 −0.6 −11.2 −0.5 −0.2 −2.7 −0.4 0.6 −2.1 1.28 1.2 −0.2 −5 0.4 −0.2 −1.9 4.7 0.8 −0.7 0.5 0.1 −0.7 −9.5 −3.4 −0.5 −4.2 4 0.4 −0.7

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Table 5 Inhibition eﬀect of diosmetin on drug transporters.

Transporter Substrate Drug Concentration (μM) Inhibition (%) IC50 (μM)

NTCP Taurocholic acid-d5 sodium salt Cyclosporin A 20 98.99 ± 1.08 Diosmetin 100 46.19 ± 4.2 ≥100 OATP Rosuvastatin Rifamycin 200 94.97 ± 1.29 Diosmetin 100 72.27 ± 2.58 21.51 OCT1 1- methyl 4- phenyl pyridine ion Verapamil 20 83.64 ± 2.29 Diosmetin 100 74.05 ± 3.56 44.98 species shared the same substrate of CYP2B, as well as CYP3A. So we and CYP3A4 with diosmetin was not observed. Moreover, a high con- determined CYP1A2, CYP2B and CYP3A in rat hepatocytes. The results centration of diosmetin had a minimal inhibitory effect on OATP and showed diosmetin has no inductive effect on CYPs in the concentration OCT1. Although further studies will also be needed to confirm the range from 0.5 μMto20μM in human or rat hepatocytes. However, potential effect of its active metabolites on drug biotransformation as- 50 μM diosmetin had an inhibitory effect on CYPs in rat hepatocytes. sociated enzymes or transporters, the present study suggested that This result in rat hepatocytes is different from that in human hepato- drug-drug interactions induced by diosmetin may occur when co-ad- cytes, which may be due to differences in drug sensitivity in different ministering diosmetin with drugs metabolized by CYP1A2. cell types. Moreover, in our experiments, we found that the activity of human hepatocytes is stronger than rat hepatocytes in vitro. Accord- Acknowledgements ingly, we speculated that the high concentration of diosmetin might injure rat hepatocytes in vitro. However, much more research should be This study was supported by the Traditional Chinese Medicine carried out to assess the possible toxicity of high dosage of diosmetin on Scientific Research Fund of the Shanghai Municipal Health Bureau (No: hepatocyte. Our research on CYPs indicated possible DDI interactions ZY3-RCPY-3-1036), Science and Technology Support Projects of the and the occurrence of adverse effects with co-administration of drugs Shanghai Science and Technology Committee (No: 14401972002) and metabolized by CYP1A2. the National Natural Science Foundation of China (No: 81773949). Primary human hepatocytes in vitro are widely used to investigate UGT activity and predict DDI (Schaefer et al., 2012). Several UGTs, Conflicts of interest such as UGT1A6, UGT1A9, UGT1A1, UGT1A10, UGT2B7 and UG- T2B15, participate in metabolizing 7-hydroxycoumarin into 7-hydro- The authors declare that they have no conflicts of interest. xycoumarin-D-glucuronide (Wang et al., 2006). Determination of the 7- hydroxycoumarin-D-glucuronide production could justify the inhibitory References effect of diosmetin on UGTs. Although the IC50 value does not reflect the suppression effect of diosmetin on each UGT isoform, 7-hydro- Androutsopoulos, V., Wilsher, N., Arroo, R.R., Potter, G.A., 2009. Bioactivation of the – xycoumarin-D-glucuronide can be used as a probe substrate to evaluate phytoestrogen diosmetin by CYP1 cytochromes P450. Cancer Lett. 274, 54 60. Androutsopoulos, V.P., Papakyriakou, A., Vourloumis, D., Tsatsakis, A.M., Spandidos, UGT activity (den Braver-Sewradj et al., 2016). Some researches also D.A., 2010. Dietary flavonoids in cancer therapy and prevention: substrates and in- use human liver microsome (HLM) and alamethicin to complete the hibitors of cytochrome P450 CYP1 enzymes. Pharmacol. Ther. 126, 9–20. UGT experiment. In my opinion, the use of HLM is sometimes a bit Bi, Y.A., Kimoto, E., Sevidal, S., Jones, H.M., Barton, H.A., Kempshall, S., Whalen, K.M., Zhang, H., Ji, C., Fenner, K.S., El-Kattan, A.F., Lai, Y., 2012. In vitro evaluation of easier and more economy than human hepatocyte. Microsomes are hepatic transporter-mediated clinical drug-drug interactions: hepatocyte model op- vesicle-like artifacts re-formed from pieces of the endoplasmic re- timization and retrospective investigation. Drug Metab. Dispos. 40, 1085–1092. ticulum (ER) when eukaryotic cells are broken-up in the laboratory. den Braver-Sewradj, S.P., den Braver, M.W., Vermeulen, N.P., Commandeur, J.N., Microsomes contain a large amount of drug metabolites, but they are Richert, L., Vos, J.C., 2016. Inter-donor variability of phase I/phase II metabolism of three reference drugs in cryopreserved primary human hepatocytes in suspension and not present in healthy, living cells. Primary human hepatocytes used in monolayer. Toxicol. in Vitro 33, 71–79. our study preserve the normal cellular structure to maintain the organic Campanero, M.A., Escolar, M., Perez, G., Garciaquetglas, E., Sadaba, B., Azanza, J.R., characteristics of drug metabolism. The primary human hepatocyte 2010. Simultaneous determination of diosmin and diosmetin in human plasma by ion trap liquid chromatography-atmospheric pressure chemical ionization tandem mass could ensure the assessment of the drug metabolism enzyme. Our study spectrometry: application to a clinical pharmacokinetic study. J. Pharmaceut. suggested that diosmetin had a weak inhibitory effect on human he- Biomed. 51, 875–881. Doostdar, H., Burke, M.D., Mayer, R.T., 2000. Bioflavonoids: selective substrates and patocyte UGTs, indicating that diosmetin is rarely a cause of DDI as- – ff inhibitors for cytochrome P450 CYP1A and CYP1B1. Toxicology 144, 31 38. sociated with UGTs. However, the exact e ects of diosmetin in clinical Godoy, P., Hewitt, N.J., Albrecht, U., Andersen, M.E., Ansari, N., Bhattacharya, S., Bode, use require further verification. J.G., Bolleyn, J., Borner, C., Böttger, J., Braeuning, A., Budinsky, R.A., Burkhardt, B., To explore the hepatocyte transporter significance for explaining Cameron, N.R., Camussi, G., Cho, C.S., Choi, Y.J., Rowlands, C.J., Dahmen, U., Damm, G., Dirsch, O., Donato, M.T., Dong, J., Dooley, S., Drasdo, D., Eakins, R., drug exposure, clearance and drug interactions in vivo (Keogh, 2012), Ferreira, K.S., Fonsato, V., Fraczek, J., Gebhardt, R., Gibson, A., Glanemann, M., we report on the maintenance of hepatic uptake transporters in human Goldring, C.E., Gómez-Lechón, M.J., Groothuis, G.M., Gustavsson, L., Guyot, C., hepatocytes, NTCP, OATP and OCT1. Our results showed that diosmetin Hallifax, D., Hammad, S., Hayward, A., Häussinger, D., Hellerbrand, C., Hewitt, P., Hoehme, S., Holzhütter, H.G., Houston, J.B., Hrach, J., Ito, K., Jaeschke, H., Keitel, rarely inhibited NTCP, OATP or OCT1, suggesting that diosmetin had V., Kelm, J.M., Kevin Park, B., Kordes, C., Kullak-Ublick, G.A., LeCluyse, E.L., Lu, P., little risk of causing hepatic transporter-mediated DDI. Luebke-Wheeler, J., Lutz, A., Maltman, D.J., Matz-Soja, M., McMullen, P., Merfort, I., In conclusion, our study comprehensively evaluated the effects of Messner, S., Meyer, C., Mwinyi, J., Naisbitt, D.J., Nussler, A.K., Olinga, P., diosmetin on the complex process of drug metabolism, including me- Pampaloni, F., Pi, J., Pluta, L., Przyborski, S.A., Ramachandran, A., Rogiers, V., Rowe, C., Schelcher, C., Schmich, K., Schwarz, M., Singh, B., Stelzer, E.H., Stieger, B., tabolizing enzymes related to absorption, distribution and transporter- Stöber, R., Sugiyama, Y., Tetta, C., Thasler, W.E., Vanhaecke, T., Vinken, M., Weiss, based metabolism and excretion. Meanwhile, primary rat and human T.S., Widera, A., Woods, C.G., Xu, J.J., Yarborough, K.M., Hengstler, J.G., 2013. hepatocytes were used together, increasing the clinical value of our Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative fi hepatocyte sources and non-parenchymal liver cells and their use in investigating ndings. The present study demonstrated that diosmetin had a strong mechanisms of hepatotoxicity, cell signaling and ADME. Arch. Toxicology 87, inhibitory effect on CYP1A2; a moderate inhibitory effect on CYP2C8, 1315–1530. fi fl CYP2C9, CYP2C19 and CYP2E1 and a minimal inhibitory effect on Hnátek, L., 2015. Therapeutic potential of micronized puri ed avonoid fraction (MPFF) ff of diosmin and hesperidin in treatment chronic venous disorder. Vnitr. Lek. 61, UGTs, while it had little e ect on CYP2A6, CYP2B6, CYP2D6 and 807–814. CYP3A4. The induction of rat and human hepatocyte CYP1A2, CYP2B6 Jancova, P., Anzenbacher, P., Anzenbacherova, E., 2010. Phase II drug metabolizing

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