http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, Early Online: 1–9 ! 2015 Informa UK Ltd. DOI: 10.3109/00498254.2015.1056283

RESEARCH ARTICLE

Interaction of six protoberberine alkaloids with human organic cation transporters 1, 2 and 3 Liping Li*, Siyuan Sun*, Yayun Weng, Feifeng Song, Sisi Zhou, Mengru Bai, Hui Zhou, Su Zeng, and Huidi Jiang

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Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, P.R. China Abstract

Keywords

1. Organic cation transporters (OCTs) play an important role in drug safety and efficacy. Protoberberine alkaloids are ubiquitous organic cations or weak bases with remarkable biological actives. This study was to elucidate the potential interaction of alkaloids (coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine and corydaline) with OCTs using Madin–Darby canine kidney (MDCK) cells stably expressing human OCT1, OCT2 and OCT3. 2. All the tested alkaloids significantly inhibited the uptake of MPP+, a model OCT substrate, in MDCK-hOCTs cells with the IC50 of 0.931–9.65 lM. Additionally, coptisine, jatrorrhizine and epiberberine were substrates of all the hOCTs with the Km of 0.273–5.80 lM, whereas berberrubine was a substrate for hOCT1 and hOCT2, but not for hOCT3, the Km values were 1.27 and 1.66 lM, respectively. The transport capacity of coptisine in MDCK cells expressing the variants of hOCT1-P341L or hOCT2-A270S was significantly higher than that in wild-type (WT) cells with the Clint (Vmax/Km) of 379 ± 7.4 and 433 ± 5.7 ll/mg protein/min, respectively. 3. The above data indicate that the tested alkaloids are potent inhibitors, and coptisine, jatrorrhizine, epiberberine and berberrubine are substrates of hOCT1, hOCT2 and/or hOCT3 with high affinity. In addition, the variants (OCT1-P341L and OCT2-A270S) possess higher transport capacity to coptisine than WT hOCTs.

Cells, inhibitor, interaction, organic cation transporters, protoberberine alkaloids, substrate

Introduction Protoberberine alkaloids, such as coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine and corydaline (Figure 1), belong to isoquinoline alkaloids mainly found in the plants of Berberidaceae, Fumariaceae and Papaveraceae families. They have many pharmacological and biological activities, such as anti-diarrhoeal (Taylor & Greenough, 1989), antibacterial (Yan et al., 2008), hypoglycaemic, lipid-lowering (Yuan et al., 2006) and antioxidant (Jung et al., 2009), and are commonly used alone or presented in some traditional Chinese medicines. For instance, Fuzi Xiexin Tang (Xu et al., 1999), composed of Rhei Radix et Rhizoma, Scutellariae Radix, Coptidis Rhizoma and Aconiti Lateralis Radix Preparata, is a classic traditional Chinese medicine formula containing berberine, jatrorrhizine, palmatine and coptisine, which has been applied to treat massive alimentary tract *These authors contributed equally to this work. Address for correspondence: Huidi Jiang, Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, Zhejiang, P.R. China. Tel: +86 571 88208408. E-mail: [email protected]

History Received 8 April 2015 Revised 24 May 2015 Accepted 26 May 2015 Published online 2 July 2015

bleeding, gastrointestinal diseases and chronic renal failure for >1800 years. Similarly, Huanglian Jiedu decoction (Choi et al., 2014) is an ancient prescription with R. coptidis, R. Scutellaria, C. phellodendri and F. gardeniae jasminoidis in the ratio of 3:2:2:3, which contained berberine, palmatine and jatrorrhizine (Sun et al., 2006), and has been used for heatclearing and detoxification in China for 2000 years. Recently, coptisine (Zhang et al., 2014) and palmatine (Ali & Dixit, 2013) were found to exert anti-tumor activity. As combination therapy of herbal formulations with prescription medications is widely applied, the safety of herb–drug interactions, especially via inhibition of transporters, has aroused intense concerns (Li et al., 2014; Lin et al., 2012). Organic cation transporters (OCTs), including OCT1, OCT2 and OCT3, mediate the transport of a broad variety of positively charged endogenous and exogenous organic substances in vivo, and the substrates of OCT1, OCT2 and OCT3 are partially overlapping (Courousse & Gautron, 2014; Yonezawa & Inui, 2011). In human, OCT1 is predominantly expressed in the sinusoidal membrane (blood side) of hepatocytes, OCT2 is mainly localized at the basolateral membrane of the proximal tubular cells in the kidney and OCT3 is broadly distributed in various organs, such as placenta, brain and lung (Koepsell et al., 2003; Yonezawa &

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Figure 1. Chemical structures of six protoberberine alkaloids.

Inui, 2011). These hepatic and renal influx transporters may not only be responsible for the toxicity of drugs but also for the potential drug–drug interactions (DDIs), which may result in changes of hepatic or renal excretion parameters and/or modification of pharmacokinetics (Cho et al., 2011; Tanihara et al., 2009). Therefore, it is vital to predict the DDI caused by inhibitors or substrates of OCTs. Based on the structures of protoberberine alkaloids (Figure 1), they might be inhibitors or substrates of OCTs, for instance, it was reported that berberine, a typical protoberberine alkaloid, was a high-affinity substrate for OCT1 and OCT2 (Nies et al., 2008), besides, jatrorrhizine was identified as a substrate of rat OCT2 (Tan et al., 2013). However, only limited information is available on protoberberine alkaloids with OCTs. Thus, the first aim of the present study was to elucidate the interaction of coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine, corydaline with human OCTs using Madin–Darby canine kidney (MDCK)-hOCTs cells established by our lab (Sun et al., 2014; Tu et al., 2013; Wang et al., 2014), and to provide a clue revealing the possible role of OCTs in the disposition of these alkaloids. Population genetics analyses has identified numerous single-nucleotide polymorphisms (SNPs) in the OCTs gene (Sakata et al., 2004; Wang et al., 2008), the allele frequencies of non-synonymous SNPs in OCT1 and OCT2 genes are different among different ethnic groups, such as OCT1-P341L variant, a significant higher allele frequency, which was found in African–Americans and Asian–Americans (8% and 17%) compared with Caucasians (up to 2%) (Nies et al., 2009). While, the variant of OCT2-A270S with the highest allelic frequency has a prevalence of 12.7% among all the different ethnic groups (Gorboulev et al., 1997). Some of these SNPs have been found to be associated with the transport activity of OCTs, for instance, the variant of OCT2-A270S exhibited significantly reduced metformin uptake, an important oral

anti-diabetic drug (Choi & Song, 2012). Furthermore, the genetic variation in OCTs may be associated with variation in response to metformin in vivo (Shu et al., 2007). Moreover, the positions of the mutations and their effects on transport activity and specificity may provide insight into the molecular mechanism, by which substrates are recognized and transported through OCTs. Thus, the second aim of the present study was to clarify whether genetic polymorphisms in hOCT1 (hOCT1-P341L) or hOCT2 (hOCT2-A270S) gene affected cellular uptake of coptisine, an important member of the protoberberine alkaloids, the results will help us to understand the interindividual differences in drug responses.

Materials and methods Chemicals and reagents Coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine and corydaline (purity of all:  98%) were purchased from Nanjing Zelang Pharmaceutical Technology Ltd. Company (Nanjing, China). Loratadine, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+), 1-methyl-4-phenylpyridinium (MPP+), verapamil, desipramine, cimetidine and quinidine were obtained from Sigma Chemical (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO (Invitrogen Life Technologies, Frederick, MD). Bicinchoninic acid (BCA) protein assay kit was obtained from Beyotime Institute of Biotechnology (Beyotime, Shanghai, China). Acetonitrile was obtained from Tedia (Fairfield, TX). All other chemicals were purchased from commercial sources and were of analytical grade. Cell culture Madin–Darby canine kidney cell line was obtained from Peking Union Medical College (Beijing, China).

Interaction of the alkaloids with hOCTs

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DOI: 10.3109/00498254.2015.1056283

MDCK cells stably transfected with plasmid pcDNA3.1(+) vector-containing human OCT1-coding DNA sequence (MDCK-hOCT1), human OCT2-coding DNA sequence (MDCK-hOCT2), human OCT3-coding DNA sequence (MDCK-hOCT3) and empty vector pcDNA3.1(+) (mock cells) were constructed in our lab (Sun et al., 2014; Tu et al., 2013; Wang et al., 2014). To obtain mutant plasmids that carried single-nucleotide substitution mutations in human OCT1 and OCT2 gene (pcDNA3.1-hOCT1-P341L and pcDNA3.1-hOCT2-A270S), mutagenesis reactions were carried out using the quick-change site-directed mutagenesis kit according to the manufacturer’s instructions (Takara Bio. Inc., Shiga, Japan). The double-stranded oligonucleotides used for site-directed mutagenesis of the OCT1 and OCT2 gene were as follows: OCT1-P341L, forward 50 -ACCTGTTCCGCA CGCTGCGCCTGAGGAAGCGC-30 and reverse 50 0 GCGCTTCCTCAGGCGCAGCGTGCGGAACAGGT-3 and OCT2-A270S, forward 50 -TTGCAGTTCACAGTTTCTCT GCCCAACTTCT-30 and reverse 50 -AGAAGTTGGGCAGA GAAACTGTGAACTGCAA-30 . MDCK-hOCT1-P341L and MDCK-hOCT2-A270S cells were established by the same method. The activity of hOCTs in the stably transfected cells was validated by the model substrates such as ASP+ (Schophuizen et al., 2013) and MPP+ (Sala-Rabanal et al., 2013), with or without OCTs inhibitors (quinidine for OCT1/ OCT3, cimetidine for OCT2) (Ahlin et al., 2008; Hasannejad et al., 2004; Yonezawa & Inui, 2011). The mRNA expression of OCT1 in MDCK-hOCT1 and MDCK-hOCT1-P341L cells, OCT2 in MDCK-hOCT2 and MDCK-hOCT2-A270S cells were confirmed to be at approximate level by real-time reverse-transcription polymerase chain reaction (real-time RT-PCR), and showed a 150 000- and 13 000-fold increase in the OCT1 and OCT2 mRNA expression compared to that in mock cells. Cells were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37  C with 5% CO2 and 95% humidity. The cells were subcultured after being 90% confluent with 0.25% trypsin. In the present study, cells were used between the 10th and 30th passages. Uptake assays Cells were seeded in 24-well plates coated with poly-D-lysine (Costar Corning Inc., Corning, NY) at a destiny of 2  105/ well, cultured for 3 days and washed twice with Hank’s balanced salt solution (HBSS, pH 7.4), then the uptake studies were performed as our previous method (Sun et al., 2014). Briefly, the cells were pre-incubated with HBSS (37  C, 10 min), then the uptake was initiated by adding 200 ll of HBSS-containing MPP+ or the alkaloids (coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine and corydaline) in the absence or presence of different inhibitors at 37  C. The uptake was terminated by removing the incubation buffer and adding ice-cold buffer quickly at the designated time. Then the cells were washed 3 times with ice-cold buffer and lysed with 100 ll of 0.1% sodium dodecyl sulfonate. hOCTs-mediated uptake was obtained by subtracting the uptake in mock cells from that in MDCK cells overexpressing wild-type (WT) hOCTs or genetic variants (hOCT1-P341L and hOCT2-A270S).

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The uptake conditions for kinetic study were optimized by performing the uptake-concentration and uptake-time course in our preliminary experiments, and the uptake time and substrate concentration in the initial linear phase (i.e. initial uptake rates) were selected. All the experiments were performed in triplicate. The uptake of MPP+ and alkaloids in the cells was quantified with LC–MS/MS and normalized to the total protein content in the lysates with a BCA protein assay kit (Beyotime, Shanghai, China). The uptake in presence of inhibitors was expressed as the percentage of the vehicle group (% of control). LC-MS/MS analysis The intracellular concentrations of MPP+ or the protoberberine alkaloids in the uptake samples were determined by modified LC-MS/MS methods (Sun et al., 2014; Zhang et al., 2008) using a Waters ACQUITY UPLC-MS (Waters, Manchester, UK) with a triple quadrupole mass spectrometer. Twice volume of acetonitrile containing 180 ng/ml loratadine as internal standard (IS) was added into the cell lysates to precipitate the protein, after centrifugation at 15 000g for 20 min, 7 ll of the supernatant was analyzed by LC-MS/MS. The HPLC separation was performed on an Agilent XDB C18 column (2.1 mm  50 mm, 3.5 mm) with a gradient mobile phase consisted of 0.1% formic acid-water and 0.1% formic acid–acetonitrile at a flow rate of 0.3 ml/min. The MS/MS analysis was performed in the positive ion mode using the electrospray ionization and the quantification was obtained using multiple-reaction monitoring (MRM) mode. Conditions for MS analysis were as following: source temperature 140  C, desolvation temperature 350  C, cone gas flow 50 l/ h, desolvation gas flow 904 l/h and collision gas flow 0.15 ml/min. Other parameters are listed in Table 1. Data analysis The half-maximal inhibitory concentration (IC50) values were estimated by the sigmoidal curve fitting of the log10 inhibitor concentrations versus the uptake of MPP+ (% of control) using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA). The Michaelis–Menten constants Vmax and Km were calculated by fitting the data to the Michaelis–Menten equation V ¼ Vmax/(1 + (Km/[S])), where V is the uptake velocity and [S] is the concentration of substrate. The intrinsic clearance for the transport (Clint) was estimated from Vmax/Km. Data are expressed as mean ± SD derived from at least three independent studies. Statistical differences were determined using one-way analysis of variance (ANOVA) with Bonferroni test for selected pairs of groups, p values 50.05 were considered statistically significant.

Results Inhibitory effect of the alkaloids on hOCTs As shown in Figure 2, all of the tested alkaloids including coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine and corydaline significantly reduced the uptake of MPP+ with the concentration-dependent manner in MDCK-hOCT1, MDCK-hOCT2 and MDCK-hOCT3 cells, and the IC50 values were 0.931–2.61, 2.27–8.30 and 2.27–9.65 mM,

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respectively, which were approximated to that of the typical inhibitors, such as quinidine (an inhibitor for OCT1 and OCT3, the IC50 values were 3.62 and 1.52 lM, respectively) and cimetidine (for OCT2, the IC50 was 2.51 mM) (Table 2). The above results indicate these protoberberine alkaloids are the potent inhibitors of hOCTs. Intracellular accumulation of the alkaloids The intracellular accumulation of the alkaloids was determined in MDCK-hOCT1, MDCK-hOCT2, MDCK-hOCT3 (MDCK-hOCTs) and mock cells. The uptake of coptisine, jatrorrhizine, epiberberine and berberrubine in MDCKhOCTs cells showed significant time-dependent, the uptake

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Table 1. Mass spectrum parameters. Detected substances

m/z (Parent and product)

CE (V)

Cone (V)

Capillary (kV)

Coptisine Jatrorrhizine Epiberberine Berberrubine Palmatine Corydaline MPP+ Loratadine (IS)

320.3/292.2 338.3/322.2 336.4/320.0 322.4/307.1 352.4/336.3 370.4/192.1 170.0/128.0 383.1/267.1

30 28 35 30 30 35 30 40

55 45 40 47 52 53 63 17

0.5 3.0 0.8 4.4 3.2 4.1 3.9 3.2

time of 2 min was in the linear phase and chosen for subsequent experiments. Coptisine, jatrorrhizine and epiberberine were accumulated in MDCK-hOCTs cells in concentration-dependent manner and significantly higher than that in mock cells, indicating the three alkaloids might be the substrates of hOCTs. Additionally, the cellular accumulation of berberrubine in MDCK-hOCT1 and MDCK-hOCT2 cells, but not in MDCK-hOCT3 cells, were also markedly higher than that in mock cells (Figure 3), suggesting berberrubine was a selective substrate of hOCT1 and hOCT2, but not of hOCT3. In contrast, the cellular accumulation of palmatine (1, 5 lM) and corydaline (1, 5 lM) in MDCK-hOCTs cells were almost equal to that in mock cells, meanwhile quinidine and cimetidine, inhibitors of OCTs, did not reduce their cellular accumulation (data not shown), indicating palmatine and corydaline were not substrates of hOCTs. The kinetic studies revealed that the accumulation of coptisine, jatrorrhizine, epiberberine and berberrubine in mock cells was linear within the tested concentration range, while the hOCTsmediated uptake followed Michaelis–Menten kinetics with the Km values were 1.27–5.80 lM (Table 3), suggesting that they were high-affinity substrates of hOCT1, hOCT2 and/or hOCT3. The other kinetic parameters, including Vmax and intrinsic clearance (Clint ¼ Vmax/Km), are listed in Table 3. Moreover, the known inhibitors (100 lM), such as quinidine and verapamil (for OCT1), cimetidine and desipramine (for

Figure 2. Inhibitory effect of the tested alkaloids and the typical inhibitors on hOCT1-, hOCT2- and hOCT3-mediated MPP+ uptake. The MDCKhOCTs cells were incubated with MPP+ (1 lM, 3 min) in the absence or presence of coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine, corydaline and quinidine, cimetidine within the concentration of 0.001–1000 lM at 37  C. The uptake was expressed as the percentage of MPP+ uptake without inhibitors (% of control). Data are expressed as mean ± SD, n ¼ 3.

Interaction of the alkaloids with hOCTs

DOI: 10.3109/00498254.2015.1056283

OCT2), quinidine and decynium22 (for OCT3), significantly reduced the hOCTs-mediated uptake of the above alkaloids in MDCK-hOCT1, MDCK-hOCT2 and/or MDCK-hOCT3 cells (p50.001) (Figure 4), which further demonstrated

that coptisine, jatrorrhizine, epiberberine and berberrubine were the substrates of hOCT1, hOCT2 and/or hOCT3.

Table 2. IC50 values of the alkaloids and the known inhibitors for MPP+ uptake mediated by hOCTs.

It is reported that non-synonymous variant P341L, one of the variants of OCT1, was extensively related to clinical drug response (Choi & Song, 2008), while OCT2-A270S variant has been identified as a common SNP with a high allele frequency (10%) in all the ethnic groups examined. To elucidate the influence of gene polymorphisms on cellular transport of protoberberine alkaloids, the uptake of coptisine was performed in MDCK-hOCT1-P341L and MDCK-hOCT2-A270S cells. The results revealed that the uptake of coptisine was in concentration-dependent manner, and the values of Km and Vmax were 0.522 ± 0.15 lM and 198 ± 8.6 pmol/mg protein/min for hOCT1-P341L, 0.198 ± 0.026 lM and 85.8 ± 2.4 pmol/mg protein/min for hOCT2-A270S, respectively (Figure 5). Compared with that

IC50 (lM)

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Compounds

hOCT1

hOCT2

hOCT3

Coptisine Jatrorrhizine Epiberberine Berberrubine Palmatine Corydaline Quinidine Cimetidine

0.931 ± 0.09 0.932 ± 0.09 1.31 ± 0.35 1.26 ± 0.19 2.30 ± 0.49 2.61 ± 1.3 3.63 ± 1.5 /

2.27 ± 0.35 2.31 ± 0.21 3.11 ± 0.53 8.30 ± 1.6 5.25 ± 0.78 2.76 ± 0.74 / 2.51 ± 0.97

2.27 ± 0.65 4.09 ± 1.2 2.27 ± 0.66 4.88 ± 0.93 6.63 ± 0.85 9.65 ± 2.0 1.52 ± 0.28 /

Data are expressed as mean ± SD, n ¼ 3.

Effects of OCT1 and OCT2 genetic polymorphisms on coptisine uptake

Figure 3. Concentration-dependent hOCTs-mediated cellular uptake of coptisine, jatrorrhizine, epiberberine and berberrubine. Cells were incubated with coptisine, jatrorrhizine, epiberberine and berberrubine for 2 min at 37  C. Data are expressed as mean ± SD, n ¼ 3.

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Table 3. Kinetic parameters for the uptake of coptisine, jatrorrhizine, epiberberine and berberrubine in MDCK-hOCT1, MDCK-hOCT2 and MDCKhOCT3 cells. MDCK-hOCT1

MDCK-hOCT2

MDCK-hOCT3

Compounds

Km

Vmax

Clint

Km

Vmax

Clint

Km

Vmax

Clint

Coptisine Jatrorrhizine Epiberberine Berberrubine

5.80 ± 1.0 4.46 ± 0.40 4.37 ± 0.42 1.27 ± 0.23

609 ± 38 251 ± 7.5 281 ± 6.7 225 ± 22

105 ± 14 56.3 ± 3.7 64.3 ± 4.4 177 ± 16

0.814 ± 0.051 0.358 ± 0.12 3.78 ± 0.10 5.66 ± 1.7

128 ± 2.5 9.54 ± 0.76 52.9 ± 4.8 101 ± 6.3

157 ± 9.2 26.6 ± 5.2 14.0 ± 1.7 17.8 ± 3.6

0.351 ± 0.034 0.453 ± 0.042 0.273 ± 0.032 /

57.5 ± 1.8 16.5 ± 0.38 4.04 ± 0.16 /

164 ± 4.7 36.4 ± 3.2 14.8 ± 0.81 /

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Km, lM; Vmax, pmol/mg protein/min; Clint, Vmax/Km: ll/mg protein/min; /, Berberrubine is not the substrate of hOCT3. Data are expressed as mean ± SD, n ¼ 3.

Figure 4. Inhibitory effect of OCT inhibitors on (A) hOCT1-, (B) hOCT2- and/or (C) hOCT3-mediated uptake of the four alkaloids. Cells were incubated with 1 lM of coptisine, jatrorrhizine, epiberberine and berberrubine in the absence or presence of OCT inhibitors for 2 min at 37  C. Data are expressed as mean ± SD, n ¼ 3. Compared with the vehicle group (0.2% DMSO), ***p50.001.

of in cells expressing WT hOCTs, the Km for hOCT1-P341L and hOCT2-A270S were much lower, indicating that the affinity of hOCT1-P341L or hOCT2-A270S to coptisine was markedly higher, meanwhile the Clint of coptisine increased to 3.6-fold in MDCK-hOCT1-P341L cells and to 2.8-fold in MDCK-hOCT2-A270S cells, respectively (Table 4).

Discussion Organic cation transporters have been increasingly recognized as key determinants in drug disposition, therefore, they are considered as one of the major factors involved in drug–drug/ herb interactions. The major findings of the current study are as follows: (i) the six alkaloids tested are potent inhibitors of hOCTs with low IC50 values, (ii) coptisine, jatrorrhizine and epiberberine are high-affinity substrates of hOCTs, and berberrubine is a selective substrate for hOCT1 and hOCT2 but not for hOCT3 and (iii) the variants of hOCT1-P341L and hOCT2-A270S significantly influence the cellular transport of coptisine, the Km values became significantly lower and the Clint values of coptisine were increased to 3.6- and 2.8-fold of the WT hOCTs, respectively.

Based upon the data generated in this study, the alkaloids tested showed potent inhibitory effect on hOCT1, hOCT2 and hOCT3 with low IC50 (Table 2), furthermore, coptisine, jatrorrhizine and epiberberine were proved to be the substrates of hOCTs with high affinity. Considering that OCTs are primarily expressed in the liver and kidney and involved in drug uptake or excretion (Koepsell et al., 2007; Nies et al., 2011b), we deduced these herbal alkaloids might interfere with OCTs-mediated hepatic uptake and/or renal excretion of other drugs, such as metformin (Nies et al., 2011a), on the other hand, other inhibitors or substrates of OCTs might affect their disposition in liver and kidney. For instance, Ma et al. (2014) found that the maximum plasma concentrations (Cmax) of jatrorrhizine and epiberberine were 3.49 lg/ml (10.3 lM) and 8.85 lg/ml (26.3 lM) after a single oral dose of Huanglian Jiedu decoction extract at 7.5 g/kg in rats, respectively, whereas 0.852 lg/ml (2.42 lM) for palmatine, suggesting that the plasma concentrations of these alkaloids were much higher than or close to the IC50 values evaluated in the present study. Otherwise, Wang et al. (2010) reported that jatrorrhizine and coptisine were primarily distributed in the liver followed by lung and kidney, while

Interaction of the alkaloids with hOCTs

DOI: 10.3109/00498254.2015.1056283

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Figure 5. Effects of genetic polymorphisms in (A) OCT1 (hOCT1-P341L) and (B) OCT2 (hOCT2-A270S) on the uptake of coptisine. Cells were incubated with coptisine (0.2–10 lM) for 2 min at 37  C, respectively. Data are expressed as mean ± SD, n ¼ 3.

Table 4. Kinetic parameters for the uptake of coptisine.

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Genetic polymorphisms hOCT1 hOCT1-P341L hOCT2 hOCT2-A270S

Km

Vmax

Clint

5.80 ± 1.0 0.522 ± 0.15 0.814 ± 0.051 0.198 ± 0.026

609 ± 38 198 ± 8.6 128 ± 2.5 85.8 ± 2.4

105 ± 14 379 ± 7.4 157 ± 9.2 433 ± 5.7

Km, lM; Vmax, pmol/mg protein/min; Clint, Vmax/Km: ll/mg protein/min. Data are expressed as mean ± SD, n ¼ 3.

palmatine was primarily distributed in the lung and liver, after oral administration of the total alkaloids (800 mg/kg) extracted from Rhizoma Coptidis to rats, indicating that these alkaloids have high concentrations in lung, kidney and liver. To our knowledge, these tested alkaloids alone, or natural medicine as well as traditional Chinese medicine containing coptisine, jatrorrhizine, palmatine, epiberberine, berberrubine or corydaline were widely applied in China. Therefore, DDI of these alkaloids with other drug/herb mediated by OCTs should be considered. Moreover, DDI mediated by transporters depends on the concentration of inhibitor, further study is needed to predict the possibility of DDI in vivo. More remarkably, the exposure of coptisine, epiberberine, jatrorrhizine and palmatine was higher observed in diabetic rats after oral administration of 1.3 g/kg Coptidis Rhizoma extract, the AUC0–8 was increased by 6.9-, 6.7-, 7.5- and 8.6-fold compared to that of age-matched control rats, and Cmax was increased by 7.1-, 7.2-, 9.5- and 7.2-fold, respectively (Yu et al., 2010), indicating that diabetic conditions might lead to higher risk of DDI mediated by transporters or CYP450 enzymes. It was proved that coptisine, jatrorrhizine and epiberberine were substrates of hOCTs with high transport efficacy, in contrast, palmatine and corydaline possessed highly passive permeability, thus their cellular accumulation were not governed by OCTs. Substrates of hOCTs are typically organic cations with one or two positive charges, or weak bases positively charged at physiological pH (Nies & Schwab, 2010), while non-charged compounds, such as cimetidine at alkaline pH, may be transported as well (Barendt & Wright, 2002). One explanation may be that selectivity of cation binding versus cation transport is determined by different criteria, which implies that binding and transport are different things (Schmitt & Koepsell, 2005). This could explain why

many large, hydrophobic compounds, such as palmatine and corydaline in the present study, were found to be strong inhibitors but not substrates of OCTs. The Km (Table 3) of coptisine, jatrorrhizine and epiberberine were much lower than that of MPP+ (Km for OCT1: 15–32 lM; OCT2: 3–19 lM; OCT3: 47–83 lM) (Nies et al., 2011a) and metformin (Km for OCT1: 2.1 mM; OCT2: 1.4 mM; OCT3: 2.3 mM) (Bednarczyk et al., 2003; Kimura et al., 2005; Zomorodi et al., 1995), indicating that the above alkaloids exhibited higher affinity for OCTs than the model substrates. This phenomenon might attribute to the presence of a highly hydrophobic aromatic isoquinoline ring in these alkaloids molecules, whereas the model substrates of OCTs, such as metformin and MPP+, have a hydrophilic second guanidine structure or aphenyl-pyridinium iodide structure instead of the isoquinoline ring (Figure 1). Since all the available pharmacophores for OCTs substrates or inhibitors contain at least one hydrophobic moiety of the molecule next to a positive charge (Moaddel et al., 2007; Nies et al., 2011a; Zhang et al., 2011), coptisine, jatrorrhizine and epiberberine fit best in the structural requirements for OCTs substrates. It is estimated that genetics can account for 20–95% of variability in drug disposition and effects (Ciarimboli, 2011). Polymorphisms of OCTs can be relevant in determining how patients respond to pharmacological therapy and further predicting the potential drug adverse effects. Obviously, altered transport activity was observed with the two common functionally relevant genetic variants of hOCT1 (hOCT1P341L) and hOCT2 (hOCT2-A270S) (Figure 5), and the kinetic studies (Table 4) demonstrated that hOCT1-P341L and hOCT2-A270S showed much lower values of Vmax and of Km, inferring that these amino acid changes resulted in altered transport capacity to coptisine, which is in good agreement with the finding by Shu et al. (2003) that two non-synonymous SNPs (P283L and P341L) of OCT1 had lower transport activity for MPP+ than WT OCT1.

Conclusion In conclusion, we demonstrated that the protoberberine alkaloids tested, including coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine and corydaline, exhibited potent inhibitory effect on OCT1, OCT2 and OCT3. Coptisine, jatrorrhizine, epiberberine are proved to be high-affinity substrates of OCTs, while berberrubine is a selective substrate for hOCT1 and hOCT2 but not for hOCT3. Two variants

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L. Li et al.

(hOCT1-P341L and hOCT2-A270S) show higher transport capacity to coptisine compared to the WT hOCTs. The present results provide us the helpful information to understand the pharmacological effect of the above alkaloids or the herb medicine containing these alkaloids, and also help us to predict the DDI between alkaloids and other substrates or inhibitors of OCTs.

Declaration of interest This work was supported by the Zhejiang Nature Science Foundation of China (LY14H310007), by the National Natural Science Foundation of China (81373474) and National Major Projects for Science and Technology Development of Ministry Science and Technology of China (2012ZX09506001-004).

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Interaction of six protoberberine alkaloids with human organic cation transporters 1, 2 and 3.

1. Organic cation transporters (OCTs) play an important role in drug safety and efficacy. Protoberberine alkaloids are ubiquitous organic cations or w...
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