Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 193–198

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Metabolism of palmatine by human hepatocytes and recombinant cytochromes P450 Jiri Vrba a , Barbora Papouskova b , Michaela Pyszkova a , Martina Zatloukalova a , Karel Lemr b , Jitka Ulrichova a , Jan Vacek a,∗ a

Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, Olomouc 77515, Czech Republic Regional Centre of Advanced Technologies and Materials, Department of Analytical Chemistry, Faculty of Science, Palacky University, 17, listopadu 12, Olomouc 77146, Czech Republic b

a r t i c l e

i n f o

Article history: Received 26 May 2014 Received in revised form 5 September 2014 Accepted 10 September 2014 Available online 19 September 2014 Keywords: Palmatine Mass spectrometry Metabolism Cytochrome P450 CYP2D6

a b s t r a c t In this study, we developed a new liquid chromatography–mass spectrometry (LC–MS) method for analysis of the protoberberine alkaloid palmatine and its metabolites with separation performed on a cyanopropyl-modified stationary phase. Palmatine (10 ␮M) was metabolized using suspensions of human hepatocytes and human recombinant cytochrome P450 (CYP) enzymes. Our analyses using electrospray ionization-quadrupole time-of-flight mass spectrometry revealed that palmatine was relatively resistant to the metabolic activity of human hepatocytes and recombinant CYP enzymes. However, we found that the biotransformation of palmatine in human hepatocytes included O-demethylation or hydroxylation, and that the product of palmatine demethylation was conjugated by glucuronidation or sulfation. Moreover, we found that human recombinant CYP2D6 and, to a lesser extent, CYP1A2 can mediate Odemethylation of palmatine. These results provide fundamental insights into the biotransformation of palmatine in human in vitro models and, together with the LC–MS method, can be applied for further studies on the biotransformation of palmatine and other protoberberine alkaloids. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Palmatine (Fig. 1) is a quaternary protoberberine alkaloid which is found in a number of medicinal plants such as Coptis and Corydalis species [1,2]. The structure of palmatine consists of 5,6dihydrodibenzo[a,g]quinolizinium, a tetracyclic skeleton common to all quaternary protoberberines, with four methoxy groups at positions 2, 3, 9 and 10. At physiological pH, protoberberine alkaloids exist solely in the form of iminium cations (Fig. 1, left) since their conversion to free bases, i.e. 8-hydroxy adducts (Fig. 1, right), occurs only in a strongly alkaline environment [3]. Palmatine exerts a wide range of biological activities arising from its ability to interact with proteins and nucleic acids [4]. Phytopreparations containing palmatine and other protoberberine alkaloids, e.g. berberine, coptisine and jatrorrhizine, are mainly used in traditional Chinese medicine, for instance, for the treatment of dysentery, jaundice, diabetes mellitus and rheumatic arthritis [4–6]. The bioavailability of protoberberine alkaloids is

∗ Corresponding author. Tel.: +420 585632303; fax: +420 585632302. E-mail address: [email protected] (J. Vacek). 0731-7085/© 2014 Elsevier B.V. All rights reserved.

presumably rather low [2]. Pharmacokinetic data for palmatine in humans are not available but experiments in rats administered perorally multicomponent herbal extracts have shown that palmatine can only reach submicromolar peak concentrations in plasma [5,7–9]. The highest plasma concentration of palmatine, 98.3 ng/ml (i.e. 0.28 ␮M), has been found using HPLC on a C18 column and electrospray ionization (ESI)-triple quadrupole mass spectrometry (MS) in rats after oral administration of Rhizoma Corydalis Decumbentis extract at a dose equivalent to 17.6 mg of palmatine per kg of body weight [10]. The research on possible herb–drug interactions has revealed that palmatine inhibits in vitro several human drug-metabolizing enzymes, namely, cytochrome P450 (CYP) 1A1, CYP1B1 [11], CYP2D6 [12] and CYP3A4 [13]. This aside, palmatine has been shown to induce the expression of CYP1A1 via activation of the aryl hydrocarbon receptor in human hepatoma HepG2 cells [14]. Using human colon adenocarcinoma Caco-2 cells, palmatine has also been recognized as a substrate but not an inhibitor of the multidrug efflux transporter P-glycoprotein [15]. The biotransformation of palmatine has been investigated in more detail in rats [16,17]. After oral administration of palmatine (20 mg/kg) to rats, the parent compound and thirteen metabolites were identified in urine using HPLC on a C18 column and ESI-tandem MS. Most of the metabolites but


J. Vrba et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 193–198

Fig. 1. Chemical structures of palmatine and its free base (8-hydroxy adduct).

not all were also found in plasma and faeces. In addition, in vitro experiments have demonstrated that the intestinal microflora and liver microsomes were responsible for the metabolism of palmatine in rats [16]. In a study with healthy volunteers who received a freeze-dried Rhizoma Coptidis decoction (750 mg/day) for 5 days orally, 11 metabolites of protoberberine alkaloids were isolated, using preparative chromatography techniques from urine and their structures were identified by ESI-MS and NMR. Nonetheless, Rhizoma Coptidis contains a mixture of structurally similar alkaloids, and hence it was impossible to recognize metabolites formed from palmatine [6]. Since the metabolic fate of palmatine in humans remains unclear, the aim of the present study was to analyze, using a new LC–MS method, palmatine metabolites produced by human hepatocytes and to identify human CYP enzymes involved in palmatine biotransformation. 2. Experimental 2.1. Chemicals Palmatine chloride (No. 361615), dimethyl sulfoxide (DMSO), and solvents for HPLC and extractions were obtained from Sigma–Aldrich (St. Louis, MO, USA). Stock solutions of palmatine in DMSO or methanol were prepared freshly before use. 2.2. Isolation of human hepatocytes Human tissue samples were obtained from multi-organ donors according to the protocols approved by the local ethics committee of the University Hospital, Olomouc, Czech Republic. Hepatocytes were isolated as described [18] and resuspended in serum-free medium containing Williams’s medium E, Ham’s F-12 medium and additives according to Isom et al. [19]. Hepatocyte cultures used in this study were prepared from liver samples of eight donors: a 62-year-old woman (culture LH44), a 46-year-old man (culture LH45), a 17-year-old woman (culture LH48), a 38-year-old man (culture LH49), a 55-year-old woman (culture LH50), a 58-year-old woman (culture LH51), a 60-year-old woman (culture LH52) and a 58-year-old woman (culture LH53). 2.3. Biotransformation of palmatine in human hepatocytes Suspensions of human hepatocytes in serum-free medium (4 × 106 cell/ml) were incubated for 2 h with 0.1% DMSO (control) or 10 ␮M palmatine, rotating at 80 rpm and 37 ◦ C in an Environmental Shaker-Incubator ES-20 (Biosan, Riga, Latvia). Samples of cell suspensions were taken after 2 h of incubation, centrifuged for 3 min at 50 × g and 4 ◦ C, and the cell pellets and media were separately stored at −80 ◦ C until being analyzed by LC–MS. Viability of hepatocytes at the end of incubations was checked by the trypan blue exclusion test [20] and the viability was higher than 75%. For LC–MS analysis, the cell pellets were washed with phosphate buffered saline, resuspended in methanol containing 5% (v/v)

acetic acid and sonicated 10 times (amplitude 50%, cycle 0.5 s) using an Ultrasonic Processor UP200s equipped with a sonicator probe Sonotrode Microtip S2 (Hielscher, Berlin-Teltow, Germany). The samples were centrifuged for 5 min at 12,000 × g and the supernatants were analyzed. The samples of media were mixed 1:1 (v/v) with methanol containing 5% (v/v) acetic acid, centrifuged for 5 min at 12,000 × g, and the supernatants were analyzed. 2.4. Biotransformation of palmatine by recombinant human CYP enzymes To identify CYP enzymes involved in palmatine metabolism, the alkaloid was incubated with bactosomes, i.e. bacterial membranes containing human recombinant CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 or 3A4 coexpressed with human NADPH-CYP reductase (Cypex, Dundee, UK). The incubation mixture consisted of 0.25 ml 50 mM potassium phosphate–HCl buffer, pH 7.4, containing 5 mM MgCl2 , 4 pmol of individual CYP enzymes, 1 mM NADPH and 10 ␮M palmatine (stock solution prepared in methanol). The final concentration of methanol was 0.1%. After 30 min of incubation at 37 ◦ C in a Thermomixer Comfort (Eppendorf, Hamburg, Germany), the samples were stored at −80 ◦ C until being analyzed. For LC–MS analysis, the samples were mixed 1:1 (v/v) with methanol containing 5% (v/v) acetic acid, centrifuged for 5 min at 12,000 × g, and the supernatants were analyzed. Control samples were prepared by incubating palmatine in the absence of CYP enzymes or in the absence of CYP enzymes and NADPH. 2.5. HPLC/MS analysis An Acquity I-Class UPLC system (Waters, Milford, MA, USA) equipped with a binary solvent manager, sample manager, column manager and photodiode array detector was used. The chromatographic separation was performed in Agilent Zorbax Eclipse XDB-CN column (100 mm × 2.1 mm i.d., 5 ␮m; Agilent Technologies, Santa Clara, CA, USA). The injection volume was 5 ␮l, the mobile phase flow rate was 0.4 ml/min, the temperature of the autosampler was 4 ◦ C and the column oven was set to 30 ◦ C. The mobile phase consisted of methanol (solvent B) and a mixture of water/methanol/glacial acetic acid (89:10:1, v/v; solvent A). The linear gradient profile was as follows: 0–9 min 10–55% B, 9–12 min 55–60% B, 12–12.1 min 60–10% B and 12.1–16 min 10% B. A Waters Synapt G2-S Mass Spectrometer (Waters, Manchester, UK) was connected to the UPLC system via an electrospray ionization (ESI) interface. The ESI source operated in positive ionization mode with the capillary voltage at 2.85 kV and the sampling cone voltage at 35 V. For in-source fragmentation experiments, the voltage at the sampling cone was raised to 150 V. The source temperature and the desolvation temperature were set to 110 ◦ C and 200 ◦ C, respectively. The cone and desolvation gas flows were 40 l/h and 450 l/h, respectively. Data were acquired from 50 to 1200 Da with a 0.2 s scan time and processed using Masslynx V4.1 software (Waters). The mass spectrometer was calibrated across

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the mass range of 50–1200 Da using a solution of sodium formate in acetonitrile. Data were automatically centroided and mass corrected during acquisition using an external reference leucineenkephalin (20 ␮g/l in a mixture of water/acetonitrile/formic acid (100:100:0.2, v/v), flow rate of 5 ␮l/min). Data acquisition was achieved using two interleaved scan functions (MSE experiments) which enabled simultaneous acquisition of both low collision energy (CE) and high collision energy mass spectra from a single experiment. The low trap CE was set to 6 V and the low transfer CE was set to 2 V for Function 1. In the case of Function 2 (high CE), the trap CE was off (default value 4 V) and the transfer CE ramped in the range of 20–60 V. The product ion spectra were primarily measured for the molecular ion of palmatine. To confirm the fragmentation pattern, in-source fragmentation of palmatine was done followed by MS/MS experiments of the selected fragment ions. Post-acquisition processing of the data was performed using Metabolynx XS V4.1 software (Waters).

3. Results and discussion 3.1. Chromatographic separation and MS fragmentation of palmatine This study was designed to examine hepatic biotransformation of the alkaloid palmatine under in vitro conditions using a newly proposed LC–MS method. For separation of both the parent compound and its metabolites, we used a cyanopropyl (CNP)-modified stationary phase. The reversed-phase system, based on a more polar stationary phase than C18 , may be used with advantage for separation of polar metabolites including products of phase II biotransformation. The CNP stationary phase has been applied successfully for separation of metabolites of benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine [21,22] and for separation of electrochemically generated oxidation products of berberine, a protoberberine alkaloid structurally related to palmatine [23,24]. In this study, the LC–MS analyses with positive electrospray ionization provided accurate masses and fragment ions. Putative structures of metabolites were proposed using the Metabolynx V4.1 software. Prior to the metabolic studies, we optimized the LC–MS method with 2.6 ␮M standard solution of palmatine to get its sufficient retention and well-shaped peak. The developed system with mobile phase gradient was then used for analyses of real samples where the chromatographic separations lasted for 12 min with palmatine eluted approximately in half of the time, i.e. 5.9 min. This performance is effective for separation of both polar and less polar metabolites, e.g. polar conjugates versus methyl derivatives as described in ref. [25]. Using positive ESI, a molecular ion (M+ ) of palmatine was observed at m/z 352, corresponding to the cationic form of palmatine (Fig. 1, left) which is spontaneously present in acidic mobile phase [3]. The full MS spectrum of palmatine is shown in Fig. 2A. MSE fragmentation acquired at higher collision energy (ramp 20–60 V) gave a typical fragmentation pattern of palmatine (Fig. 2B). The proposed fragmentation scheme for palmatine is shown in Fig. 3. The putative structures of palmatine fragmentation products were proposed on the basis of the accurate mass measurement of full scan MS, MS/MS, and the information available in the literature. We found that the main fragmentation processes were in accordance with previously published data [26]. In addition, our fragmentation scheme newly disclosed two structural options for the fragment ion with m/z 336.1236, which allow formation of the previously described ion m/z 334.1079 [26], and also formation of the ion with m/z 308.1287. Its further fragmentation gives rise to the most intense ion in the spectrum at m/z 278.0817 that also stems from the ions m/z 322.1079 and 294.1130. These results were confirmed using the in-source fragmentation of palmatine followed

Fig. 2. Full (A) and fragmentation (B) ESI-QqTOF MS spectra of palmatine.

by MS/MS of the observed fragment ions: 337 Da, 336 Da, 322 Da, 308 Da, 294 Da and 278 Da. Moreover, the scheme reveals a characteristic feature of the collision spectrum of palmatine, namely, a series of two hydrogen atom losses. Thus, the following ions are dehydrogenated: 322 Da to 320 Da, 320 Da to 318 Da, 294 Da to 292 Da, and 292 Da to 290 Da. 3.2. Biotransformation of palmatine in human hepatocytes Human hepatocytes represent an excellent in vitro model for toxicological and biotransformation studies [27–29]. In this study, suspensions of human hepatocytes prepared from livers of eight donors were incubated for 2 h with 10 ␮M palmatine. At this concentration, palmatine has been found to be nontoxic to human hepatocytes [14]. After incubation, hepatocytes and culture medium were separated, homogenized with acidified methanol (see Section 2.3), and the extracts were analyzed by the LC–MS method described above. It should be mentioned that during incubation (i.e. at physiological pH) as well as during sample preparation and analysis (i.e. at acidic pH), palmatine dominantly exists in its cationic form (Fig. 1, left) [3]. Analyses of cell extracts showed that phase I biotransformation of palmatine in human hepatocytes involved either O-demethylation (m/z 338.1398;  1.8 ppm) or hydroxylation (m/z 368.1504;  1.6 ppm) with the corresponding metabolites found at tR of 5.5 and 4.3 min, respectively (Fig. 4). Palmatine was also found to undergo phase II biotransformation in human hepatocytes. The product of palmatine O-demethylation was conjugated by sulfation (m/z 418.0954;  −1.4 ppm) or glucuronidation (m/z 514.1703;  −1.9 ppm) and the resulting metabolites were eluted at tR of 4.8 and 3.3 min, respectively (Fig. 4). The same metabolites were also detected in the culture medium (data not shown). Three of the above palmatine metabolites (m/z 338, 368 and 514) have been reported in rats [17]. In addition, it has been shown in vitro that O-demethylation and hydroxylation of palmatine may be mediated by rat liver microsomes as well as by rat intestinal microflora [16]. These findings suggest that palmatine may undergo similar biotransformation in rats and humans.


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Fig. 3. MS fragmentation scheme of palmatine (putative structures).

Fig. 4. LC–MS chromatograms of palmatine metabolites in homogenate of human hepatocytes. Hepatocytes were incubated with 10 ␮M palmatine for 2 h at 37 ◦ C.

The full MS spectra and putative structures of products of palmatine biotransformation are shown in Fig. 5. In general, the lower retention of all palmatine metabolites in comparison with the parent compound indicated their increased polarity. To determine the position of metabolic transformation in palmatine molecule, we used a MassFragmentTM tool, an IsoCount Metabolite Localization procedure of Metabolynx XS software, which enabled us to calculate the percentage of positional preference on the basis of the presence of confirming and contradicting spectral peaks in MSE spectra of metabolites. Although the software analyses do not provide 100% confirmation of the metabolite structure, our data revealed that the A ring of palmatine was preferentially prone to

metabolic transformations. The O-demethylation showed 63% preference for the A ring but only 42% preference for the D ring. In case of phase II biotransformation, the preference for O-demethylation and glucuronidation was 50% for the A ring and 20% for the D ring. For O-demethylation and sulfation, the preference for the A ring was 50% and for the D ring 0%. We also used the LC–MS data to evaluate semi-quantitatively, the relative amounts of individual metabolites according to the published procedure [22,25] where the sum of the parent compound plus all metabolites represented 100%. After 2 h of incubation with human hepatocytes, the products of phase I and phase II biotransformation of palmatine did not exceed 5% and 2%, respectively. Similar results were obtained in all of eight hepatocyte cultures used in the study. These results show that the extent of palmatine biotransformation is relatively low in comparison with other types of isoquinoline alkaloids. For instance, the extent of biotransformation of benzo[c]phenantridine alkaloids has been shown to amount to several tens of per cent under the same experimental conditions [21,22]. 3.3. Involvement of human CYP enzymes in palmatine biotransformation To identify CYP enzymes involved in the biotransformation of palmatine, we incubated palmatine with bactosomes containing major human recombinant drug-metabolizing enzymes, including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 [30]. After incubation, the reaction mixtures were extracted and the extracts were analyzed by the above LC–MS technique. Our analyses revealed that CYP2D6 and, to a lesser extent, CYP1A2 were able to catalyze O-demethylation of palmatine, giving the metabolite with m/z 338. In accordance with the low palmatine-metabolizing

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Fig. 5. Full ESI-QqTOF MS spectra of palmatine metabolites extracted from corresponding chromatographic peaks shown in Fig. 4. Putative structures are presented with modification at C3 in all cases. Positional isomerism C2 vs. C3 has to be taken into account.

activity of human hepatocytes, the activity of both recombinant enzymes was also relatively low. After 30 min of incubation, CYP2D6 and CYP1A2 converted only 4.6% and 1.4% of palmatine, respectively. In contrast, the other tested CYP enzymes showed no catalytic activity towards palmatine (data not shown). Finally it should be mentioned that CYP2D6 and CYP1A2 may also play a role in the metabolism of other protoberberine alkaloids. Up to now, both enzymes have been found to be involved in the biotransformation of berberine [31] and CYP1A2 has also been shown to metabolize jatrorrhizine [32]. 4. Conclusions Advanced analytical methods based on mass spectrometry and reproducible experimental protocols are prerequisites for metabolic screening. Here, we proposed and optimized the LC–MS method, based on separation with CNP-column connected with (+)ESI-qTOF-MS, which is applicable for metabolic studies of palmatine. We found that the biotransformation of palmatine in human hepatocytes includes O-demethylation or hydroxylation with the demethylated product being conjugated by glucuronidation or sulfation. Using bactosomes containing human recombinant drug-metabolizing enzymes, we also found that CYP2D6 and CYP1A2 are involved in the metabolism of palmatine. We assume that the methodology and results described here are useful for future metabolic studies on palmatine and other protoberberine alkaloids. Acknowledgements This work was supported by grant from the Czech Science Foundation (No. P303/12/G163), by grant from Palacky University (No. LF 2014 014), by Operational Programme Research and Development for Innovations – European Regional Development Fund (No. CZ.1.05/2.1.00/03.0058) and by Operational Program Education for Competitiveness – European Social Fund (project CZ.1.07/2.3.00/20.0058 of the Ministry of Education, Youth and

Sports of the Czech Republic). We thank to Dr. Petr Bachleda (University Hospital Olomouc) for the donation of human liver samples, to Drs. Martin Modriansky and Eva Gabrielova (Faculty of Medicine and Dentistry, Palacky University) for the isolation of human hepatocytes, and to Dr. Alexander Oulton for providing linguistic assistance.

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Metabolism of palmatine by human hepatocytes and recombinant cytochromes P450.

In this study, we developed a new liquid chromatography-mass spectrometry (LC-MS) method for analysis of the protoberberine alkaloid palmatine and its...
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