http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, Early Online: 1–10 ! 2013 Informa UK Ltd. DOI: 10.3109/00498254.2013.845707
RESEARCH ARTICLE
In vitro metabolism of monensin A: microbial and human liver microsomes models Bruno A. Rocha1, Marilda D. Assis1, Ana P. F. Peti1, Luiz A. B. Moraes1, Fernanda L. Moreira2, Norberto P. Lopes2, Stanislav Pospı´sˇil3, Paul J. Gates4, and Anderson R. M. de Oliveira1
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Departamento de Quı´mica, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo (FFCLRP-USP), Ribeira˜o Preto, Brazil, 2Nu´cleo de pesquisas em Produtos Naturais e Sinte´ticos, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo (FCFRP-USP), Ribeira˜o Preto, Brazil, 3Institute of Microbiology, Academy of Sciences of the Czech Republic (ASCR), Vı´denˇska´, Prague, Czech Republic, and 4School of Chemistry, University of Bristol, Bristol, UK Abstract
Keywords
1. Monensin A, an important antibiotic ionophore that is primarily employed to treat coccidiosis, selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. 2. In this study, we evaluated the fungi Cunninghamella echinulata var. elegans ATCC 8688A, Cunninghamella elegans NRRL 1393 ATCC 10028B and human hepatic microsomes as CYPP450 models to investigate the in vitro metabolism of monensin A and compare the products with the metabolites produced in vivo. 3. Mass spectrometry analysis of the products from these model systems revealed the formation of three metabolites: 3-O-demethyl monensin A, 12-hydroxy monensin A and 12hydroxy-3-O-demethyl monensin A. We identified these products by tandem mass spectrometry and through comparison with the in vivo metabolites. 4. This analysis demonstrated that the model systems produce the same metabolites found in in vivo studies, thus they could be used to predict the metabolism of monensin A. Furthermore, we verified that liquid chromatography coupled to mass spectrometry is a powerful tool to study the in vitro metabolism of drugs, because it allows the successful identifications of several derivatives from different metabolic models.
Cytochrome P450, LC–ESI-MS/MS, mass spectrometry, microbial transformation, polyether ionophore
Introduction Drug metabolism studies constitute an important and necessary step when evaluating drug efficacy and safety (Asha & Vidyavathi, 2009; Pupo et al., 2008). Although these studies can rely on the use of in vivo animal systems as models, these models pose a number of issues such as the high costs and time associated with animal breeding as well as the ethical aspects of experimentation on animals. Therefore, conducting such studies in vitro has become an important tool for drug development and metabolite production (Asha & Vidyavathi, 2009; Pekala et al., 2012). Fungi have extensively been used to biotransform organic compounds, because they are considered an economically and ecologically viable technology (Faber, 2011; Pupo et al., 2008). In addition, fungi are a useful tool to structurally modify bioactive compounds and drugs. Using fungi, it is possible to add target groups at certain positions of the molecule of interest, which would be difficult to accomplish Address for correspondence: Anderson Rodrigo Moraes de Oliveira, Departamento de Quı´mica, Faculdade de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, USP, Avenida dos Bandeirantes, 3900, 14040-901, Ribeira˜o Preto, SP, Brazil. Tel: +55-16-3602-0388. Fax: +55-16-36024838. E-mail: [email protected]
History Received 15 August 2013 Revised 5 September 2013 Accepted 13 September 2013 Published online 17 October 2013
via conventional synthesis methods (Asha & Vidyavathi, 2009; Borges et al., 2009a,b; Faber, 2011; Pupo et al., 2008). Moreover, some microorganisms can simulate the mammalian cytochrome P450 (CYP-P450) metabolism of many pharmacologically important molecules such as drugs (Barth et al., 2012; Bocato et al., 2012; Borges et al., 2009b; Carrao et al., 2011; Fortes et al., 2013; Hilario et al., 2012) and natural products (Capel et al., 2011; Liu & Yu, 2010; Parshikov et al., 2012; Rocha et al., 2012; Verza et al., 2009). Among fungi, the Cunninghamella species can regio- and stereoselectively metabolize a wide range of xenobiotics, resembling reactions that occur in mammalian enzymatic systems. This genus has specific properties that make these microorganisms very useful in drug metabolism studies (Asha & Vidyavathi, 2009; Pekala et al., 2012). The Cunninghamella genus has enzymes that are synonymous to those involved in xenobiotic detoxification in mammals (Asha & Vidyavathi, 2009; Sun et al., 2004). Indeed, evidences show that the Cunninghamella genus can predict the fate of the drug in the mammalian organism better than other microorganisms (Asha & Vidyavathi, 2009; Pekala et al., 2012; Sun et al., 2004). Because it is impractical to isolate metabolites from dosed animals, researchers have turned to producing large quantities of the major and minor
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metabolites. This avoids the concerns often associated with chemical synthesis, such as the use of toxic reagents and drastic reaction conditions (Borges et al., 2009a; Faber, 2011; Pupo et al., 2008). An effective in vitro screening strategy to estimate the metabolic fate of drug in humans is to use a human liverderived experimental system (Asha & Vidyavathi, 2010; Li, 2001, 2004). The past few decades have witnessed the development of several in vitro human liver models, with one of the most commonly used systems being liver microsomes (Asha & Vidyavathi, 2010). Liver microsomes models offer many advantages: they are inexpensive, reproducible and easy to prepare (Asha & Vidyavathi, 2010; Raucy & Lasker, 1991; Wu et al., 2012) and they are one of the most fully characterized in vitro systems for drug metabolism research (Asha & Vidyavathi, 2010; Messiano et al., 2013; Moreira et al., 2013). Monensin A (MonA) is representative of a large group of naturally occurring polyether ionophore antibiotics. In veterinary medicine, it is currently the most widely used coccidiostatic and non-hormonal growth-promoting agent (Huczyn´ski et al., 2012; Lowici & Huczyn´ski, 2013). Its properties arise from its ability to form complexes with sodium cations and transport the resulting complexes across cell membranes, thereby disturbing the natural Naþ/Kþ concentration gradient and leading to cell death among Gram-positive bacteria (Huczyn´ski et al., 2008, 2011; Huczyn´ski, 2012). To identify which CYP-P450 isoenzyme participates in the biotransformation of this ionophore, Nebbia et al. (1999, 2001) used rat liver microsomes. They concluded that CYP-P450 3A (the major CYP-P450 enzyme in human liver) plays an important role in the MonA oxidative metabolism, and that compounds able to bind or inhibit this isoenzyme could elicit toxic interactions with MonA (Nebbia et al., 1999, 2001). Therefore, the possibility that humans could ingest food of animal origin containing MonA residues should not be ignored, in fact it should be avoided, to prevent the onset of possible interaction or health problems (e.g. antibiotic resistance) (Nebbia et al., 1999, 2001). MonA presents a narrow safety margin and animals have experienced several accidental poisonings (Harris et al., 1998; Nebbia et al., 2001). These two separate issues have encouraged researchers to design studies to better understand the mechanism of action of MonA and its metabolites. The present study investigated the in vitro metabolism of MonA using a microbial model and human liver microsomes. We identified the metabolites identification by low- and highresolution liquid chromatography–electrospray ionizationtandem mass spectrometry (LC–ESI-MS/MS) and analyzed the LC retention times on the basis of the corresponding reference standards.
Materials and methods Chemicals MonA, 95% purity, was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Solvents such as chloroform (CHCl3) and methanol (MeOH) were high-performance liquid chromatography HPLC grade. All the compounds used in this study were commercially available from Aldrich
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or Sigma and were of analytical grade purity unless otherwise stated. Microorganisms The microorganisms used in this study, Cunninghamella echinulata var. elegans ATCC 8688A and Cunninghamella elegans NRRL 1393 ATCC 10028B were purchased from American Type Culture Collection (ATCCÕ ) (Manassas, VA). Fungal cultures were maintained on potato dextrose agar slants and stored at 4 C. Microbial biotransformation procedure Screening for this experiment was performed in 30 mL of fermentation medium in 125 mL Erlenmeyer flasks, to select efficient conditions to perform the biotransformations of MonA. The procedures were performed as previously described by our group (Barth et al., 2012; Bocato et al., 2012; Carrao et al., 2011; Hilario et al., 2012), with modifications. Five disks of 0.5 cm diameter containing the fungal mycelia were aseptically transferred to 9.0 cm diameter Petri dishes containing potato dextrose agar, the mycelia were allowed to grow for 5 days at 30 C. After that, three disks of 0.5 cm diameter containing the mycelia were transferred using a Transfer tube (Fisher Scientific, Pittsburgh, PA) and inoculated in 50 mL Falcon tubes containing 20 mL of prefermentative medium (10 g of malt extract, 10 g of dextrose, 5 g of triptone, 3 g of yeast extract and deionized water to 1 L). The pre-fermentative medium was used to grow the microorganism for 3 days, with shaking at 100 rpm, at 30 C. After that, the mycelia were transferred to 125 mL Erlenmeyer flasks containing 30 mL of modified Czapek medium (25 g of sucrose, 2 g of NaNO3, 1 g of KH2PO4, 0.5 g of MgSO4 7H2O, 0.5 g of KCl, 0.01 g of FeSO4 7H2O and deionized water to 1 L; the pH was adjusted to 5.0 with 1 mol/L HCl). After 2 days of cultivation, 3.0 mg of MonA was dissolved in 300 mL of N,N-dimethylformamide and added to Czapek medium. The cultures were incubated for 8 days at 30 C, with shaking at 120 rpm. Control flasks containing culture medium with the fungus but without MonA, culture medium with the fungus and N,N-dimethylformamide and without MonA, culture medium with MonA and without the fungus, or only the culture medium were analyzed. A time-course study was carried out as follows: (i) one Erlenmeyer flask was taken every 24 h; (ii) the product was extracted with chloroform and then analyzed by (iii) thin layer chromatography (TLC) and (iv) LC–MS, to check the degree of MonA transformation. The time-course of these biotransformations were followed by LC–MS and the relative ratio between the substrate and its transformed products was determined on the basis of their peak area. Extraction procedure and chromatographic analysis Aliquots of 2 mL of biotransformation medium were centrifuged at 1000 rpm (180 g) for 5 min. Then, 1 mL was transferred to a conical tube, extracted with 3 mL of chloroform, and agitated for 15 min at 1000 rpm by employing the Vibrax VXRÕ (IKA, Staufen, Germany). After that, 2 mL of the organic phase was collected, transferred to the tube, and allowed to evaporate until complete dryness under
In vitro metabolism of monensin A
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DOI: 10.3109/00498254.2013.845707
compressed air. The residues were dissolved in the mobile phase and analyzed by chromatographic systems. The recovery of the MonA extracted from the biotransformation samples was determined using a calibration curve obtained from the data of the analytes not submitted to extraction. By employing this solvent, it was possible to extract 100% of MonA from Czapek liquid culture medium. The TLC analysis was carried out on silica gel [GF254 Merck, Brazil, Category No. 7730; mobile phase: CHCl3/ MEOH (93:7, v/v) plates 0.25 mm thickness], and the spots were visualized after spraying the plates with vanillin–sulfuric acid (1% H2SO4 in ethanol). The LC–ESI-MS analysis was performed in a Varian LC–MS 1200L triple quadrupole apparatus coupled to a mass spectrometer with ESI ionization in the positive mode (Varian Medical Systems Inc., Palo Alto, CA). The chromatographic analysis was accomplished by an injection volume of 10 mL, sample concentration 50 mg/mL in an XterraÕ analytical column MS C-18 (150 2.1 mm, 5 mm) (Waters, Milford, MA) and following gradient (MeOH:H2O): 0.1 min 70% MeOH, 20.0 min 98% MeOH, 21.0 min 30% MeOH, 30.0 min 70% MeOH. The MS conditions were: capillary voltage of 3.2 kV, cone voltage of 40 V, source temperature of 40 C, and N2 desolvation temperature of 350 C. The sample ion was monitored for m/z values ranging from 610 to 800. MonA metabolism was determined by comparing the metabolites retention time and unit mass. Metabolites elucidation in tandem mass spectrometry The fragmentation studies were conducted according to Lopes et al. (2002a,b). The ion spectrum of the products were obtained using the micrOTOF-Q IITM ESI-Qq-TOF Mass Spectrometer (Bruker Daltonics Inc., Billerica, MA) operating in the positive mode, equipped with a hybrid quadrupole and a time-of-flight analyzer. The samples were directly introduced into the spectrometer through an infusion pump (Cole-Palmer, Vernon Hills, IL), at a flow rate of 300 mL/h. The capillary temperature and voltage were maintained at 250 C and 4.0 kV, respectively. The ion was extracted from the ionization source using a cone potential between 5 and 30 kV. Nitrogen was used to spray and dry the sample solution; argon was employed as the collision gas. MS/MS analysis of the precursor ion was extracted into the mass spectrometer for the fragmentation; it was activated by collision with argon (energy 5–80 eV). In vitro assay with human liver microsomes Pooled human liver microsomes (pooled mixed sex, fifty individual donors, protein concentration: 20 mg/mL, stored at 80 C), NADPH-regenerating system solution: solution A (glucose-6-phosphate and NADPþ) and solution B (glucose6-phosphate dehydrogenase) and potassium phosphate buffer 0.5 M pH 7.4 were purchased from BD GentestTM (Woburn, MA) and used throughout this study. The in vitro metabolism using human liver microsomes was investigated by adding 1.4 mmol/L (1.0 mg/mL) MonA to the incubation medium (1.5 mg/mL human liver microsomal protein, potassium phosphate buffer 0.5 mol/L pH 7.4, NADPH-regenerating system). An aliquot of 20 mL of the
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MonA solution was transferred to a 10-mL conical glass tube, to which 18 mL of potassium phosphate buffer 0.5 mol/L pH 7.4, 10 mL of solution A containing NADPþ and glucose6-phosphate, and 2 mL of solution B containing glucose-6phosphate dehydrogenase were added. After 60 min of incubation with 1.5 mg/mL human liver microsomal, at 37 C with agitation, the reaction was terminated by adding chloroform (1.0 mL). This mixture was agitated for 10 min at 1000 rpm in a Vibrax VXRÕ agitator and centrifuged for 5 min at 4000 rpm (2900g). Finally, 750 mL of the organic phase was collected and allowed to evaporate to complete dryness, under compressed air. After that, the residue was reconstituted in the mobile phase and injected into the chromatographic system. Control incubations were performed in the absence of the cofactor solution and in the absence of the microsomal preparation. The difference between ‘‘with’’ and ‘‘without’’ NADPH was considered CYP-P450-mediated metabolism.
Results Fungal biotransformation studies The biotransformation of MonA results showed that both C. echinulata and C. elegans were able to metabolize MonA into polar derivatives, as shown by appearance of spots with lower retention factors that MonA spot in TLC plates (data not shown) and with lower retention time that the MonA peak in the chromatograms of LC–MS experiments (Figure 1) and showed that the MonA was stable in the blank culture medium with the concentration remaining unchanged over the whole 8-day period. The structure MonA and its metabolites are showed in Figure 2. Comparing the MonA ESI-MS spectrum with that of the biotransformed product mixture (data not shown), oxidation of MonA can be detected. The mass spectrum showed that O-demethylated (m/z 679), hydroxylated (m/z 709) and probably O-demethylated-hydroxylated (m/z 695) compounds were the major products of the biotransformation process. All three biotransformed products still acted as ionophores as they were observed to preferentially coordinate to sodium. This suggests that the central structure of monensin remained unaltered after biotransformation (Lopes et al., 2001). Additionally, the intensity of the ion at m/z 679 in the spectrum of the oxidation products increased proportionally in relation to the MonA spectrum, indicating a product originated from O-demethylation, the main reaction observed in the in vivo metabolism of MonA (Nebbia et al., 1999, 2001). From Figure 1 is can be seen that there are more polar peaks that MonA in the chromatogram related to the biotransformation process which were absent from the chromatogram of the control samples. The retention time of MonA was 16.4 min, whereas the metabolites with m/z 679, m/z 709 and m/z 695 appeared at 13.1, 11.8 and 8.9 min, respectively. The metabolite with m/z 679 and retention time 13.7 min was identified as monensin B (MonB), found as a contaminant of MonA (the identity of the peaks was obtained by employing the multiple reaction monitoring (MRM) acquisition mode during the spectrometric analysis, as shown in the case of the human microsome study).
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Figure 1. LC–MS analysis of the biotransformation culture of C. echinulata var. elegans ATCC 8688A and C. elegans NRRL 1393 ATCC 10028B incubated with MonA showing the formation of MonA metabolites: Biot’s Medium ¼ MonA incubated with fungus for 8 days; Control ¼ MonA incubated with only Czapek medium; Blank ¼ Fungus incubated with Czapek without MonA. In this blank, the major peak in the chromatogram has m/z 659.
Figure 2. Structure of MonA and its metabolites found in this study: metabolite 1 (3-O-demethyl MonA, m/z 679); metabolite 2 (12-hydroxy MonA, m/z 709) and metabolite 3 (12-hydroxy-3-O-demethyl MonA, m/z 695).
The time-course for the biotransformation of MonA by C. echinulata and C. elegans was investigated and the ratio of substrate/metabolite (on the basis of the peak area) was obtained by LC–MS (Figure 3). Around 80% of MonA had been transformed after 2 days of incubation. The maximum concentration obtained for the metabolites 1, 2 and 3 after biotransformation by C. echinulata was reached (13.1, 2.7 and 3.2%, respectively) on the sixth day and the maximum concentration obtained for the metabolites 1, 2 and 3 after biotransformation by C. elegans was reached (9.6, 5.8 and 3.2%, respectively) on the eighth day of incubation.
Structural identification of the biotransformation products A previous systematic ESI-MS/MS investigation carried out with MonA demonstrated that it is possible to detect [M þ H]þ only at low pH and high cone voltage and that increasing the energy of the collision-induced dissociation (CID) process enables extensive fragmentation of the sodium salts. The fragmentation of protonated and sodiated MonA produces a substantial number of ions (Lopes et al., 2001). This study revealed that employing the protonated precursor
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Figure 3. Time-course of the microbial transformation of MonA by C. echinulata and C. elegans: (diamond) MonA; (square) metabolite 1, m/z 679; (triangle) metabolite 2, m/z 709 and (cross) metabolite 3, m/z 695. Table 1. Formula, observed mass, calculated mass, mass error (parts per million) and identity in high-resolution accuratemass spectrum of MonA and its metabolites. Formula C36H62NaO11 C35H60NaO11 C35H60NaO11 C36H62NaO12 C35H60NaO12
Observed mass
Calculated mass
Error
Identity
693.4200 679.4026 679.4008 709.4134 695.3977
693.4184 679.4028 679.4028 709.4139 695.3977
2.3 2.6 þ2.9 0.1 0.0
MonA MonB 3-O-Demethyl MonA 12-Hydroxy MonA 12-Hydroxy-3-O-demethyl MonA
ion in ESI-MS/MS studies posed limitations, and that it is important to understand the fragmentation pathways of sodiated salts. Key ions result from Grob–Wharton type fragmentation or pericyclic rearrangements as well as simple neutral losses (Lopes et al., 2002a,b). The same fragmentation mechanisms occurred for the metabolites produced in the present in vitro metabolism studies. The low-resolution product ion spectrum of metabolites 1, 2 and 3 (Supplementary Figure S1) displayed structural fragments that possibly stemmed from the Grob–Wharton type fragmentation mechanism (Grob, 1969; Grob & Baumann, 1955; Wharton & Hiegel, 1965) and/or H2O elimination, as previously proposed (Lopes et al., 2002a,b) for MonA. High-resolution accurate-mass MS was used to determine the molecular formula of these ions. Table 1 contains the accurate masses and theoretical formulae with errors for these fragments and Table 2 shows the fragment identity for all the ions observed in the product ion spectrum of MonA and its metabolites. The proposed fragmentation for metabolites 1, 2 and 3 are shown in Figures 4–7. In this proposal, the acid function undergoes protonation, because it is the most available position for this event, in agreement with all the data published on the 3D structure of MonA in the solid state (Duax et al., 1980; Martinek et al., 2000; Paz et al., 2003). Figure 4 shows elimination of 172 Da (for metabolites 1 and 2) and of 186 Da (for metabolite 3) via the Grob–Wharton type mechanism. This elimination involved ring A (O-4), to yield fragment ‘‘B’’ (m/z 507, 523 and 523 for metabolites 1, 2 and 3, respectively). Fragment ‘‘B’’ lost CO and H2O, which resulted in ion ‘‘C’’ (m/z 479, 495 and 495 for metabolites 1, 2 and 3, respectively) and ion ‘‘D’’ (m/z 461,
Table 2. Fragment identity for all the ions observed in the product ion spectrum of MonA and its metabolites.
Ion
MonA
MonB
3-O-Demethyl MonA
A B C D E F G H I J L M N O P
693 507 479 461 443 – 343 303 675 657 – 675 501 483 383
679 493 465 447 429 – 329 289 661 643 – 661 487 469 369
679 507 479 461 443 – 343 303 661 643 – 661 501 483 383
12-Hydroxy MonA
12-Hydroxy-3O-demethyl MonA
709 523 495 477 459 441 343 303 691 673 655 691 517 499 441
695 523 495 477 459 441 343 303 677 659 641 677 517 499 441
477 and 477 for metabolites 1, 2 and 3, respectively). Ion ‘‘D’’ then lost H2O, to give fragment ‘‘E’’ (m/z 443, 459 and 459 for metabolites 1, 2 and 3, respectively). The ions with m/z 343 (‘‘G’’) and m/z 303 (‘‘H’’) (Figure 5) originated from product ion ‘‘E’’ via two possible mechanistic pathways. The first would involve loss of H2O from the tetrahydrofuran ring, generating the product ion ‘‘F’’ (m/z 441), which in turn might give the product ion ‘‘H’’ (m/z 303) by a Grob–Wharton-type fragmentation resulting in loss of 82 Da (for metabolite 1) and 98 Da (for metabolites 2 and 3), followed by a loss of 40 Da (elimination of 1,4-propyne in a charge remote mechanism). In the second step of this
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Figure 4. The proposed fragmentation route showing the formation of product ion ‘‘E’’ for metabolites 1, 2 and 3, as previously discussed for metabolite 2 by Sousa-Junior et al. (2013).
Figure 5. The proposed fragmentation route showing the formation of product ion m/z 303 for metabolites 1, 2 and 3.
pathway, the product ion with m/z 303 would be originated from loss of 140 Da (for metabolite 1) and loss of 156 Da (for metabolites 2 and 3). Both ions derived from the product ion ‘‘E’’ would stem from a pericyclic rearrangement analogous to the one observed for the dihydropyran fragment with elimination of propyne. This pathway, together with MonA fragmentation studies in the gas phase; led to the proposal that hydroxylation had occurred at the OH position in the MonA structure (for both metabolites 2 and 3). All these fragmentation pathways fully agree with the data previously published for MonA and MonB (Lopes et al., 2002a,b). The second pathway proposed for the metabolites fragmentation would be loss of water involving the oxygen atom
O-11 (Figure 6), to produce the product ion ‘‘I’’. Another possibility would be a neutral loss of water involving the oxygen atoms O-10 and O-11 with formation of the fragment ‘‘L’’ (with m/z 655 and 641 for metabolites 2 and 3, respectively). Migration of a hydrogen in the b-position would favor this third water elimination involving the O-12 atom, culminating in the conjugated -system that prevents elimination of 116 Da by pericyclic rearrangement. The relationship of the hydroxyl group O-5 and the ether oxygen (O-4) in ring ‘‘A’’ may lead to neutral loss of water, producing the intermediate ion ‘‘M’’ and giving rise to a third possible route for ion ‘‘A’’ fragmentation (Figure 7). This ion could fragment, to give the product ion ‘‘N’’ m/z 501 (for
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Figure 6. The proposed fragmentation route showing the formation of product ion ‘‘L’’ for metabolites 2 and 3.
Figure 7. Proposed fragmentation mechanism involving the dihydropyran system of MonA and subsequent formation of fragment ion ‘‘O’’’ 441. This production is key to location of hydroxylation in metabolites 2 and 3.
metabolite 1) and m/z 517 (for metabolites 2 and 3) by the loss of 160, 174 or 160 Da, respectively. The elimination of an H2O molecule involving the dihydropyran system is shown in Figure 7 suggesting the formation of the product ion ‘‘O’’. Consequently, product ion ‘‘O’’ could fragment by migration of a hydrogen in position a to the carbonyl group, producing the ion ‘‘O0 ’’ (m/z 441) by loss of 58 Da from the hydroxyl derivative (Figure 7). For metabolite 1, ‘‘O’’ could undergo loss of 100 Da, resulting in the ion ‘‘P’’, the same ion observed in MonA fragmentation. The existence of the product ion with m/z 441 as a fragment of OH-MonA might represent a key product ion to justify a hydroxylation position in the MonA molecule. The structural identification of these products shown that metabolite 1 is 3-O-demethyl MonA, metabolite 2 is 12-hydroxy MonA and metabolite 3 is 12-hydroxy-3-O-demethyl MonA (Figure 2). Human liver microsome study To establish correlations between the microbial model employed in this study and the MonA human drug metabolism, an in vitro metabolism assay employing human liver microsomes was also conducted. It is usual for this in vitro process to yield low amounts of the metabolites. Therefore, the LC–ESI-MS/MS MRM mode of analysis was employed,
to monitor the following transitions: m/z 693 ! 461 for MonA, m/z 679 ! 465 for MonB, m/z 679 ! 461 for 3-Odemethyl MonA, m/z 709 ! 477 for 12-hydroxy MonA and m/z 695 ! 477 for 12-hydroxy-3-O-demethyl MonA. The microsomal metabolism furnished metabolites 3-O-demethyl MonA and 12-hydroxy MonA, but not metabolite 12hydroxy-3-O-demethyl MonA. The metabolites showed the same retention times as those observed in the microbial transformation, as well as the same ion product spectrum (Figure S2). In addition, the total yield of the MonA metabolism was estimated to be 20% by measuring the rate of substrate disappearance by LC–ESI-MS. Metabolites structure confirmation We confirmed the structure of metabolite 1, 3-O-demethyl MonA, according to Pospı´sˇil et al. (Pospı´sˇil et al., 1987; Pospı´sˇil & Zima, 1987). In this article, 3-O-demethyl MonA was isolated from Streptomyces cinnamonensis cultures in the presence of a methylation inhibitor (Pospı´sˇil et al., 1987; Pospı´sˇil & Zima, 1987), which served a standard. They verified its chemical structure by MS/MS and 1H NMR analyses. The chromatographic behavior (retention time) and MS spectra of this standard was compared with those of the metabolite in this study. LC–ESI-MS/MS analysis revealed
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that both substances presented identical product ion spectra and retention times (Supplementary Figure S3). 12-Hydroxy MonA was obtained by performing bioinorganic catalysis with metalloporphyrins (Sousa-Junior et al., 2013). In this study, the formation of 12-hydroxy MonA was demonstrated with full characterization by high-resolution accurate-mass MS/MS studies performed in a Fouriertransform ion cyclotron resonance mass spectrometer (Sousa-Junior et al., 2013). Once again, we compared the chromatographic behavior (retention time) and mass spectrum of this reported product with those of the metabolite obtained here. Both substances displayed the same ESI-MS/MS spectra and retention time during LC–ESI-MS/MS analysis (Supplementary Figure S3).
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Discussion Various metabolism and biotransformation studies on MonA have been performed by various laboratories and these results have shown that this drug undergoes extensive metabolization to a great number of products that are qualitatively similar among species but not quantitatively (Davison, 1984; Donoho et al., 1978; Kiel et al., 1998; Nebbia et al., 1999). Some metabolites have been identified; that seem to result from an O-demethylation and/or hydroxylation at various positions along the carbon backbone of the ionophore molecule (Davison, 1984; Donoho et al., 1978; Kiel et al., 1998; Nebbia et al., 1999). Numerous fungi (and other microorganisms) have hydrolytic and reductive ability to metabolize foreign organic substances as mammalian systems do; therefore, they are applicable as microbial models as proxies to study mammalian metabolism. Among fungi, the genus Cunninghamella can regio- and stereo-selectively metabolize a wide variety of xenobiotics. These reactions resemble those taking place in mammalian systems (Asha & Vidyavathi, 2009; Pupo et al., 2008). Vaufrey et al. (1990) reported one of the first studies involving the microbial transformation of MonA using the soil bacterium Sebekia benihana NRRL 11111. This microorganism efficiently converted MonA to three major compounds that do not occur in mammalian species. This study showed that the three metabolites contained an open terminal ring, and that additional oxidation took place for two metabolites (Vaufrey et al., 1990). Despite the report of Vaufrey et al. (1990) on the biotransformation of MonA by a microorganism, this study is the first to describe that a microorganism produces the same metabolites as those found in mammalian species, which makes the fungal microbial model very attractive to obtain in vivo metabolites of this ionophore. The aim of this present study was to identify a microorganism that can metabolize MonA and to explore the similarities between this microbial model and the reported mammalian systems in terms of MonA metabolism. On the basis of the success previously observed for the Cunninghamella genus, we evaluated the potential of two species of the genus Cunninghamella (C. echinulata and C. elegans) to oxidize MonA. During the MonA biotransformation study, we observed the production of three major compounds. We identified these compounds by comparing
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their ESI-MS/MS with that of MonA: 3-O-demethyl MonA, which originated from O-demethylation (m/z 679), 12-hydroxy MonA, which resulted from hydroxylation (m/z 709) and 12-hydroxy-3-O-demethyl MonA which stemmed from O-demethylation and a hydroxylation (m/z 695), had fragmentation patterns similar to that of the substrate, as described in the ‘‘Results and discussion’’ section. These three metabolites had been previously detected in studies involving MonA metabolism (Davison, 1984; Donoho et al., 1978; Kiel et al., 1998). MonA is safe and effective when used at the recommended dosages. The therapeutic index of this drug is rather narrow and several accidental poisononings have been described in the literature not only involving animals (Henri et al., 2008; Nebbia et al., 1999, 2001) but also man (Caldeira et al., 2001; Kouyoumdjian et al., 2001; Souza et al., 2005). Recent reports of significant levels of MonA residues in broiler meat, chicken meat and eggs (Rose´n, 2001; Moloney et al., 2012; Rokka & Peltonen, 2006) have leading to possible/eventual interaction with other drugs or possible human health problems such as antibiotic resistance and intoxications. Moreover, recent studies involving their biological properties for cancer therapy have called for studies involving drug metabolism in humans (Lowici & Huczyn´ski, 2013; Huczyn´ski, 2012). Therefore, the metabolism of MonA using a human liver microsome model was also investigated because it represents an effective strategy to estimate drug metabolic fates in humans (Asha & Vidyavathi, 2010; Li, 2001, 2004). Additionally, these microsomes constitute one of the best characterized in vitro systems for drug metabolism research (Asha & Vidyavathi, 2010). Our preliminary results showed the formation of two metabolites, 3-O-demethyl MonA and 12-hydroxy MonA; hence, one can use this model to predict human metabolism of MonA. In addition, studies involving the biological activities of these metabolites have shown that they are less active than MonA; thus, MonA biotransformation corresponds to a classic detoxification pathway: the resulting molecules are more polar, facilitating their elimination into the culture medium (Pospı´sˇil et al., 1987; Pospı´sˇil & Zima, 1987; Vaufrey et al., 1990).
Conclusions In conclusion, our results have demonstrated the ability of the C. echinulata and C. elegans to mimic the action of CYPP450 in MonA metabolism. This species produces the same metabolites found in vivo studies. The ability of these models to mimic mammalian metabolism, to perform biotransformation reactions, and to produce significant amounts of drug metabolites suggest that this microbial system represents a suitable alternative for drug biotransformation studies. This system can also complement in vivo metabolism studies, thereby dismissing the need to use large quantities of animals in experimental research. The MonA metabolites were elucidated by ESI-MS/MS; the fragmentation studies showed that LC–ESI-MS/MS is a powerful tool to study drug metabolism in vitro, because it can identify metabolites in different models.
DOI: 10.3109/00498254.2013.845707
Declaration of interest This research was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP Process 2011/ 05800-0, 2011/17508-1 and 2009/51812-0), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) for financial support and for granting research fellowships. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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RESEARCH ARTICLE
In vitro metabolism of monensin A: microbial and human liver microsomes models Bruno A. Rocha1, Marilda D. Assis1, Ana P. F. Peti1, Luiz A. B. Moraes1, Fernanda L. Moreira2, Norberto P. Lopes2, Stanislav Pospı´sˇil3, Paul J. Gates4, and Anderson R. M. de Oliveira1
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1
Departamento de Quı´mica, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo (FFCLRP-USP), Ribeira˜o Preto, Brazil, 2Nu´cleo de pesquisas em Produtos Naturais e Sinte´ticos, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo (FCFRP-USP), Ribeira˜o Preto, Brazil, 3Institute of Microbiology, Academy of Sciences of the Czech Republic (ASCR), Vı´denˇska´, Prague, Czech Republic, and 4School of Chemistry, University of Bristol, Bristol, UK Abstract
Keywords
1. Monensin A, an important antibiotic ionophore that is primarily employed to treat coccidiosis, selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. 2. In this study, we evaluated the fungi Cunninghamella echinulata var. elegans ATCC 8688A, Cunninghamella elegans NRRL 1393 ATCC 10028B and human hepatic microsomes as CYPP450 models to investigate the in vitro metabolism of monensin A and compare the products with the metabolites produced in vivo. 3. Mass spectrometry analysis of the products from these model systems revealed the formation of three metabolites: 3-O-demethyl monensin A, 12-hydroxy monensin A and 12hydroxy-3-O-demethyl monensin A. We identified these products by tandem mass spectrometry and through comparison with the in vivo metabolites. 4. This analysis demonstrated that the model systems produce the same metabolites found in in vivo studies, thus they could be used to predict the metabolism of monensin A. Furthermore, we verified that liquid chromatography coupled to mass spectrometry is a powerful tool to study the in vitro metabolism of drugs, because it allows the successful identifications of several derivatives from different metabolic models.
Cytochrome P450, LC–ESI-MS/MS, mass spectrometry, microbial transformation, polyether ionophore
Introduction Drug metabolism studies constitute an important and necessary step when evaluating drug efficacy and safety (Asha & Vidyavathi, 2009; Pupo et al., 2008). Although these studies can rely on the use of in vivo animal systems as models, these models pose a number of issues such as the high costs and time associated with animal breeding as well as the ethical aspects of experimentation on animals. Therefore, conducting such studies in vitro has become an important tool for drug development and metabolite production (Asha & Vidyavathi, 2009; Pekala et al., 2012). Fungi have extensively been used to biotransform organic compounds, because they are considered an economically and ecologically viable technology (Faber, 2011; Pupo et al., 2008). In addition, fungi are a useful tool to structurally modify bioactive compounds and drugs. Using fungi, it is possible to add target groups at certain positions of the molecule of interest, which would be difficult to accomplish Address for correspondence: Anderson Rodrigo Moraes de Oliveira, Departamento de Quı´mica, Faculdade de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, USP, Avenida dos Bandeirantes, 3900, 14040-901, Ribeira˜o Preto, SP, Brazil. Tel: +55-16-3602-0388. Fax: +55-16-36024838. E-mail: [email protected]
History Received 15 August 2013 Revised 5 September 2013 Accepted 13 September 2013 Published online 17 October 2013
via conventional synthesis methods (Asha & Vidyavathi, 2009; Borges et al., 2009a,b; Faber, 2011; Pupo et al., 2008). Moreover, some microorganisms can simulate the mammalian cytochrome P450 (CYP-P450) metabolism of many pharmacologically important molecules such as drugs (Barth et al., 2012; Bocato et al., 2012; Borges et al., 2009b; Carrao et al., 2011; Fortes et al., 2013; Hilario et al., 2012) and natural products (Capel et al., 2011; Liu & Yu, 2010; Parshikov et al., 2012; Rocha et al., 2012; Verza et al., 2009). Among fungi, the Cunninghamella species can regio- and stereoselectively metabolize a wide range of xenobiotics, resembling reactions that occur in mammalian enzymatic systems. This genus has specific properties that make these microorganisms very useful in drug metabolism studies (Asha & Vidyavathi, 2009; Pekala et al., 2012). The Cunninghamella genus has enzymes that are synonymous to those involved in xenobiotic detoxification in mammals (Asha & Vidyavathi, 2009; Sun et al., 2004). Indeed, evidences show that the Cunninghamella genus can predict the fate of the drug in the mammalian organism better than other microorganisms (Asha & Vidyavathi, 2009; Pekala et al., 2012; Sun et al., 2004). Because it is impractical to isolate metabolites from dosed animals, researchers have turned to producing large quantities of the major and minor
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metabolites. This avoids the concerns often associated with chemical synthesis, such as the use of toxic reagents and drastic reaction conditions (Borges et al., 2009a; Faber, 2011; Pupo et al., 2008). An effective in vitro screening strategy to estimate the metabolic fate of drug in humans is to use a human liverderived experimental system (Asha & Vidyavathi, 2010; Li, 2001, 2004). The past few decades have witnessed the development of several in vitro human liver models, with one of the most commonly used systems being liver microsomes (Asha & Vidyavathi, 2010). Liver microsomes models offer many advantages: they are inexpensive, reproducible and easy to prepare (Asha & Vidyavathi, 2010; Raucy & Lasker, 1991; Wu et al., 2012) and they are one of the most fully characterized in vitro systems for drug metabolism research (Asha & Vidyavathi, 2010; Messiano et al., 2013; Moreira et al., 2013). Monensin A (MonA) is representative of a large group of naturally occurring polyether ionophore antibiotics. In veterinary medicine, it is currently the most widely used coccidiostatic and non-hormonal growth-promoting agent (Huczyn´ski et al., 2012; Lowici & Huczyn´ski, 2013). Its properties arise from its ability to form complexes with sodium cations and transport the resulting complexes across cell membranes, thereby disturbing the natural Naþ/Kþ concentration gradient and leading to cell death among Gram-positive bacteria (Huczyn´ski et al., 2008, 2011; Huczyn´ski, 2012). To identify which CYP-P450 isoenzyme participates in the biotransformation of this ionophore, Nebbia et al. (1999, 2001) used rat liver microsomes. They concluded that CYP-P450 3A (the major CYP-P450 enzyme in human liver) plays an important role in the MonA oxidative metabolism, and that compounds able to bind or inhibit this isoenzyme could elicit toxic interactions with MonA (Nebbia et al., 1999, 2001). Therefore, the possibility that humans could ingest food of animal origin containing MonA residues should not be ignored, in fact it should be avoided, to prevent the onset of possible interaction or health problems (e.g. antibiotic resistance) (Nebbia et al., 1999, 2001). MonA presents a narrow safety margin and animals have experienced several accidental poisonings (Harris et al., 1998; Nebbia et al., 2001). These two separate issues have encouraged researchers to design studies to better understand the mechanism of action of MonA and its metabolites. The present study investigated the in vitro metabolism of MonA using a microbial model and human liver microsomes. We identified the metabolites identification by low- and highresolution liquid chromatography–electrospray ionizationtandem mass spectrometry (LC–ESI-MS/MS) and analyzed the LC retention times on the basis of the corresponding reference standards.
Materials and methods Chemicals MonA, 95% purity, was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Solvents such as chloroform (CHCl3) and methanol (MeOH) were high-performance liquid chromatography HPLC grade. All the compounds used in this study were commercially available from Aldrich
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or Sigma and were of analytical grade purity unless otherwise stated. Microorganisms The microorganisms used in this study, Cunninghamella echinulata var. elegans ATCC 8688A and Cunninghamella elegans NRRL 1393 ATCC 10028B were purchased from American Type Culture Collection (ATCCÕ ) (Manassas, VA). Fungal cultures were maintained on potato dextrose agar slants and stored at 4 C. Microbial biotransformation procedure Screening for this experiment was performed in 30 mL of fermentation medium in 125 mL Erlenmeyer flasks, to select efficient conditions to perform the biotransformations of MonA. The procedures were performed as previously described by our group (Barth et al., 2012; Bocato et al., 2012; Carrao et al., 2011; Hilario et al., 2012), with modifications. Five disks of 0.5 cm diameter containing the fungal mycelia were aseptically transferred to 9.0 cm diameter Petri dishes containing potato dextrose agar, the mycelia were allowed to grow for 5 days at 30 C. After that, three disks of 0.5 cm diameter containing the mycelia were transferred using a Transfer tube (Fisher Scientific, Pittsburgh, PA) and inoculated in 50 mL Falcon tubes containing 20 mL of prefermentative medium (10 g of malt extract, 10 g of dextrose, 5 g of triptone, 3 g of yeast extract and deionized water to 1 L). The pre-fermentative medium was used to grow the microorganism for 3 days, with shaking at 100 rpm, at 30 C. After that, the mycelia were transferred to 125 mL Erlenmeyer flasks containing 30 mL of modified Czapek medium (25 g of sucrose, 2 g of NaNO3, 1 g of KH2PO4, 0.5 g of MgSO4 7H2O, 0.5 g of KCl, 0.01 g of FeSO4 7H2O and deionized water to 1 L; the pH was adjusted to 5.0 with 1 mol/L HCl). After 2 days of cultivation, 3.0 mg of MonA was dissolved in 300 mL of N,N-dimethylformamide and added to Czapek medium. The cultures were incubated for 8 days at 30 C, with shaking at 120 rpm. Control flasks containing culture medium with the fungus but without MonA, culture medium with the fungus and N,N-dimethylformamide and without MonA, culture medium with MonA and without the fungus, or only the culture medium were analyzed. A time-course study was carried out as follows: (i) one Erlenmeyer flask was taken every 24 h; (ii) the product was extracted with chloroform and then analyzed by (iii) thin layer chromatography (TLC) and (iv) LC–MS, to check the degree of MonA transformation. The time-course of these biotransformations were followed by LC–MS and the relative ratio between the substrate and its transformed products was determined on the basis of their peak area. Extraction procedure and chromatographic analysis Aliquots of 2 mL of biotransformation medium were centrifuged at 1000 rpm (180 g) for 5 min. Then, 1 mL was transferred to a conical tube, extracted with 3 mL of chloroform, and agitated for 15 min at 1000 rpm by employing the Vibrax VXRÕ (IKA, Staufen, Germany). After that, 2 mL of the organic phase was collected, transferred to the tube, and allowed to evaporate until complete dryness under
In vitro metabolism of monensin A
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DOI: 10.3109/00498254.2013.845707
compressed air. The residues were dissolved in the mobile phase and analyzed by chromatographic systems. The recovery of the MonA extracted from the biotransformation samples was determined using a calibration curve obtained from the data of the analytes not submitted to extraction. By employing this solvent, it was possible to extract 100% of MonA from Czapek liquid culture medium. The TLC analysis was carried out on silica gel [GF254 Merck, Brazil, Category No. 7730; mobile phase: CHCl3/ MEOH (93:7, v/v) plates 0.25 mm thickness], and the spots were visualized after spraying the plates with vanillin–sulfuric acid (1% H2SO4 in ethanol). The LC–ESI-MS analysis was performed in a Varian LC–MS 1200L triple quadrupole apparatus coupled to a mass spectrometer with ESI ionization in the positive mode (Varian Medical Systems Inc., Palo Alto, CA). The chromatographic analysis was accomplished by an injection volume of 10 mL, sample concentration 50 mg/mL in an XterraÕ analytical column MS C-18 (150 2.1 mm, 5 mm) (Waters, Milford, MA) and following gradient (MeOH:H2O): 0.1 min 70% MeOH, 20.0 min 98% MeOH, 21.0 min 30% MeOH, 30.0 min 70% MeOH. The MS conditions were: capillary voltage of 3.2 kV, cone voltage of 40 V, source temperature of 40 C, and N2 desolvation temperature of 350 C. The sample ion was monitored for m/z values ranging from 610 to 800. MonA metabolism was determined by comparing the metabolites retention time and unit mass. Metabolites elucidation in tandem mass spectrometry The fragmentation studies were conducted according to Lopes et al. (2002a,b). The ion spectrum of the products were obtained using the micrOTOF-Q IITM ESI-Qq-TOF Mass Spectrometer (Bruker Daltonics Inc., Billerica, MA) operating in the positive mode, equipped with a hybrid quadrupole and a time-of-flight analyzer. The samples were directly introduced into the spectrometer through an infusion pump (Cole-Palmer, Vernon Hills, IL), at a flow rate of 300 mL/h. The capillary temperature and voltage were maintained at 250 C and 4.0 kV, respectively. The ion was extracted from the ionization source using a cone potential between 5 and 30 kV. Nitrogen was used to spray and dry the sample solution; argon was employed as the collision gas. MS/MS analysis of the precursor ion was extracted into the mass spectrometer for the fragmentation; it was activated by collision with argon (energy 5–80 eV). In vitro assay with human liver microsomes Pooled human liver microsomes (pooled mixed sex, fifty individual donors, protein concentration: 20 mg/mL, stored at 80 C), NADPH-regenerating system solution: solution A (glucose-6-phosphate and NADPþ) and solution B (glucose6-phosphate dehydrogenase) and potassium phosphate buffer 0.5 M pH 7.4 were purchased from BD GentestTM (Woburn, MA) and used throughout this study. The in vitro metabolism using human liver microsomes was investigated by adding 1.4 mmol/L (1.0 mg/mL) MonA to the incubation medium (1.5 mg/mL human liver microsomal protein, potassium phosphate buffer 0.5 mol/L pH 7.4, NADPH-regenerating system). An aliquot of 20 mL of the
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MonA solution was transferred to a 10-mL conical glass tube, to which 18 mL of potassium phosphate buffer 0.5 mol/L pH 7.4, 10 mL of solution A containing NADPþ and glucose6-phosphate, and 2 mL of solution B containing glucose-6phosphate dehydrogenase were added. After 60 min of incubation with 1.5 mg/mL human liver microsomal, at 37 C with agitation, the reaction was terminated by adding chloroform (1.0 mL). This mixture was agitated for 10 min at 1000 rpm in a Vibrax VXRÕ agitator and centrifuged for 5 min at 4000 rpm (2900g). Finally, 750 mL of the organic phase was collected and allowed to evaporate to complete dryness, under compressed air. After that, the residue was reconstituted in the mobile phase and injected into the chromatographic system. Control incubations were performed in the absence of the cofactor solution and in the absence of the microsomal preparation. The difference between ‘‘with’’ and ‘‘without’’ NADPH was considered CYP-P450-mediated metabolism.
Results Fungal biotransformation studies The biotransformation of MonA results showed that both C. echinulata and C. elegans were able to metabolize MonA into polar derivatives, as shown by appearance of spots with lower retention factors that MonA spot in TLC plates (data not shown) and with lower retention time that the MonA peak in the chromatograms of LC–MS experiments (Figure 1) and showed that the MonA was stable in the blank culture medium with the concentration remaining unchanged over the whole 8-day period. The structure MonA and its metabolites are showed in Figure 2. Comparing the MonA ESI-MS spectrum with that of the biotransformed product mixture (data not shown), oxidation of MonA can be detected. The mass spectrum showed that O-demethylated (m/z 679), hydroxylated (m/z 709) and probably O-demethylated-hydroxylated (m/z 695) compounds were the major products of the biotransformation process. All three biotransformed products still acted as ionophores as they were observed to preferentially coordinate to sodium. This suggests that the central structure of monensin remained unaltered after biotransformation (Lopes et al., 2001). Additionally, the intensity of the ion at m/z 679 in the spectrum of the oxidation products increased proportionally in relation to the MonA spectrum, indicating a product originated from O-demethylation, the main reaction observed in the in vivo metabolism of MonA (Nebbia et al., 1999, 2001). From Figure 1 is can be seen that there are more polar peaks that MonA in the chromatogram related to the biotransformation process which were absent from the chromatogram of the control samples. The retention time of MonA was 16.4 min, whereas the metabolites with m/z 679, m/z 709 and m/z 695 appeared at 13.1, 11.8 and 8.9 min, respectively. The metabolite with m/z 679 and retention time 13.7 min was identified as monensin B (MonB), found as a contaminant of MonA (the identity of the peaks was obtained by employing the multiple reaction monitoring (MRM) acquisition mode during the spectrometric analysis, as shown in the case of the human microsome study).
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Figure 1. LC–MS analysis of the biotransformation culture of C. echinulata var. elegans ATCC 8688A and C. elegans NRRL 1393 ATCC 10028B incubated with MonA showing the formation of MonA metabolites: Biot’s Medium ¼ MonA incubated with fungus for 8 days; Control ¼ MonA incubated with only Czapek medium; Blank ¼ Fungus incubated with Czapek without MonA. In this blank, the major peak in the chromatogram has m/z 659.
Figure 2. Structure of MonA and its metabolites found in this study: metabolite 1 (3-O-demethyl MonA, m/z 679); metabolite 2 (12-hydroxy MonA, m/z 709) and metabolite 3 (12-hydroxy-3-O-demethyl MonA, m/z 695).
The time-course for the biotransformation of MonA by C. echinulata and C. elegans was investigated and the ratio of substrate/metabolite (on the basis of the peak area) was obtained by LC–MS (Figure 3). Around 80% of MonA had been transformed after 2 days of incubation. The maximum concentration obtained for the metabolites 1, 2 and 3 after biotransformation by C. echinulata was reached (13.1, 2.7 and 3.2%, respectively) on the sixth day and the maximum concentration obtained for the metabolites 1, 2 and 3 after biotransformation by C. elegans was reached (9.6, 5.8 and 3.2%, respectively) on the eighth day of incubation.
Structural identification of the biotransformation products A previous systematic ESI-MS/MS investigation carried out with MonA demonstrated that it is possible to detect [M þ H]þ only at low pH and high cone voltage and that increasing the energy of the collision-induced dissociation (CID) process enables extensive fragmentation of the sodium salts. The fragmentation of protonated and sodiated MonA produces a substantial number of ions (Lopes et al., 2001). This study revealed that employing the protonated precursor
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Figure 3. Time-course of the microbial transformation of MonA by C. echinulata and C. elegans: (diamond) MonA; (square) metabolite 1, m/z 679; (triangle) metabolite 2, m/z 709 and (cross) metabolite 3, m/z 695. Table 1. Formula, observed mass, calculated mass, mass error (parts per million) and identity in high-resolution accuratemass spectrum of MonA and its metabolites. Formula C36H62NaO11 C35H60NaO11 C35H60NaO11 C36H62NaO12 C35H60NaO12
Observed mass
Calculated mass
Error
Identity
693.4200 679.4026 679.4008 709.4134 695.3977
693.4184 679.4028 679.4028 709.4139 695.3977
2.3 2.6 þ2.9 0.1 0.0
MonA MonB 3-O-Demethyl MonA 12-Hydroxy MonA 12-Hydroxy-3-O-demethyl MonA
ion in ESI-MS/MS studies posed limitations, and that it is important to understand the fragmentation pathways of sodiated salts. Key ions result from Grob–Wharton type fragmentation or pericyclic rearrangements as well as simple neutral losses (Lopes et al., 2002a,b). The same fragmentation mechanisms occurred for the metabolites produced in the present in vitro metabolism studies. The low-resolution product ion spectrum of metabolites 1, 2 and 3 (Supplementary Figure S1) displayed structural fragments that possibly stemmed from the Grob–Wharton type fragmentation mechanism (Grob, 1969; Grob & Baumann, 1955; Wharton & Hiegel, 1965) and/or H2O elimination, as previously proposed (Lopes et al., 2002a,b) for MonA. High-resolution accurate-mass MS was used to determine the molecular formula of these ions. Table 1 contains the accurate masses and theoretical formulae with errors for these fragments and Table 2 shows the fragment identity for all the ions observed in the product ion spectrum of MonA and its metabolites. The proposed fragmentation for metabolites 1, 2 and 3 are shown in Figures 4–7. In this proposal, the acid function undergoes protonation, because it is the most available position for this event, in agreement with all the data published on the 3D structure of MonA in the solid state (Duax et al., 1980; Martinek et al., 2000; Paz et al., 2003). Figure 4 shows elimination of 172 Da (for metabolites 1 and 2) and of 186 Da (for metabolite 3) via the Grob–Wharton type mechanism. This elimination involved ring A (O-4), to yield fragment ‘‘B’’ (m/z 507, 523 and 523 for metabolites 1, 2 and 3, respectively). Fragment ‘‘B’’ lost CO and H2O, which resulted in ion ‘‘C’’ (m/z 479, 495 and 495 for metabolites 1, 2 and 3, respectively) and ion ‘‘D’’ (m/z 461,
Table 2. Fragment identity for all the ions observed in the product ion spectrum of MonA and its metabolites.
Ion
MonA
MonB
3-O-Demethyl MonA
A B C D E F G H I J L M N O P
693 507 479 461 443 – 343 303 675 657 – 675 501 483 383
679 493 465 447 429 – 329 289 661 643 – 661 487 469 369
679 507 479 461 443 – 343 303 661 643 – 661 501 483 383
12-Hydroxy MonA
12-Hydroxy-3O-demethyl MonA
709 523 495 477 459 441 343 303 691 673 655 691 517 499 441
695 523 495 477 459 441 343 303 677 659 641 677 517 499 441
477 and 477 for metabolites 1, 2 and 3, respectively). Ion ‘‘D’’ then lost H2O, to give fragment ‘‘E’’ (m/z 443, 459 and 459 for metabolites 1, 2 and 3, respectively). The ions with m/z 343 (‘‘G’’) and m/z 303 (‘‘H’’) (Figure 5) originated from product ion ‘‘E’’ via two possible mechanistic pathways. The first would involve loss of H2O from the tetrahydrofuran ring, generating the product ion ‘‘F’’ (m/z 441), which in turn might give the product ion ‘‘H’’ (m/z 303) by a Grob–Wharton-type fragmentation resulting in loss of 82 Da (for metabolite 1) and 98 Da (for metabolites 2 and 3), followed by a loss of 40 Da (elimination of 1,4-propyne in a charge remote mechanism). In the second step of this
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Figure 4. The proposed fragmentation route showing the formation of product ion ‘‘E’’ for metabolites 1, 2 and 3, as previously discussed for metabolite 2 by Sousa-Junior et al. (2013).
Figure 5. The proposed fragmentation route showing the formation of product ion m/z 303 for metabolites 1, 2 and 3.
pathway, the product ion with m/z 303 would be originated from loss of 140 Da (for metabolite 1) and loss of 156 Da (for metabolites 2 and 3). Both ions derived from the product ion ‘‘E’’ would stem from a pericyclic rearrangement analogous to the one observed for the dihydropyran fragment with elimination of propyne. This pathway, together with MonA fragmentation studies in the gas phase; led to the proposal that hydroxylation had occurred at the OH position in the MonA structure (for both metabolites 2 and 3). All these fragmentation pathways fully agree with the data previously published for MonA and MonB (Lopes et al., 2002a,b). The second pathway proposed for the metabolites fragmentation would be loss of water involving the oxygen atom
O-11 (Figure 6), to produce the product ion ‘‘I’’. Another possibility would be a neutral loss of water involving the oxygen atoms O-10 and O-11 with formation of the fragment ‘‘L’’ (with m/z 655 and 641 for metabolites 2 and 3, respectively). Migration of a hydrogen in the b-position would favor this third water elimination involving the O-12 atom, culminating in the conjugated -system that prevents elimination of 116 Da by pericyclic rearrangement. The relationship of the hydroxyl group O-5 and the ether oxygen (O-4) in ring ‘‘A’’ may lead to neutral loss of water, producing the intermediate ion ‘‘M’’ and giving rise to a third possible route for ion ‘‘A’’ fragmentation (Figure 7). This ion could fragment, to give the product ion ‘‘N’’ m/z 501 (for
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Figure 6. The proposed fragmentation route showing the formation of product ion ‘‘L’’ for metabolites 2 and 3.
Figure 7. Proposed fragmentation mechanism involving the dihydropyran system of MonA and subsequent formation of fragment ion ‘‘O’’’ 441. This production is key to location of hydroxylation in metabolites 2 and 3.
metabolite 1) and m/z 517 (for metabolites 2 and 3) by the loss of 160, 174 or 160 Da, respectively. The elimination of an H2O molecule involving the dihydropyran system is shown in Figure 7 suggesting the formation of the product ion ‘‘O’’. Consequently, product ion ‘‘O’’ could fragment by migration of a hydrogen in position a to the carbonyl group, producing the ion ‘‘O0 ’’ (m/z 441) by loss of 58 Da from the hydroxyl derivative (Figure 7). For metabolite 1, ‘‘O’’ could undergo loss of 100 Da, resulting in the ion ‘‘P’’, the same ion observed in MonA fragmentation. The existence of the product ion with m/z 441 as a fragment of OH-MonA might represent a key product ion to justify a hydroxylation position in the MonA molecule. The structural identification of these products shown that metabolite 1 is 3-O-demethyl MonA, metabolite 2 is 12-hydroxy MonA and metabolite 3 is 12-hydroxy-3-O-demethyl MonA (Figure 2). Human liver microsome study To establish correlations between the microbial model employed in this study and the MonA human drug metabolism, an in vitro metabolism assay employing human liver microsomes was also conducted. It is usual for this in vitro process to yield low amounts of the metabolites. Therefore, the LC–ESI-MS/MS MRM mode of analysis was employed,
to monitor the following transitions: m/z 693 ! 461 for MonA, m/z 679 ! 465 for MonB, m/z 679 ! 461 for 3-Odemethyl MonA, m/z 709 ! 477 for 12-hydroxy MonA and m/z 695 ! 477 for 12-hydroxy-3-O-demethyl MonA. The microsomal metabolism furnished metabolites 3-O-demethyl MonA and 12-hydroxy MonA, but not metabolite 12hydroxy-3-O-demethyl MonA. The metabolites showed the same retention times as those observed in the microbial transformation, as well as the same ion product spectrum (Figure S2). In addition, the total yield of the MonA metabolism was estimated to be 20% by measuring the rate of substrate disappearance by LC–ESI-MS. Metabolites structure confirmation We confirmed the structure of metabolite 1, 3-O-demethyl MonA, according to Pospı´sˇil et al. (Pospı´sˇil et al., 1987; Pospı´sˇil & Zima, 1987). In this article, 3-O-demethyl MonA was isolated from Streptomyces cinnamonensis cultures in the presence of a methylation inhibitor (Pospı´sˇil et al., 1987; Pospı´sˇil & Zima, 1987), which served a standard. They verified its chemical structure by MS/MS and 1H NMR analyses. The chromatographic behavior (retention time) and MS spectra of this standard was compared with those of the metabolite in this study. LC–ESI-MS/MS analysis revealed
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that both substances presented identical product ion spectra and retention times (Supplementary Figure S3). 12-Hydroxy MonA was obtained by performing bioinorganic catalysis with metalloporphyrins (Sousa-Junior et al., 2013). In this study, the formation of 12-hydroxy MonA was demonstrated with full characterization by high-resolution accurate-mass MS/MS studies performed in a Fouriertransform ion cyclotron resonance mass spectrometer (Sousa-Junior et al., 2013). Once again, we compared the chromatographic behavior (retention time) and mass spectrum of this reported product with those of the metabolite obtained here. Both substances displayed the same ESI-MS/MS spectra and retention time during LC–ESI-MS/MS analysis (Supplementary Figure S3).
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Discussion Various metabolism and biotransformation studies on MonA have been performed by various laboratories and these results have shown that this drug undergoes extensive metabolization to a great number of products that are qualitatively similar among species but not quantitatively (Davison, 1984; Donoho et al., 1978; Kiel et al., 1998; Nebbia et al., 1999). Some metabolites have been identified; that seem to result from an O-demethylation and/or hydroxylation at various positions along the carbon backbone of the ionophore molecule (Davison, 1984; Donoho et al., 1978; Kiel et al., 1998; Nebbia et al., 1999). Numerous fungi (and other microorganisms) have hydrolytic and reductive ability to metabolize foreign organic substances as mammalian systems do; therefore, they are applicable as microbial models as proxies to study mammalian metabolism. Among fungi, the genus Cunninghamella can regio- and stereo-selectively metabolize a wide variety of xenobiotics. These reactions resemble those taking place in mammalian systems (Asha & Vidyavathi, 2009; Pupo et al., 2008). Vaufrey et al. (1990) reported one of the first studies involving the microbial transformation of MonA using the soil bacterium Sebekia benihana NRRL 11111. This microorganism efficiently converted MonA to three major compounds that do not occur in mammalian species. This study showed that the three metabolites contained an open terminal ring, and that additional oxidation took place for two metabolites (Vaufrey et al., 1990). Despite the report of Vaufrey et al. (1990) on the biotransformation of MonA by a microorganism, this study is the first to describe that a microorganism produces the same metabolites as those found in mammalian species, which makes the fungal microbial model very attractive to obtain in vivo metabolites of this ionophore. The aim of this present study was to identify a microorganism that can metabolize MonA and to explore the similarities between this microbial model and the reported mammalian systems in terms of MonA metabolism. On the basis of the success previously observed for the Cunninghamella genus, we evaluated the potential of two species of the genus Cunninghamella (C. echinulata and C. elegans) to oxidize MonA. During the MonA biotransformation study, we observed the production of three major compounds. We identified these compounds by comparing
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their ESI-MS/MS with that of MonA: 3-O-demethyl MonA, which originated from O-demethylation (m/z 679), 12-hydroxy MonA, which resulted from hydroxylation (m/z 709) and 12-hydroxy-3-O-demethyl MonA which stemmed from O-demethylation and a hydroxylation (m/z 695), had fragmentation patterns similar to that of the substrate, as described in the ‘‘Results and discussion’’ section. These three metabolites had been previously detected in studies involving MonA metabolism (Davison, 1984; Donoho et al., 1978; Kiel et al., 1998). MonA is safe and effective when used at the recommended dosages. The therapeutic index of this drug is rather narrow and several accidental poisononings have been described in the literature not only involving animals (Henri et al., 2008; Nebbia et al., 1999, 2001) but also man (Caldeira et al., 2001; Kouyoumdjian et al., 2001; Souza et al., 2005). Recent reports of significant levels of MonA residues in broiler meat, chicken meat and eggs (Rose´n, 2001; Moloney et al., 2012; Rokka & Peltonen, 2006) have leading to possible/eventual interaction with other drugs or possible human health problems such as antibiotic resistance and intoxications. Moreover, recent studies involving their biological properties for cancer therapy have called for studies involving drug metabolism in humans (Lowici & Huczyn´ski, 2013; Huczyn´ski, 2012). Therefore, the metabolism of MonA using a human liver microsome model was also investigated because it represents an effective strategy to estimate drug metabolic fates in humans (Asha & Vidyavathi, 2010; Li, 2001, 2004). Additionally, these microsomes constitute one of the best characterized in vitro systems for drug metabolism research (Asha & Vidyavathi, 2010). Our preliminary results showed the formation of two metabolites, 3-O-demethyl MonA and 12-hydroxy MonA; hence, one can use this model to predict human metabolism of MonA. In addition, studies involving the biological activities of these metabolites have shown that they are less active than MonA; thus, MonA biotransformation corresponds to a classic detoxification pathway: the resulting molecules are more polar, facilitating their elimination into the culture medium (Pospı´sˇil et al., 1987; Pospı´sˇil & Zima, 1987; Vaufrey et al., 1990).
Conclusions In conclusion, our results have demonstrated the ability of the C. echinulata and C. elegans to mimic the action of CYPP450 in MonA metabolism. This species produces the same metabolites found in vivo studies. The ability of these models to mimic mammalian metabolism, to perform biotransformation reactions, and to produce significant amounts of drug metabolites suggest that this microbial system represents a suitable alternative for drug biotransformation studies. This system can also complement in vivo metabolism studies, thereby dismissing the need to use large quantities of animals in experimental research. The MonA metabolites were elucidated by ESI-MS/MS; the fragmentation studies showed that LC–ESI-MS/MS is a powerful tool to study drug metabolism in vitro, because it can identify metabolites in different models.
DOI: 10.3109/00498254.2013.845707
Declaration of interest This research was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP Process 2011/ 05800-0, 2011/17508-1 and 2009/51812-0), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) for financial support and for granting research fellowships. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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