Use of Electrochemical Oxidation and Model Peptides To Study

Article pubs.acs.org/crt

Use of Electrochemical Oxidation and Model Peptides To Study Nucleophilic Biological Targets of Reactive Metabolites: The Case of Rimonabant Annika Thorsell,* Emre M. Isin, and Ulrik Jurva DMPK Design and Biotransformation, CVMD iMed DMPK, AstraZeneca R&D Mölndal, Sweden, Pepparedsleden 1, SE-431 83, Mölndal, Sweden S Supporting Information *

ABSTRACT: Electrochemical oxidation of drug molecules is a useful tool to generate several different types of metabolites. In the present study we developed a model system involving electrochemical oxidation followed by characterization of the oxidation products and their propensity to modify peptides. The CB1 antagonist rimonabant was chosen as the model drug. Rimonabant has previously been shown to give high covalent binding to proteins in human liver microsomes and hepatocytes and the iminium ion and/or the corresponding aminoaldehyde formed via P450 mediated α-carbon oxidation of rimonabant was proposed to be a likely contributor. This proposal was based on the observation that levels of covalent binding were significantly reduced when iminium species were trapped as cyanide adducts but also following addition of methoxylamine expected to trap aldehydes. Incubation of electrochemically oxidized rimonabant with peptides resulted in peptide adducts to the N-terminal amine with a mass increment of 64 Da. The adducts were shown to contain an addition of C5H4 originating from the aminopiperidine moiety of rimonabant. Formation of the peptide adducts required further oxidation of the iminium ion to shortlived intermediates, such as dihydropyridinium species. In addition, the metabolites and peptide adducts generated in human liver microsomes were compared with those generated by electrochemistry. Interestingly, the same peptide modification was found when rimonabant was coincubated with one of the model peptides in microsomes. This clearly indicated that reactive metabolite(s) of rimonabant identical to electrochemically generated species are also present in the microsomal incubations. In summary, electrochemical oxidation combined with peptide trapping of reactive metabolites identified a previously unobserved bioactivation pathway of rimonabant that was not captured by traditional trapping agents and that may contribute to the in vitro covalent binding.

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INTRODUCTION The covalent binding (CVB) of drug molecules to biological macromolecules was reported more than 60 years ago,1 and the involvement of reactive metabolites derived from drug molecules covalently binding to proteins in adverse drug reactions has been extensively studied in recent years.2−5 Although a correlation between levels of CVB body burden and drugs causing adverse drug reactions has been shown, the biological targets of reactive metabolites and their role in idiosyncratic adverse drug reactions are not well understood.5 Comprehensive characterization of protein targets of reactive drug metabolites in complex biological samples, such as tissue and cells, is an analytical challenge. Several researchers have successfully identified the adducted amino acids but a complete understanding of pathways leading to CVB has been complicated.6−10 In recent years, different less complex model systems have been employed in proteomic studies to overcome some of the predicaments of a global proteomic analysis. They © 2014 American Chemical Society

all have in common that the amino acid sequence of the target peptides or proteins were known, facilitating characterization of the adduction products and identification of the modification sites. These model systems include reconstituted cytochrome P450s (P450)11−13 and incubation of reactive species with peptides or single proteins.8,14−19 The majority of the efforts have been focused on the interaction of soft electrophiles such as epoxides, quinones, quinoneiminies etc. with proteins,9−12,14,15,17,18 but the reactivity of hard electrophiles like iminium ions and aldehydes with proteins has been less extensively studied.6,7,16,20 Electrochemistry (EC) is a useful tool to generate several different types of metabolites.21−28 Examples of reactive drug metabolites that have been generated by EC to study interactions with biological macromolecules include quinones, Received: June 30, 2014 Published: September 11, 2014 1808

dx.doi.org/10.1021/tx500255r | Chem. Res. Toxicol. 2014, 27, 1808−1820

Chemical Research in Toxicology

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2.67 kBq/nmol, 31, 23 and 46% for none, one and two [14C]-labels) were provided by AstraZeneca R&D (Mölndal, Sweden). Human liver microsomes (20 mg protein/mL, pooled, 10 donors, male and female) were obtained from BD Gentest (Woburn, MA). 1,5-pentanedial, angiotensin II, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, reduced glutathione (GSH), leucine-enkephalinamide, methoxylamine hydrochloride and NADP were purchased from Sigma-Aldrich (St. Louis, MO). Potassium cyanide (KCN) was obtained from Acros Organics (Geel, Belgium) and bovine serum albumin digestion standard from Waters (Milford, MA). All organic solvents were obtained from commercial sources. Electrochemical Oxidation of Rimonabant and Trapping Studies. The EC/ESI/MS system was set up as previously reported.22 Briefly, samples were infused through an ESA Coulochem 5011 analytical cell (ESA Inc., Bedford, MA) via a syringe pump at a flow rate of 5 μL/min. A makeup flow, consisting of 50% acetonitrile and 0.05% aqueous formic acid, was added before the EC cell at a flow rate of 50 μL/min. The EC cell was controlled by an ESA Coulochem II potentiostat (ESA Inc.). The ESA working electrode was porous graphite, and all the reported cell potentials were measured versus a palladium reference electrode. The outlet from the ESA cell was connected to a Sciex API 4000 QTrap mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a TurboIonSpray interface. For online EC/ESI/MS studies, the rimonabant concentration in the syringe was 5 μM. The EC oxidation potential was varied between 0 and 1000 mV and full-scan mass spectra were acquired continuously. In the subsequent trapping experiments the rimonabant concentration in the syringe was 100 μM. Methoxylamine, GSH, and KCN were dissolved in 1 M potassium phosphate buffer (pH 7.4) to a concentration of 11 mM and 10 μL aliquots were transferred into a 96well plate. Rimonabant was oxidized at various potentials (0, 300, 500, 700, 900 mV) and 100 μL of eluent from the ESA cell was collected into the wells containing the trapping agents or phosphate buffer. The final concentrations of oxidized rimonabant sample and trapping agents were 9.1 μM and 1 mM, respectively in 50% acetonitrile in potassium phosphate buffer (pH 7.4, 38 mM). There was an approximate 100-fold molar excess of trapping agent to oxidized rimonabant. The samples collected into wells only containing buffer were used as controls and for profiling of stable EC oxidation products. Incubations with trapping agents or buffer only were performed for 4 h at 37 °C with shaking (500 rpm) followed by dilution with 100 μL of water and analyzed by LC/MS. Rimonabant oxidized at 900 mV was also analyzed prior to the incubation. Samples were separated by a liquid chromatography system (Waters ACQUITY UPLC) on a Waters ACQUITY UPLC BEH (C18, 1.7 μm, 2.1 × 100 mm) column, at a flow rate of 500 μL/min with the column temperature set to 45 °C. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B was 100% acetonitrile. At the start of the gradient, the acetonitrile content was 5% and was then linearly increased to 15% over a period of 4 min and to 90% over 11 min. This condition was held for 1 min, and finally the initial mobile phase composition was restored over 0.01 min. The UPLC system was coupled to a Waters Synapt G2S Q-TOF instrument equipped with an ESI source. Leucine-enkephalin was used as the lock mass (m/z 556.2771) for accurate mass calibration and was introduced using the Lock Spray interface. All samples were diluted with water 1:1, and aliquots of 10 μL were injected onto the LC/MS system. Spectra were acquired with MSE in the positive ionization mode at a capillary potential of 0.5 kV. The cone voltage was held constant at 35 V for all experiments. The mass range was m/z 50−1200. The collision energy was set to 4 V in low energy scans and a 20−45 V ramp in high energy scans. For acquisition of MS/MS spectra the same collision energy ramp (20−45 V) was used. The data were evaluated manually using MassLynx v.4.1 (Waters). Human Liver Microsomal Incubations. Human liver microsomes (1 mg protein/mL) were incubated with 10 μM rimonabant or coincubated with 20 μM rimonabant and 20 μM leucineenkephalinamide. The incubations (250 μL) were conducted in 100 mM potassium phosphate buffer (pH 7.4) containing 3.3 mM MgCl2

quinoneimines, quinonemethides, imine methides, iminium ions, dihydropyridinium species and aldehydes.15,19,21,26,29−31 EC oxidation of drugs facilitates the generation of relatively large amounts of reactive metabolites in the absence of biological matrices. An advantage of this purely instrumental system is that highly reactive and labile metabolites can be detected that otherwise would rapidly bind covalently to proteins. Additionally, the approach can be used to study the propensity of a reactive drug metabolite to form covalent adducts with selected peptides and proteins and to identify specific binding sites. Rimonabant is an N-acylaminopiperidinyl derivative with cannabinoid type 1 receptor (CB1r) antagonist properties that has been reported to be effective in appetite control and smoking cessation therapy.32,33 Rimonabant was approved in 2006 for the treatment of obesity but was withdrawn in 2008 due to serious drug-related psychiatric disorders, including anxiety and depression.34,35 The structure of rimonabant is given in Figure 1. Biotransformation studies of rimonabant in

Figure 1. Structure of rimonabant.

vitro in rat and human liver microsomes (HLM) have revealed high levels of reactive metabolite formation and NADPH dependent CVB to microsomal proteins.36 Similarly high levels of CVB were also observed in incubations with human hepatocyes3 as well as SV40-T antigen-immortalized human liver epithelial derived (THLE) cells selectively expressing P450 3A4.37 Furthermore, rimonabant has been shown previously to exhibit toxicity in the THLE-3A4 cells37 possibly due to CVB of rimonabant. Rimonabant also exhibit time dependent inhibition of recombinant P450 3A4.36 The electrophilic iminium ion and/or the corresponding aminoaldehyde formed via P450 mediated α-carbon oxidation of rimonabant were proposed to be likely contributors to CVB. Levels of CVB were significantly reduced when the iminium species was trapped as a cyanide adduct and were also reduced following the addition of methoxylamine. Although methoxylamine can be expected to trap aldehydes, putative methoxyiminyl adduct was not detected under the experimental conditions. To what extent the iminium species or aminoaldehyde contribute to the CVB is unknown. However, it is crucial to understand the pathways leading to covalent modification of proteins so that the necessary drug design efforts can be focused on modification of structural moieties ultimately leading to covalent modification of proteins.38 The aims of the present study were to characterize the EC oxidation products of rimonabant and the mechanism by which they covalently bind to model peptides. To assess the biological relevance of this approach, the EC oxidation products and peptide adducts were compared with those generated in HLM incubations.

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EXPERIMENTAL SECTION

Chemicals and Reagents. Rimonabant (purity of 99%) and [14C]-rimonabant (radiochemical purity of >99%, specific activity of 1809



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with an NADPH generating system (1.3 mM NADP, 3.3 mM glucose 6-phosphate, and 0.4 U/mL of glucose 6-phosphate dehydrogenase) for 4 h at 37 °C with shaking (500 rpm). The reactions were initiated by the addition of rimonabant after a 5 min preincubation period at 37 °C. The incubations were carried out in triplicates and aliquots were taken after 2 and 4 h and protein precipitated with ice-cold methanol in a [1:2] ratio. The samples were stored at −20 °C for 20 min and were then centrifuged (4000g, 4 °C) for 20 min followed by 1:1 dilution of supernatant with water. Incubations in the absence of NADP were used as negative controls in the metabolite profiling study, while in the peptide trapping experiments aliquots taken at the start of the incubation (0 min) served as controls. Samples for metabolite profiling were analyzed on the UPLC system coupled to a Waters Synapt G2S Q-TOF instrument as described above with the following exceptions. The column was a Waters ACQUITY UPLC HSS (T3, 1.8 μm, 2.1 × 100 mm). At the start of the gradient, the acetonitrile content was 1% for the first 5 min and then linearly increased to 10% over a period of 4 min and to 95% over 20 min. This condition was held for 1 min, and finally the initial mobile phase composition was restored over 0.01 min. In the peptide trapping experiments the UPLC system was coupled to a Waters Xevo TQ-S instrument and separations were performed on a Waters ACQUITY UPLC BEH (C18, 1.7 μm, 2.1 × 50 mm) column, at a flow rate of 500 μL/min (the column temperature was set at 40 °C). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B was 100% acetonitrile. At the start of the gradient, the acetonitrile content was 5% and linearly increased to 95% over a period of 6 min and held for 0.6 min, and finally the initial mobile phase composition was restored within 0.01 min. All of the samples were diluted with water 1:1, and aliquots of 3 μL were injected onto the LC/MS system. Capillary potential was set to 3.0 kV and the cone voltage to 39 V. The multiple-reaction monitoring (MRM) transitions were as follows: 619.32/200.10, 619.32/342.14, 619.32/489.21 and 619.32/602.30. The collision energies were set to 50, 30, 20, and 20 V for the different transitions. All the selected product ions were ions containing the N-terminal modification. Incubation of Peptides from a Tryptic Bovine Serum Albumin Digest with EC Oxidized Rimonabant. The EC oxidation potential was adjusted to maximize the generation of the iminium ion and with lower amounts of other oxidation products. Rimonabant (100 μM) was infused through the EC cell at 500 mV and collected in glass vials on ice. BSA digestion standard (40 μM) was mixed with oxidized rimonabant to final concentrations of 9.1 μM and 18.1 μM in 50% acetonitrile in phosphate buffer (pH 7.4, 38 mM). Theoretically there was an approximate 2-fold molar excess of each peptide in the digest to oxidized rimonabant. Oxidized rimonabant incubated with phosphate buffer or the peptide digest incubated with oxidized makeup flow served as control incubations. Incubations were performed for 4 h at 37 °C with shaking (500 rpm). The UPLC system was coupled to a Waters Xevo G2S Q-TOF instrument equipped with an ESI source, and samples were separated on a Waters ACQUITY UPLC BEH300 (C18, 1.7 μm, 2.1 × 100 mm) column, at a flow rate of 200 μL/min (the column temperature was set at 40 °C). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B was 100% acetonitrile. At the start of the gradient, the acetonitrile content was 3% for 3 min and then linearly increased to 20% over a period of 60 min and to 40% within 15 min. The acetonitrile content was then increased to 95% within 5 min. This condition was held for 1 min, and finally the initial mobile phase composition was restored within 0.01 min. All samples were diluted with water 1:1, and aliquots of 5 μL were injected onto the LC/MS system. Leucine-enkephalin was used as the lock mass (m/z 556.2771) for accurate mass calibration and was introduced using the Lock Spray interface. Spectra were acquired in MSE mode, where the collision energy was set to 4 V in low energy scans and a 25−65 V ramp in high energy scans. The capillary potential was 3.0 kV, and the cone voltage was held constant at 40 V for all experiments. The mass range was m/z 50−2000. For acquisition of MS/MS spectra, the same collision energy ramp (25−65 V) was used.

Data were processed with BioPharmaLynx v.1.3.3 (Waters), a software program for analysis of peptide mass maps from known protein sequences. Default peptide mass map analysis criteria of 30 and 50 ppm mass error in low and high collision energy mode were specified. Trypsin was specified as the digestion enzyme with one missed cleavage allowed. The bovine serum albumin sequence was P02769 (www.uniprot.org/uniprot/P02769), where the signal and propeptides were removed. Modifications, such as the iminium ion, were entered into the search, but none of these rimonabant modifications could be found by the software. Instead, identification of unpredicted modifications included (1) alignment and comparison of peptide mass maps from analyte and control samples, (2) search for modified peptide where the observed m/z and chromatographic retention time had to be unique to analyte samples and not present in controls, and, from a doubly charged ion, (3) confirmation that the modified peptides were unique for the analyte samples by extracting their m/z from raw data using MassLynx, and (4) MS/MS analysis and manually sequencing using BioLynx v.4.1 (Waters) to identify the tryptic peptides, the site, and molecular mass of adduction. The finding of an N-terminal additional mass of 64 Da allowed us to enter this modification into BioPharmaLynx and process the data again. Identification of other modified peptides proceeded as above but also included the following steps: (1) the mass error of the observed modified peptides was required to be less than 25 ppm; (2) MS/MS data containing bn- and yn-type ions consisting of the assigned sequence and modification were acquired. Incubation of Leucine-Enkephalinamide and Angiotensin II with EC Oxidized Rimonabant. Leucine-enkephalinamide and angiotensin II were diluted from a 1 mM stock solution in 1 M potassium phosphate buffer (pH 7.4) to a concentration of 200 μM and incubated individually with oxidized rimonabant as described above for the trapping agents. As control samples, the peptides were also incubated with eluent from the ESA-cell in the absence of rimonabant as well as oxidized rimonabant incubated with buffer. Final concentrations were 18 μM and 9.1 μM for peptides and oxidized rimonabant in 50% acetonitrile in phosphate buffer (pH 7.4, 38 mM). There was an approximate 2-fold molar excess of peptide to oxidized rimonabant. Incubations containing the model peptide and rimonabant as well as controls were diluted with 100 μL of water. Samples were analyzed and data evaluated as described above for the profiling of the EC oxidation products. Incubation of Leucine-Enkephalinamide and Angiotensin II with Pentanedial. Leucine-enkephalinamide and angiotensin II were diluted in 1 M potassium phosphate buffer (pH 7.4) to a concentration of 200 μM. Pentanedial was diluted to a concentration of 2 μM in 50% acetonitrile in water. For the incubations, 10 μL peptide aliquots were mixed with 6, 20, 50, or 100 μL of pentanedial solution and 50% acetonitrile to a final volume of 110 μL. Final concentrations were 18 μM for peptides and 0.1, 0.4, 0.9, and 1.8 μM for pentanedial in 50% acetonitrile and potassium phosphate buffer (pH 7.4, 38 mM). Control samples consisted of peptides or pentanedial incubated in 50% acetonitrile and potassium phosphate buffer (pH 7.4, 38 mM). Samples were diluted with 100 μL of water. Samples were analyzed and data evaluated as described above.

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RESULTS Comparison of EC Oxidation with P450 Mediated Oxidation of Rimonabant. Electrochemistry has proven useful to generate several different types of metabolites.21,24−26 In order to evaluate whether an EC model system could be applied to study the P450 mediated metabolism of rimonabant, the oxidation products generated by EC oxidation were compared with those produced during HLM incubations. Extracted ion chromatograms of rimonabant and its metabolites and EC oxidation products are presented in Figure 2, with the corresponding structures given in Scheme 1. The metabolites are denoted as in an earlier paper by Andresen-Bergstrom et al.36 In our study, the metabolites M2−M4 and M6−M9 were 1810



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Figure 2. Extracted ion chromatograms of (A) rimonabant and its oxidations products generated electrochemically at an oxidation potential of 900 mV and (B) rimonabant and its metabolites in HLM after 2 h incubations. For clarity of presentation, the extracted ion chromatograms for M2−M4, M6, M8−M9, and M11 are multiplied by 13 (A) and 2.5 (B). The tentative dimer M13 is not included in the chromatograms.

M9 was not detected online, it was observed post-EC as a major oxidation product in all oxidized rimonabant samples, both prior to incubation (900 mV) and after incubation (0− 900 mV). The highest level of M9 was observed at the highest oxidation potential, and since M9 is formed via hydrolysis of the amide moiety, we conclude that one or several of the EC oxidation products are more prone to amide hydrolysis than rimonabant and M7. The minor metabolites M6 (+16 Da) and M12 (+32 Da), originating from hydroxylation(s), were not detected as EC oxidation products. This is in line with previous findings that EC, under the conditions used here, is not able to mimic aliphatic hydroxylations.21 Three minor metabolites, M2, M4, and M8, with a net mass change of +14 Da, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms, were detected in the HLM incubations. M2 was not detected as an EC oxidation product, but the two EC oxidation products corresponding to M4 and M8 were observed. There was a distinct increase in the levels of M8 with higher potential whereas M4 was present in lower levels (Figure 3C). The minor metabolite M13, also formed as an EC oxidation product, eluted late in the chromatogram with a pseudomolecular ion at m/z 921 Da. We hypothesize that M13 is a dimer that could potentially be formed via a reaction between one molecule of M7 and one molecule of M7′. Collision induced dissociation of M13 generated a fragment ion at m/z 461 Da corresponding to M7 and/or M7′. The analysis also revealed an additional EC oxidation product denoted M11

detected and three previously unobserved metabolites and oxidation products of rimonabant were identified, denoted M10−M13. The proposed structures were confirmed by MS/ MS and by comparison with previous work.36 The formation of rimonabant EC oxidation products was monitored online with EC/ESI/MS at various oxidation potentials (Figure 3A). This online setup facilitates the detection of oxidation products that are too labile to survive the LC/MS analysis conditions. However, coeluting oxidation products with overlapping isotope clusters cannot be distinguished from each other. At an oxidation potential of 500 mV, rimonabant was oxidized to the iminium ion M7 (−2 Da) with low levels of other oxidation products. The increased formation of M10 (−4 Da) correlated with increasing potential. The formation of the oxidation product(s) corresponding to +14 Da (M2, M4, and/or M8) was also enhanced when the potential was further increased, while the levels of M7 were reduced. Thus, while M7 was further oxidized as a result of the higher potentials, it still constituted the major EC oxidation product. Comparison of stable products/metabolites from EC and HLM incubations by LC/MS analysis revealed that M7 was the major EC oxidation product as well as the major metabolite formed in HLM incubations in the presence of NADPH (Figure 2). M7 was formed at all oxidation potentials, with the highest levels formed at 500 mV (Figure 3B). In HLM two other major metabolites were M3 (+16 Da) and M9. Although 1811



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Scheme 1. Proposed Rimonabant Metabolites in HLM and EC Oxidation Products

a c

Presence in HLM previously confirmed by Andresen−Bergstrom et al.36 bPresence confirmed by trapping with cyanide resulting in CA2 (Figure 4). Dimer of M7/M7′.

(−68 Da) corresponding to a previously unobserved hydrazide metabolite that was also formed in the HLM incubations. M11 was not detected online by EC/ESI/MS but was formed postEC in all incubations in a time dependent manner and is therefore hypothesized to originate from a less stable intermediate (Figure 3C). The EC oxidation product M10 was only detected online and could not be detected following LC/MS, suggesting that it is a short-lived intermediate. When trapping agents were added to the EC oxidized samples, no adducts were detected in the incubations containing GSH or methoxylamine, while two cyanide adducts were detected (Figure 4). GSH is known to be an efficient scavenger of reactive metabolites that are soft electrophiles, whereas KCN and methoxylamine are more selective and used to trap the hard electrophiles iminium ions and aldehydes, respectively.39,40 The major cyanide adduct corresponded to CA2, while the minor adduct corresponded to CA3. The M7 oxidation product was almost completely depleted in the incubations containing cyanide, and the level of M4 was significantly reduced. The cyanide adduct CA3 corresponds to trapping of the short-lived intermediate M10. Identification of Adducted Peptides in the Tryptic BSA Digest. EC oxidation of rimonabant was used to generate reactive oxidation products in the absence of biological matrices and was followed by incubation with biologically relevant targets. The oxidation potential was optimized for generation of M7, based on the observation that cyanide trapping of M7 lowers the CVB in HLM.36 Initially, oxidized rimonabant was

Figure 3. Correlation between EC oxidation potential and the formation of oxidation products (A) voltammogram from online EC/ ESI/MS, m/z 477.1 corresponds to +14 Da oxidation products (B) LC/MS analysis of rimonabant and M7 (C) LC/MS analysis of M4, M8, M9 and M11.

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incubated with various N-acetyl amino acids but no adducted amino acids were found (data not shown). Instead, a tryptic digest of bovine serum albumin (BSA) was allowed to react with the EC oxidation products of rimonabant. The BSA digest contains approximately 50 different peptides with known amino acid sequence that may all serve as model peptides for the reaction with rimonabant EC oxidation products. The data from the LC/MS-analyses of peptides derived from the tryptic digestion of BSA incubated with EC oxidized rimonabant was evaluated manually in search of modified peptides. Prior to that, the chromatograms and spectra from the analyte and control samples were aligned using BioPharmaLynx. Two peptides were found in the analyte samples but not in the controls that were not recognized as BSA peptides by the software. These peptides were selected for MS/MS analysis and manually sequenced. Both peptides were identified by the obtained amino acid sequence tags as the tryptic peptide with the sequence TVEMFVAFVDK, where one of the peptides had an oxidized methionine. The doubly charged modified peptide ions were detected at m/z 732.289 and m/z 740.289 Da, while their corresponding unmodified peptides were detected at m/z 700.250 and m/z 708.274 Da, respectively. The mass difference of 32.0 Da for the doubly charged ions suggests peptide adducts with a mass increment of 64 Da. Product ion spectra revealed that all ions containing the N-terminal (b- and a-ions) were modified, while the y-ions extending from the C-terminus were unmodified (Figure 5). Additionally, the immonium ion corresponding to the modified N-terminal threonine (T) was also found in the spectrum. As a result of this finding, the N-terminal modification was entered into BioPharmaLynx and a larger number of adducted peptides were identified by the software. The most abundant peptides were selected for MS/MS analysis and unambiguously identified. The MS/MS product ion spectra contained bn- and yn-type ions, consistent with the assigned sequences, and the bions had mass increments of 64 Da due to the N-terminal modification. The N-terminal amino acids for the modified

Figure 4. Cyanide and peptide adducts from trapping experiments in EC oxidized sample and HLM incubation.

Figure 5. Product ion spectrum of the +64 Da modified tryptic peptide with the sequence TVMENFVAFVDK. * indicates a modified ion with a mass increment of 64 Da. 1813



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Figure 6. Representative MS spectra of the model peptides. Leucine-enkephalinamide incubated with (A) oxidized [14C]-labeled rimonabant (B) oxidized unlabeled rimonabant (C) unmodified peptide, and angiotensin II incubated with (D) oxidized [14C]-labeled rimonabant (E) oxidized unlabeled rimonabant (F) unmodified peptide. The [M + H]+ and [M+2H]2+ ions are shown for leucine-enkephalinamide and angiotensin II, respectively. The ratio for [14C]-incorporation in rimonabant is 31, 23 and 46% for none, one or two [14C]-atoms, respectively, in the piperidine-ring.

peptides (Figure S1−S3). MS/MS analysis of the leucineenkephalinamide +64 Da adduct clearly showed that the modification was exclusively located on the N-terminal tyrosine (Figure 7). All b-ions (b2−b4) including the N-terminal tyrosine (Y), and their corresponding a-ions (a2−a4) appear with a mass increment of 64.03 Da for the modified peptide when compared with the unmodified peptide. No y-ions were detected for this peptide. Fragmentation data from angiotensin II also confirmed the N-terminus as the site of modification (Figure S4). Incubation with oxidized [14C]-rimonabant labeled on both α-carbons of the piperidine ring resulted in a shift in the isotopic pattern of the modified peptides, and the +4 Da isotope became the most abundant (Figure 6A and D). This is in agreement with the [14C]-labeling on the piperidine ring, where the isotope containing two [14C]-labels is most abundant (31, 23, and 46% for none, one, and two [14C]-labels, respectively). Additionally, product ion spectra revealed an isotopic shift for the N-terminal tyrosine (Y) after incubations with [14C]-rimonabant (Figure S5). All together, the results from the [14C]-rimonabant incubations support the finding that the adduction site is N-terminal and that the piperidine ring of rimonabant is involved in the modification, consistent with structure PX in Figure 4. To examine whether a correlation exists between the oxidation products and the formation of the modified peptides, rimonabant was oxidized at increasing EC oxidation potentials and incubated with the model peptides. LC/MS analysis of the incubations demonstrated that the level of modified peptides increased with the increasing oxidation potential (Figure 8). At the lower oxidation potentials, where rimonabant is oxidized to M7 with low levels of other oxidation products, no adducted

peptides were aspartic acid, glutamic acid, glutamine, histidine, leucine, threonine, and tyrosine, providing evidence that the Nterminal amine and not the side chains is the target of covalent modification. This result is also in accordance with previous publications where the N-terminus has been shown to be adducted with various reactive species.17,41−44 Based on the elemental composition, a modification of 64 Da is likely to originate from the piperidine ring of rimonabant (see Discussion). Incubation of EC Oxidized Rimonabant with Model Peptides. Based on the observation that a number of peptides in the BSA digest were modified on the N-terminus, two model peptides were selected for individual incubations with EC oxidized rimonabant and characterization of the corresponding adducts. The model peptides were leucine-enkephalinamide and angiotensin II, and they were selected based on their molecular weight and amino acid sequence. Both peptides contained N-terminal amino acids that were previously found adducted in the digested BSA sample. Angiotensin II contains putative target amino acid side chains such as histidine, while leucine-enkephalinamide does not contain any nucleophilic amino acid side chains. An advantage with the leucineenkaphalinamide is that it only appears as singly charged in the mass spectra, facilitating its characterization. [14C]rimonabant labeled on the piperidine ring was also included in the studies, since this part of the molecule was suspected to be involved in the adduct formation. Incubation of EC oxidized rimonabant with both leucineenkephalinamide and angiotensin II resulted in peptide adducts with a mass increment of 64 Da, in line with the findings from the tryptic BSA experiments (Figure 6). No other modifications by rimonabant oxidation products could be found for the 1814



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Figure 7. Product ion spectra of leucine-enkephalinamide corresponding to the sequence YGGFL-NH2 (A) adducted peptide (B) unadducted peptide. * indicates a modified ion with a mass increment of 64 Da.

samples prior to incubation at time point 0 min, but their levels increased with incubation time. Furthermore, the corresponding adducted peptides in the EC incubation sample serving as the positive control in this experiment had the same retention times. These results clearly indicate that the same reactive intermediate causing the modification of the peptide in the EC experiment is also present in the HLM incubations.

peptides could be detected. A significant increase in adduction of both peptides was found at the higher potentials where M7 is further oxidized. This result demonstrates that M7 is not directly involved in the adduction, but rather other oxidation products generated at the higher potentials. Incubation of Pentanedial with Model Peptides. EC oxidation of rimonabant was suggested to involve additional oxidation of the iminium ion (M7) after its formation to shortlived intermediates and finally M11 and pentanedial. Pentanedial was proposed to react with the N-terminal, giving rise to the mass increment of 64 Da (see Discussion). To test this hypothesis, the model peptides were incubated with pentanedial. Interestingly, the same peptide modification was observed for both peptides at all pentanedial concentrations in a concentration dependent manner (Figure S6). The adducted peptides were unambiguously identified by fragmentation analysis, and their product ion spectra were identical to the adducted peptides incubated with oxidized rimonabant (Figure S7). Human Liver Microsomal Incubation. To investigate the biological relevance of our findings, leucine-enkephalinamide was used as a trapping agent during HLM incubations of rimonabant. The supernatants from the HLM incubations were analyzed with multiple reaction monitoring (MRM) MS. The product ions in the selected transitions were all ions containing the N-terminal modification in order to obtain a high specificity in the analysis. The adducted peptide from the EC experiment was analyzed in parallel as a control. Leucine-enkephalinamide modified with the 64 Da adduct was the major peak detected in chromatograms from the microsomal incubations with all four MRM transitions (Figure 9). The later eluting peak detected in the chromatograms was the modified peptide (PX) further adducted with cyanide (vide supra and see Supporting Information). None of the peaks were present in the HLM

Figure 8. Correlation between EC oxidation potential (mV) and the levels of adducted leucine-enkephalinamide and angiotensin II.

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DISCUSSION The mechanism(s) for the CVB of reactive rimonabant species to proteins and as a consequence to what extent different reactive species contribute to the CVB are unknown. The aims of the present study were to characterize the EC oxidation products of rimonabant and to explore the mechanism by which they covalently bind to model peptides. To assess the biological relevance of this approach, the EC oxidation products and peptide adducts were compared with those generated in HLM. 1815



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Figure 9. Representative chromatograms from MRM analysis of HLM and EC experiments (A) Co-incubation of rimonabant and leucineenkephalinamide for 4 h in HLM (B) leucine-enkephalinamide incubated with electrochemically oxidized rimonabant at 900 mV. The MRM transitions were 619.3 to 200.1, 619.3 to 342.14, 619.3 to 489.2 and 619.3 to 602.3.

Scheme 2. Proposed mechanism for EC oxidation of rimonabant and suggested P450 metabolic pathways. Tentative intermediate structures are denoted I1−I8

because it was not affected during the 4 h incubation at 37 °C. In an aqueous environment, equilibrium exists between M7 and the α-hydroxy piperidine species I1, which in turn will be in equilibrium with the ring-opened aldehyde I2. This equilibrium is clearly shifted toward the iminium ion M7, as neither I1 nor I2 was detected online by EC/ESI/MS. When the EC oxidation

The proposed mechanism for EC oxidation of rimonabant is presented in Scheme 2. At a potential of +300 mV, a 2-electron oxidation of the piperidine moiety yields the iminium ion M7 as the major oxidation product with only low levels of other oxidation products formed. Clearly, M7 and/or its conjugate enamine base M7′ is very stable in phosphate buffer pH 7.4 1816



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Scheme 3. Proposed mechanism for adduction of pentanedial (I8) to the N-terminus of peptides. * indicates the positions of the [14C]-labels in the reactive species and the final PX adduct

mandatory intermediates to form α-hydroxy piperidines by EC,30 while for P450 the initial product is generally an αhydroxy piperidine that then may lose water to give the corresponding iminium ion.46−49 Despite this mechanistic difference, the stable metabolites/EC oxidation products M4, M7, M8, M9, M11, and M13, as well as the cyanide adducts are formed in both systems (Scheme 1, Figures 1 and 3). The metabolites M3, M6, and M12, originating from hydroxylation(s), were not detected as EC oxidation products. This is in line with previous findings that EC, under the conditions used here, is not able to mimic aliphatic hydroxylations.30 We find it reasonable to believe that, for rimonabant, the EC oxidation and the P450 metabolism share common intermediates on the path to these stable metabolites. Products with biologically relevant nucleophiles and EC generated reactive intermediates may therefore be of importance to understand the metabolic fate leading to CVB. Since the N-terminal modification of +64 Da (PX) was proven to contain C5H4 originating from the aminopiperidine moiety of rimonabant, a reasonable assumption is that one or several of the metabolites and/or intermediates in Scheme 2 may be involved in the formation of this adduct. This is supported by the finding that the level of modified peptides significantly increased with increasing EC oxidation potential (Figure 8). At lower EC oxidation potentials where mainly M7/ M7′ is generated and the levels of other oxidation products are low, no adducted peptides could be detected after incubation. In other words, modification of the peptides required subsequent oxidation of M7/M7′ to short-lived intermediates, providing evidence that M7/M7′ is not directly involved in the adduction. One plausible mechanism for the formation of PX is presented in Scheme 3. Addition of the peptide N-terminal NH2 to pentanedial (I8) would give the carbinolamine intermediate P1. Dehydration of P1 would give the aldehyde intermediate P2. Subsequent addition of the secondary Nterminal nitrogen to the aldehyde would close the ring and give the carbinolamine intermediate P3, which, finally, would lose a molecule of water to form the stable peptide adduct PX. This mechanism is supported by the finding that incubation of pentanedial with the model peptides resulted in the same peptide modification as when incubated with EC oxidized rimonabant. The observation of time-dependent formation of M11 in the EC sample indicates that also I8 is generated over time in the EC oxidized sample. A similar mechanism for adduction of pentanedial to the N-terminal amine of hemoglobin was proposed by Gasthuys et al.44 Pentanedial, also known as glutaraldehyde, is well-known to be toxic.50,51 Furthermore, it is as an effective protein cross-linking reagent used in a variety of applications such as enzyme immobilization.52 It should be noted that this finding does not exclude that other reactive intermediates may contribute to the stable peptide adduct PX. For example, alternative routes to initiate the formation of PX are addition of the peptide N-terminal

potential is increased, the α-hydroxy species I1 is further oxidized to the lactam M8. Because this step is not reversible under the oxidative conditions in the EC cell, M8 accumulates as a stable product at oxidation potentials of +500 mV and higher (Figure 3). Nevertheless, M7 remained as the major EC oxidation product derived from rimonabant. The iminium ion M7 does not have a lone pair of electrons on the piperidine nitrogen and is therefore a poor substrate for EC oxidation. The nitrogen of the enamine conjugate base M7′, on the other hand, carries a lone pair of electrons and can undergo a second 2-electron oxidation to form a dihydropyridinium species M10. The M10 oxidation product was unstable and could only be detected online by EC/ESI/MS. Several isomeric structures can be drawn for M10 of which two are exemplified in Scheme 2 as the 3,4-dihydropyridinium (M10) and the 2,3-dihydropyridinium (M10′). The electrochemical oxidation of M7′ is initiated by a 1-electron oxidation to an aminyl radical cation that is expected to deprotonate in the αposition to an α-carbon centered neutral radical that then undergoes a second 1-electron oxidation. The most favored product following electrochemical 2-electron oxidation of M7′ is therefore expected to be the 3,4-dihydropyridinium (M10). The detection of cyanide adduct CA3 (Figure 4) following addition of cyanide to the EC oxidized samples points to the existence of at least one dihydropyridinium form of M10. Analogous to the fate of the iminium ion M7, M10′ would in an aqueous environment be in equilibrium with the hydrated products I3 and I4. On a highly speculative note, intermediate I3/I3′ could be stable and correspond to the EC oxidation product/metabolite M4. The hydrazide product (M11) was not detected online by EC/ESI/MS but was formed following incubation of the ECoxidized samples at 37 °C. There was a clear correlation between M11 and the increasing oxidation potential (Figure 3C). Clearly, M11 is not a product of direct EC oxidation but rather a product formed from the degradation of a less stable species formed in the EC cell. We find it reasonable to believe that the unstable species M10 is involved, as illustrated by a potential mechanism for the formation of M11 from the 3,4dihydropyridinium (M10). An initial hydration of M10 would give the 1,2,3,4-tetrahydropyridin-2-ol (I5), which would be in equilibrium with the N-pentenal (I6′), which in turn would be in equilibrium with its conjugate acid, the imine I6. Hydration of I6 would give the unstable carbinolamine I7, which would spontaneously form pentanedial (I8) and the hydrazide product M11. An alternative pathway for the formation of I8 and M11 might be oxidation of the aldehyde I2. Shown in Scheme 2 are also potential P450 metabolic pathways from rimonabant to the α-hydroxy piperidine species (I1) and from the iminium ion/enamine M7/M7′ to the 1,2,3,4-tetrahydropyridin-2-ol intermediate (I5). Previous comparisons between EC oxidations and P450 catalyzed oxidations of piperidine derivatives have revealed important mechanistic differences.30,45 Iminium ions are, for example, 1817



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the utility of EC generation of reactive metabolites in combination with biologically relevant model trapping systems as a powerful tool in understanding mechanisms that lead to in vitro covalent modification of proteins.

NH2 to the imine of the 3,4-dihydropyridinium (M10) or one of the aldehyde intermediates I6, I6′, or I7 in Scheme 2 (Scheme S1 for adduction mechanisms). Whether or not or to what extent this finding contributes to the overall CVB of rimonabant in HLM is not known at this point. Clearly, the N-terminal modification of C5H4 originating from the aminopiperidine moiety of rimonabant may contribute. Importantly, the same adducted peptide was detected in HLM coincubated with leucine-enkephalinamide and rimonabant, providing direct evidence that reactive rimonabant intermediate(s) identical to EC generated species are also present in the microsomal incubations. On a more speculative note, other intermediates may be more stable in a protein environment than in aqueous solutions used in these studies. One could also imagine that, in a more complex environment, neighboring amino acids could react with several of the peptide intermediates in Scheme S1 to form more stable covalent bonds that include more than one amino acid. In a previous study of rimonabant, Andresen-Bergstrom et al.36 demonstrated that levels of CVB were significantly reduced in HLM when the iminium species were trapped as cyanide adducts but also following addition of methoxylamine. Addition of cyanide to HLM resulted in three cyanide adducts formed by trapping of M7, M2, and/or M4 as well as M10 (Figure 4), while no methoxylamine adducts were detected. No −4 Da metabolite (e.g., M10) accountable for the formation of the cyanide adduct CA3 was detected in incubations without cyanide, and they suggested that M10 is a short-lived intermediate. The authors proposed that M7 and/or its corresponding aldehyde (I2) are likely contributors to the CVB. Our data suggest an alternative pathway potentially leading to CVB, which might also explain the lowering in CVB upon addition of both cyanide and methoxylamine. Due to the equilibrium between I2, M7, and M7′, a consequence of trapping M7 with cyanide or I2 with methoxylamine is that the levels of M10 as well as I8 will be reduced. In addition, the level of M10 is further reduced due to formation of the cyanide adduct CA3. Other intermediates in Scheme 2 that can be trapped by cyanide and affect the CVB, as well as account for the formation of CA1, include I5′ and I6. The reduction in CVB in the methoxylamine experiments might also be explained by trapping of aldehydes I6, I6′, I7, and I8.

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Information Figures S1−S7 and Scheme S1, containing selected chromatograms, MS/MS-spectra, and proposed adduction mechanisms. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +46 31 77 61989. Fax: +46 31 77 63700. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Lars Weidolf and Professor Neal Castagnoli Jr. for helpful discussions and critical reading of this manuscript. We also thank Dr. Jonas Boström for help with the illustrations in the cover page.

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ABBREVIATIONS BSA, bovine serum albumin; CB1r, cannabinoid type 1 receptor; CVB, covalent binding; EC, electrochemistry; ESI, electrospray ionization; HLM, human liver microsomes; KCN, potassium cyanide; MRM, multiple-reaction monitoring; P450, cytochrome P450; THLE, SV40-T antigen-immortalized human liver epithelial derived cells

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REFERENCES

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CONCLUSIONS Trapping of EC generated reactive intermediates with model peptides led to the identification of a previously unobserved bioactivation pathway of rimonabant and a potential mechanism leading to in vitro covalent binding to proteins that was not captured by traditional trapping agents. There are several reactive oxidation products that could yield this modification, and we propose that the iminium ion is further oxidized to short-lived intermediates as the dihydropyridinium ion. Studies are being undertaken to determine whether this finding is applicable to other piperidine containing drug molecules or if it is unique to rimonabant. Alicyclic amine scaffolds such as piperidine are commonly used in drug design. It is therefore important to understand the biotransformation and bioactivation pathways to enable design of drug candidates that are devoid of structural moieties ultimately leading to covalent modification of proteins. Identification of biological targets of reactive metabolites is expected to aid in closing the gap between CVB and observed idiosyncratic adverse drug reactions. Our studies demonstrated 1818



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Use of Electrochemical Oxidation and Model Peptides To Study

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