The first-line antiepileptic drug carbamazepine: Reaction with biologically relevant free radicals

Author’s Accepted Manuscript The first-line antiepileptic drug carbamazepine: Reaction with biologically relevant free radicals Inês L. Martins, João Nunes, Catarina Charneira, Judit Morello, Sofia A. Pereira, João P. Telo, M. Matilde Marques, Alexandra M.M. Antunes www.elsevier.com

PII: DOI: Reference:

S0891-5849(18)31308-X https://doi.org/10.1016/j.freeradbiomed.2018.10.408 FRB13972

To appear in: Free Radical Biology and Medicine Received date: 30 July 2018 Revised date: 1 October 2018 Accepted date: 4 October 2018 Cite this article as: Inês L. Martins, João Nunes, Catarina Charneira, Judit Morello, Sofia A. Pereira, João P. Telo, M. Matilde Marques and Alexandra M.M. Antunes, The first-line antiepileptic drug carbamazepine: Reaction with biologically relevant free radicals, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.10.408 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The first-line antiepileptic drug carbamazepine: reaction with biologically relevant free radicals Inês L. Martins1, João Nunes1, Catarina Charneira1, Judit Morello1, Sofia A. Pereira2, João P. Telo1, M. Matilde Marques1, Alexandra M. M. Antunes*,1 1

Centro de Química Estrutural, Instituto Superior Técnico, ULisboa, 1049-001 Lisboa, Portugal

2

CEDOC, Chronic Diseases Research Centre, NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, 1169-006 Lisboa, Portugal

*

Corresponding Author: Centro de Química Estrutural, Instituto Superior Técnico,

ULisboa, 1049-001, Lisboa, Portugal, Tel: +351-21-8417627; fax: +351-21-8464455; [email protected]

Abstract Carbamazepine (CBZ) is one of the most widely used antiepileptic drugs by both adults and children. Despite its widespread use, CBZ is associated with central nervous system toxicity and severe hypersensitivity reactions, which raise concerns about its chronic use. While the precise mechanisms of CBZ-induced adverse events are still unclear, metabolic activation to the epoxide (CBZ-EP) has been thought to play a significant role. This work reports first-hand evidence that CBZ reacts readily with biologically relevant thiyl radicals with no need for bioactivation. Using liquid chromatography coupled with high resolution mass spectrometry, multiple products from direct reaction of CBZ with glutathione (GSH) and N-acetyl-L-cysteine (NAC) were unequivocally identified, including the same product obtained upon ring-opening of CBZ-EP. The product profile is complex and consistent with radical-mediated mechanisms. Importantly, side products and adducts compatible with this non-enzymatic pathway were identified in liver extracts from CBZ-treated Wistar rats. The reaction of CBZ with GSH and NAC is more extensive in the presence of oxygen. Taking into consideration that GSH conjugation is, in general, a detoxification pathway, these results suggest that under hyperoxia/oxidative stress conditions the 1

bioavailability of the parent drug may be compromised. Additionally, this non-enzymatic process can be anticipated to play, at least in part, a role in the onset of CBZ-induced adverse effects due to the concomitant generation of reactive oxygen species. Therefore, the search for causal relationships between the formation of nonenzymatically-driven CBZ products and the occurrence of CBZ-induced adverse events in human patients merits further research, aiming the translation of basic mechanistic findings into a clinical context that may ultimately lead to a safer CBZ prescription.

Abbreviations: AAED, aromatic anti-epileptic drug; CBZ, carbamazepine; CBZ-EP, 10,11-dihydro-10,11-epoxycarbamazepine; CYP, cytochrome P450; DAD, diode-array detector; DMSO, dimethyl sulfoxide; DRESS, drug rash with eosinophilia and systemic symptoms; EPHX1, epoxide hydrolase 1; ESI, electrospray ionization; FDA, U.S. Food and Drug Administration; GSH, glutathione; HLA, human leukocyte antigen; HSA, Human

Serum

Albumin;

IDR,

idiosyncratic drug

reaction;

LC-HRMS,

liquid

chromatography–high resolution mass spectrometry; LC-MS, liquid chromatography mass spectrometry; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NAC, N-acetyl-L-cysteine; NAL, N-acetyl-L-lysine; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; ROS, reactive oxygen species; SJS, Stevens-Johnson syndrome; SULT, sulfotransferase; TEN, toxic epidermal necrolysis; THF, tetrahydrofuran; TOCO, thiol-olefin cooxidation process; UGT, uridine 5'-diphospho-glucuronosyltransferase

2

Graphical Abstract:

ROS

Keywords Carbamazepine, covalent adducts, radical-based mechanism, glutathione conjugates, mercapturates, reactive oxygen species.

1. Introduction

The first-generation aromatic anti-epileptic drug (AAED) carbamazepine (5Hdibenzo[b,f]azepine-5-carboxamide, CBZ, 1, Scheme 1) is marketed since the early 1960’s. Currently, it is still a first-line drug for the treatment of partial and generalized epileptic seizures [1], a chronic brain disorder that according to the World Health Organization affects around 50 million people worldwide [2]. This dibenzazepine derivative is also used as a highly effective agent for chronic pain syndromes, trigeminal neuralgia and psychiatric diseases, such as bipolar disorder, depression, manic-depressive illness, schizophrenia and aggression due to dementia [3, 4]. Despite its widespread use, with more than 1000 tons consumed annually, up to 10% of patients on CBZ are reported to develop adverse effects [1]. Most of the CBZ-induced adverse effects are mild and dose-dependent [5]. However, its chronic therapeutic use has been associated with a variety of serious idiosyncratic drug reactions (IDRs), including cutaneous, hematological, immunological, renal and hepatic disorders [6, 7]. 3

In fact, when compared with other anti-epileptic drugs, there is a greater number of reported cases of CBZ-induced hypersensitivity reactions, namely severe cutaneous adverse reactions such as Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), or drug rash with eosinophilia and systemic symptoms (DRESS). To date, two distinct models/hypotheses have been considered to explain the mechanisms of the CBZ-induced immune-mediated adverse reactions: 1) the prohapten hypothesis, which assumes the involvement of CBZ bioactivation to metabolites capable of binding irreversibly to proteins, generating covalent adducts that act as antigens [8]; and 2) the altered peptide mechanism, which states that an immune response can be stimulated upon reversible (non-covalent) binding of the non-reactive parent drug to target proteins [9, 10]. A genetic association was also recognized in the CBZ-induced hypersensitivity reactions, which can be dangerous or even fatal [11-15]. However, CBZ-induced liver injury can also occur without immunoallergic features. This is in line with the fact that drug-induced adverse reactions frequently involve multiple nonmutually exclusive mechanisms. Accordingly, the increased risk of developing systemic lupus erythematosus, identified in women on CBZ therapy, has yet to be explained [16]. Moreover, although the association of CBZ exposure with congenital malformations has not been demonstrated, some CBZ-related teratogenic effects have been clinically observed, particularly in co-administration with other AAEDs [17, 18]. Furthermore, while CBZ-induced pulmonary toxicity is rare, interstitial pneumonitis, bronchiolitis obliterans organizing pneumonia, bronchospasm, pulmonary edema and pulmonary nodules have been associated with CBZ-based regimens [19-22]. Taking into consideration the high daily dose (up to 2000 mg) and widespread chronic use of this drug, by both adults and children, along with the severity of CBZinduced adverse reactions, further investigations toward more accurate risk/benefit estimations are needed, necessarily involving more comprehensive insights into the toxicokinetics of the drug. The role of CBZ metabolism in the excretion and toxicity of the drug has been consistently argued. CBZ metabolism is predominantly mediated by cytochrome P450 (CYP) isoforms in the liver. In Humans, approximately 70% of the dose is excreted in the urine, and only 2 - 5% are excreted as unchanged drug [23]. More than 30 different CBZ metabolites have been identified in the urine of rats [24, 25] and patients [26]. CBZ metabolic pathways include the N-glucuronidation of the parent drug [27], catalyzed by UGT2B7 [28], and the oxidation of aromatic positions to phenolic metabolites,

yielding

2-hydroxycarbamazepine

(2-OH-CBZ,

2),

3-

4

hydroxycarbamazepine (3-OH-CBZ, 3) and 2,3-dihydroxycarbamazepine (2,3-diOHCBZ, 4), which undergo subsequent O- and N-glucuronidation followed by urinary excretion (Scheme 1) [24, 27]. However, the major route of CBZ metabolism involves the formation of 10,11-dihydro-10,11-epoxycarbamazepine (CBZ-EP, 5) [27, 29]. This epoxide is a pharmacologically active anticonvulsant and is thought to have the same mechanism of action as the parent drug [30]. CBZ-EP can undergo ring-opening, catalyzed by epoxide hydrolase 1 (EPX1), yielding 10,11-dihydro-10,11-antidihydroxycarbamazepine (anti-CBZ-diol, 6a, Scheme 1) [31]. Unconjugated forms of CBZ-EP (5) and anti-CBZ-diol (6a) were identified by liquid chromatography-mass spectrometry (LC-MS) in the urine of patients on long-term CBZ regimens, on the basis of retention times and mass spectra matching those of authentic standards [27]. The formation of the syn-diol isomer 6b (Scheme 1) has also been reported in vivo, albeit its formation is unlikely to stem from the ring-opening reaction of epoxide 5 [25].

Multiple reactive metabolites (5, 7-10, Scheme 1) have been suggested to play a role in the onset of CBZ-induced adverse reactions [32-34]. In particular, the major CBZ metabolite, CBZ-EP (5), has been consistently argued to be pivotal in this context [35]. The recent identification of covalent adducts, presumably formed between CBZEP and histidine residues of Human serum albumin (HSA) isolated from the blood of patients on CBZ, is suggestive of this role [36], albeit the correlation with plasmatic CBZ-EP levels was weak. Additionally, the authors reported that CBZ-EP formed covalent protein adducts in vitro with histidine and cysteine residues of HSA and glutathione-S-transferase pi, respectively [36], along with the formation of the GSH adducts 11a from microsomally-generated CBZ-EP. Moreover, several distinct CBZderived methylthio, methylsulfinyl, and methylsulfonyl metabolites were isolated by HPLC from enzymatically hydrolyzed rat and human urine and subsequently identified by gas chromatography-mass spectrometry with plasma desorption ionization [25]. These catabolic products were proposed to stem from GSH conjugation to metabolically-generated CBZ-derived epoxides. These reports prompted us to conduct a comprehensive study on the reactivity of CBZ-EP towards bionucleophiles. Surprisingly, this led to first-hand evidence that CBZ reacts with glutathione (GSH) and N-acetyl-L-cysteine (NAC), with no need for bioactivation. The use of high resolution mass spectrometry (HRMS) provided unbiased identification of the product profile obtained upon reaction of CBZ with GSH and NAC, which is compatible with radicalmediated mechanisms. CBZ-derived products and adducts were also identified in liver

5

extracts from CBZ-treated Wistar rats. The evidence provided herein suggests that these non-enzymatic pathways may have a significant impact on CBZ toxicokinetics, particularly in hyperoxia/oxidative stress conditions.

2. Experimental Chemicals. All commercially available reagents were acquired from Sigma-Aldrich Química, S.A. (Madrid, Spain), unless specified otherwise, and were used as received. High performance liquid chromatography with diode array detection (HPLC-DAD) analyses HPLC-DAD analyses were conducted on an Ultimate 3000 Dionex system consisting of an LPG-3400A quaternary gradient pump and a diode array spectrophotometric detector (Dionex Co., Sunnyvale, CA). HPLC analyses were performed with a Luna C18 (2) column (250 mm x 4.6 mm; 5 µm; Phenomenex, Torrance, CA), at a flow rate of 1 mL/min. Semipreparative HPLC separations were conducted with a Luna C18 (2) column (250 mm x 10 mm; 5 µm; Phenomenex) at a flow rate of 3 mL/min. A 2.5 min linear gradient from 5 to 50% acetonitrile in 0.1% aqueous formic acid, followed by a 25.5 min linear gradient to 100% acetonitrile and a 7 min isocratic elution with 100% acetonitrile, was used in all instances. The UV absorbance was monitored at 254 nm. Liquid chromatography - mass spectrometry (LC-MS) analyses LC-MS analyses were conducted on an HPLC Dionex Ultimate 3000 system coupled in-line to an LCQ Fleet ion trap mass spectrometer equipped with an ESI ion source (ThermoFisher Scientific, Waltham, MA). Chromatographic separation was performed on a Luna C18(2) column (150 mm x 2 mm, 3 μm; Phenomenex) at a constant temperature of 30 ºC, using an elution gradient of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B) at a flow rate of 200 μL/min. Gradient conditions were as follows: 0-2 min, isocratic 5% B; 2-30 min, linear gradient to 70% B; 30-32 min, linear gradient to 100 % B; 32-40 min, isocratic 100% B; and 40-45 min, linear gradient to 5% B (followed by a 10 min re-equilibration time). The mass spectrometer was operated in the ESI positive mode, with the following optimized parameters: ion spray voltage, 4.5 kV; capillary voltage, 16 V; tube lens offset, 58 V; sheath gas (N2), 80 arbitrary units; auxiliary gas (N2), 5 arbitrary units; 6

capillary temperature, 270 ºC. Spectra typically corresponded to the average of 20–35 scans, and were recorded in the 100-1000 Da range. Tandem mass spectra (MS/MS) were obtained with an isolation window of 1 or 6 m/z units, 28-35 % relative collision energy and an excitation time of 30 ms. Data acquisition and processing were performed using the Xcalibur 2.2 software. Liquid chromatography - high resolution mass spectrometry (LC-HRMS) analyses LC-HRMS analyses were performed on an Ultimate 3000 RSLCnano system (ThermoFisher Scientific), interfaced with a Bruker Impact II quadrupole time-of-flight mass spectrometer equipped with an ESI source (Bruker Daltoniks, Bremen, Germany). Chromatographic separation was performed on a HypersilGold C18 column (150 mm x 2.1 mm; 1.9 μm; ThermoFisher Scientific) using an elution gradient of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B) at a flow rate of 200 μL/min. The elution conditions were as follows: 0-2.4 min, isocratic 5% B; 2.4-4.5 min, linear gradient to 25% B; 4.5-8.6 min, linear gradient to 70% B; 8.6-11.6 min, linear gradient to 100% B; 11.6-13.6 min, linear gradient to 5% B; and 13.6-19.6 min, isocratic 5% B. The injection volume was 10 L. The column and the autosampler were maintained at 40 ºC and 8 ºC, respectively. The mass spectrometric parameters were set as follows: end plate offset, 500V; capillary voltage, 4.5 kV; nebulizer, 40 psi; dry gas, 8 L/min; heater temperature, 200 ºC. Internal calibration was performed for sodium formate/acetate clusters, with a sodium formate/acetate solution introduced to the ion source via a 20 L loop at the beginning of each analysis using a six-port valve. Calibration was then performed using highprecision calibration mode (HPC). Acquisition was performed in the m/z 50-1000 range and in a data-dependent MS/MS mode with an acquisition rate of 3 Hz using a dynamic method with a fixed cycle time of 3 s. Dynamic exclusion duration was 0.4 min. Mass spectrometry data processing Mass spectrometry data were processed using the DataAnalysis 4.1 software (Bruker Daltoniks). An in-house library was built with the chemical formulas, m/z ratios, and retention times of CBZ-derived products and adducts obtained in vitro upon reaction with GSH or NAC. This library was used for targeted

screening

using

the

TargetAnalysis

Software

(Bruker

Daltoniks).

Identifications were based on three parameters: retention time, mass accuracy, and isotopic pattern. Values for retention time deviation lower than 0.3 min, mass deviation lower than 5 ppm and mSigma lower than 100 were considered acceptable for positive

7

confirmation. All spectra corresponding to CBZ-derived products and/or adducts were then manually checked. High resolution mass spectra of the adducts and CBZ-derived products identified in this work and the high resolution tandem mass spectra of their protonated molecules are presented as Supporting Information (Figures S1-S20).

Animal Experiments Male Wistar rats (6 weeks old) were obtained from the NOVA Medical School animal facility and were housed two per cage in polycarbonate cages with wire lids (Tecniplast, Italy). They were maintained under controlled environmental conditions (12 h light/dark schedule at 22 ± 2.0 °C, 60 ± 10% humidity, with food (SDS RM1, Special Diets Services, UK)) and reverse osmosis water supplied ad libitum. The animals were Specific Pathogen Free (SPF) according to FELASA recommendations [37]. Applicable institutional and governmental regulations concerning ethical use of animals were followed, according to the NIH Principles of Laboratory Animal Care (NIH Publication 86-23, revised 1985), the European guidelines for the protection of animals used for scientific purposes (European Union Directive 2010/63/EU), and the Portuguese Law nº 113/2013. Experimental procedures received prior approval by the Institutional Ethics Committee of the NOVA Medical School for animal care and use in research. All animals underwent a 7-day period of handling acclimatization. The rats were handled daily by the same individual for a period of 2 minutes each and accustomed to the gavage position, in a different animal facility procedures room. Rats (3 per group) were treated by gavage with five daily doses of CBZ (50 mg/kg) or vehicle, using a sterile polypropylene feeding tube (15 gauge; tip diameter: 3 mm; length: 78 mm; Instech Laboratories, Inc., USA) to reduce the risk of trauma, perforation and cross-contamination. On the last day of treatment, approximately 2 hours after the 5th CBZ dose, the rats were anesthetized by intraperitoneal injection with medetomidine (0.5mg/kg body weight; Domitor®, Pfizer Animal Health) and ketamine (75 mg/kg body weight; Imalgene 1000®, Merial). The livers were rapidly removed and stored at -80 °C until use.

Extraction of metabolites and adducts from rat livers 8

To the frozen rat liver tissues (200 mg), 1 mL of cold methanol/water (4:1) was added and each mixture was vortexed for 3 min. Following 60-min sonication at 4ºC, the suspension was centrifuged (5000 x g for 5 min at 4 ºC). The supernatants were then recovered and dried on a SpeedVac® concentrator. The residues were reconstituted in water containing 10% acetonitrile before LC-HRMS analysis.

In vitro Reactions Reaction of NAC with a CBZ-EP/CBZ mixture m-Chloroperbenzoic acid (256 mg, 1.5 mmol, 1.4 eq) was added dropwise to a solution of CBZ (250 mg, 1.1 mmol, 1.0 eq.) in CH2Cl2 (5 mL). The resulting solution was stirred at room temperature during 4 days, whereupon the solution was cooled in an ice bath and the ensuing precipitate subsequently filtered. Following extraction with saturated NaHCO3, the organic fraction was dried over anhydrous Na2SO4, and evaporated. The residue was analysed by 1H NMR, which indicated a 2/1 molar ratio of CBZ-EP/CBZ. A portion of this residue (15.9 mg), containing 42.9 µmol of CBZ-EP, was dissolved in THF (0.5 mL) and subsequently added to a solution of NAC (64.7 mg, 396.4 µmol, 9.2 eq.) in 50 mM phosphate buffer pH 7.4 (2.0 mL). The resulting mixture was stirred at 37 ºC for 72 h. The reaction was followed by HPLC-DAD and the products analyzed by LC-MS. Reaction of CBZ with bionucleophiles To a solution of CBZ (4.2 mM in acetonitrile, 118 µL, 1 eq.) was added a solution of GSH, NAC, NAL or ethyl L-valinate (66.8 mM in 10 mM ammonium acetate buffer pH 7.5, 374 µL, 50 eq.), and the final volume was set to 500 μL with the same buffer. The resulting CBZ (1 mM) and GSH, NAC, NAL or ethyl L-valinate (50 mM) solutions were stirred at 37 ºC for 24 h, under either an O2 or an N2 stream. Aliquots (80 µL) were collected after 2 and 24 h and then each aliquot was added to 20 µL of a 3.8 mM nevirapine solution in 10 mM ammonium acetate (internal standard). Control incubations were performed using the same conditions in the absence of bionucleophiles. The reaction mixtures were analysed by LC-MS and LC-HRMS.

Reaction of CBZ-EP with bionucleophiles

9

To a solution of CBZ-EP (4.2 mM in acetonitrile, 118 µL, 1 eq.) was added a solution of GSH, NAC, NAL or ethyl L-valinate (66.8 mM in 10 mM ammonium acetate buffer pH 7.5, 374 µL, 50 eq.), and the final volume was set to 500 μL with the same buffer. The resulting CBZ-EP (1 mM) and GSH, NAC, NAL or ethyl L-valinate (50 mM) solutions were stirred at 37 ºC for 24 h. Aliquots (80 µL) were collected after 2 and 24 h and then each aliquot was added to 20 µL of a 3.8 mM nevirapine solution in 10 mM ammonium acetate (internal standard). Control incubations were performed using the same conditions in the absence of bionucleophiles. The reaction mixtures were analysed by LC-MS and LC-HRMS. Reaction of CBZ with GSH and NAC in the presence of H2O or H218O To a solution of CBZ (1.0 eq., 0.42 µmol, 100 µg) in dry acetonitrile (10 µL) was added a solution of GSH or NAC (50.0 eq., 21 µmol) in H2O or H218O (40 µL). The reaction mixture was stirred at 37 ºC for 24 h. Two distinct assays were run in parallel: 1) under an N2 stream; and 2) under an O2 stream. The reaction mixtures were analysed by LC-MS and LC-HRMS. Reaction of CBZ with NAC in the presence of tert-butyl hydroperoxide To a solution of NAC (133.6 mM in water, 187 µL, 50 eq.), tert-butyl hydroperoxide (c.a. 70% in water, 0.5 µL, 8 eq.) and CBZ (4.2 mM in acetonitrile, 118 µL, 1 eq.) were added and the final volume was set to 500 μL with water. The resulting CBZ (1 mM), tert-butyl hydroperoxide (8 mM) and NAC (50 mM) solution was stirred at 60 ºC overnight. A control incubation was performed using the same conditions in the absence of tert-butyl hydroperoxide. The reaction mixtures were analysed by LCHRMS.

3. Results and Discussion Prior reports on the ability of CBZ-EP to yield covalent adducts with S- and Nbased bionucleophiles in vitro and in vivo [34-36] raised our interest in studying the reactivity of this CBZ metabolite towards the S-based bionucleophiles, GSH and NAC, and the N-based bionucleophiles, N-acetyl-L-lysine (NAL) and ethyl L-valinate (as a surrogate for the N-terminal valine of hemoglobin). In a preliminary approach to this study, a tetrahydrofuran (THF) solution containing CBZ-EP and CBZ in a 2:1 molar ratio, obtained following work-up of a reaction mixture from CBZ oxidation with m10

chloroperbenzoic acid, was used. The HPLC-DAD chromatograms obtained after 1 and 72 h incubation of this mixture with a 9.2-fold molar excess of NAC showed the formation of early eluting products (Figure 1). Surprisingly, only the chromatographic peak corresponding to CBZ showed a clear area decrease with time. This observation suggested that CBZ reacts with NAC with no need for bioactivation.

To clarify this issue, two reactions were conducted individually with NAC, one using CBZ and the other using pure, commercially obtained, CBZ-EP. A 50-fold molar excess of the bionucleophile (similar to the 62:1 molar ratio used by Bu et al. [35]), was used for the in vitro formation of adduct 12a. The LC-MS chromatogram of the CBZ-EP reaction mixture (Figure 2A) showed a peak at 12.2 min with m/z 416, compatible with the protonated molecule of two diastereomeric anti adducts 12a, formed upon ring opening of CBZ-EP by NAC (Figure 2A3), which co-eluted under the chromatographic conditions used. The fragmentation pattern, obtained by tandem mass spectrometry (MS/MS) analysis of this ion is compatible with the proposed structure (Scheme 2A).

Interestingly, the LC-MS chromatogram of the CBZ reaction mixture showed this same peak, thereby demonstrating that the anti adducts 12a are also formed upon direct reaction of NAC with CBZ. Additionally, two other peaks, eluting earlier at 10.4 and 10.9 min and also with m/z 416, were identified in the CBZ reaction mixture (Figure 2B3). When analysed by MS/MS, a fragmentation pattern similar to that of 12a was obtained, which suggests a structural similarity between these products. This is compatible with the formation of the two diastereomeric syn adducts 12b upon reaction of NAC with CBZ. One additional signal was observed in the CBZ reaction chromatogram at 14.5 min with m/z 414, which is 2 Da lower than the protonated molecules of adducts 12a and 12b (Figure B2). The MS/MS spectrum of this signal was fully compatible with structure 13, stemming from the incorporation of OH and NAC units in the CBZ structure (Scheme 2B). No evidence for the formation of this product from CBZ-EP was obtained (Figure 2A2).

11

These unexpected results prompted us to explore further the reaction of CBZ with bionucleophiles. Towards this goal, several reactions were conducted, to address the following questions: 1) can CBZ react with other bionucleophiles?; 2) which is the oxygen source for the formation of adducts from CBZ?; and 3) how can the formation of the syn adducts 12b and the iminostilbene adduct 13 from CBZ be explained? To address the first question, three parallel reactions were performed with CBZ and ca. 50 molar eq. of GSH, N-acetyl-L-lysine (NAL), or ethyl L-valinate. When analysed by LC-MS, following 72 h incubation, no evidence was obtained for product formation with the nitrogen-based nucleophiles, NAL and ethyl L-valinate. In contrast, the ion chromatogram obtained from the GSH reaction mixture indicated the formation of GSH analogs of the adducts identified in the NAC reaction, with peaks at m/z 558 (14, structure in Figure 3), eluting at 12.6 min, and m/z 560 (not shown). Similarly to what was observed in the NAC reaction, two other chromatographic peaks were obtained, in this case with m/z 560 and eluting at 8.8 and 10.3 min. The later (major) one corresponded to the anti adducts 11a, also obtained upon ring-opening of CBZ-EP with GSH, and the earlier one to the syn adducts, 11b. The possible oxygen source for the adduct formation between sulphur-based nucleophiles and CBZ was the next issue to be investigated. As these adducts were obtained by simply mixing a solution of the nucleophile in phosphate buffer with a CBZ solution in THF, the presence of trace amounts of peroxides in the organic solvent offered a plausible explanation to our observations. However, this possibility was discarded when the replacement of THF by acetonitrile (MeCN) caused no changes in the product profile. The two other possible oxygen sources were water and oxygen present in solution. To gain insight into the potential role of these two sources, parallel CBZ reactions with NAC and GSH were performed using dry MeCN in the presence of H2O or H218O, under two distinct atmospheric conditions: an N2 or O2 stream. The reaction mixtures were analysed by LC-HRMS and the contribution of water as the oxygen source was established on the basis of the distinct isotopic patterns of adducts 11-14, obtained in the presence of either H2O or H218O. As shown in Figure 3 for the formation of the GSH adducts 11a and 14, a clear increase of the signal corresponding to the [MH+2]+ ion was consistently observed for both adducts in all reaction mixtures conducted in the presence of H218O, regardless of the atmospheric conditions used. However, the signal corresponding to the [MH]+ ion was always observed with high intensity and the relative intensity of the [MH+2]+ ion depended on the specific atmospheric conditions.

Taken together, these data are compatible with the

12

incorporation of

18

O in the GSH-CBZ adducts but also suggest that water was not the

sole oxygen source. The use of nevirapine as an internal standard enabled the assessment of the relative efficiencies of the reactions conducted under N2 and O2 streams (Table 1). This quantification demonstrated a key role for the oxygen concentration in solution media on the product yield, which can only be explained by the participation of O2 in the reaction mechanism. Accordingly, following 24 h incubation of CBZ with NAC under O2 stream only 0.2 % of CBZ remained unreacted. This contrasts with the results obtained under N2 stream, where 94% of CBZ remained unreacted. Adduct 12a was obtained in 85 and 0.2 % yield under O2 and N2 stream, respectively. While less expressive, the same effect was observed in the GSH reaction with CBZ, with yields of 10 and 0.3% for the anti adduct, 11a, following 24 h incubation under O2 or N2 stream, respectively.

Table 1. Unreacted CBZ and relative yields (%) of NAC and GSH adducts obtained upon reaction of these thiols with CBZ under O2 or N2 for 24 h. Unreacted CBZ 12a 12b 13 11a 11b 14

CBZ+NAC N2 94 0.2 0.1 2 -

CBZ +NAC O2 0.2 85 4 9 -

CBZ+GSH N2 97 0.3 1

CBZ+GSH O2 76 10 0.7 13

Insert Figure 3 Taking these results together, we can conclude that in the absence of any additional reactant, CBZ reacts with sulphur-based bionucleophiles, with participation of water and O2. The clear role of the thiol group and O2 suggests that oxygenmediated formation of sulphur-based radicals (N-acetyl-L-cysteinyl or glutathionyl radicals) is the initiating step. In fact, the residual presence of O2 in solution is often

. responsible for the initiation of chain thiyl radical (RS ) reactions that occur in the absence of any other radical initiator [38-40]. Nonetheless, the possible contribution of trace transition metal contaminants, particularly iron, in the water used cannot be disregarded for this initiating step. Regardless of the pathway that led to thiyl radical formation under the experimental conditions used in this study, these radicals are ubiquitous in biological systems. Namely, thiyl radicals can be formed enzymatically by peroxidases and superoxide dismutase or upon reaction with reduced oxygen species 13

.. . such as the superoxide anion (O2 ), hydroxyl radicals ( OH), or peroxyl radicals ( OOR) [41]. Therefore, the occurrence of similar reactions in vivo is plausible. The lower efficiency of GSH, when compared with NAC, was consistently observed in all reactions performed with CBZ, which is compatible with the higher disulfide-forming propensity of NAC when compared with GSH, involving the formation of the corresponding thiyl radicals [42]. While the reactivity of thiyl radicals is much lower than that of oxygen- or carbon-centred radicals, thiyl radicals are able to abstract hydrogen from C-H bonds in peptides [43] and from methylene groups in polyunsaturated fatty acids. Moreover, they can add reversibly to double bonds of simple olefins and unsaturated fatty acids, and to the C5-C6 double bond of pyrimidines [44]. Actually, the addition of thiyl radicals to olefins is a particularly common reaction in the human body, which is namely involved in the cis-trans isomerization of lipids [45, 46] through the formation of a new carbon-centred radical. Thus, the cysteinyl or glutathionyl radicals formed under the reaction conditions we used (and presumably in vivo) may undergo addition to the CBZ C11-C12 double bond, via a thiol-ene reaction, forming the carbon-centered radical 15 (Scheme 3). Once formed, possible termination reactions for this radical are oxidative/reductive processes, forming adducts 16-19. Similarly to what is observed with other thiyl ene products [47], 16 and 17 can subsequently undergo thiyl radical addition followed by reduction yielding the bis adducts 20 and 21. Propagation reactions of the carboncentered radical 15 can stem from reaction with O2, forming the peroxyl radical 22, similarly to what happens during lipid peroxidation [46]. Notably, although present in trace amounts, adducts 16-21 were detected in our reaction mixtures by LC-MS and characterized by LC-HRMS (vide infra).

The peroxyl radical 22 can undergo a thiol-olefin cooxidation process (TOCO) yielding adducts 23 and 24, which were also detected and characterized (vide infra). This oxidative process, first reported by Kharasch et al. [48], involves the reaction of mercaptans with olefins in the presence of O2, leading to the formation of racemic mixtures of α-hydroxylated sulfoxides. The involvement of peroxyl radical intermediates in this reaction was subsequently demonstrated through mechanistic studies [40]. In addition, elimination of the superoxide anion (or of its conjugated acid, the hydroperoxyl radical) from peroxyl radicals is well documented in cases where the resulting carbocation (or neutral product) is stable enough [43, 49]. The loss of superoxide from 14

22 is therefore plausible, leading to the highly resonance-stabilized carbocation 25, which can undergo subsequent nucleophilic attack. Reaction with water will result in a mixture of the anti and syn adducts 11 and 12 identified in the reaction of CBZ with GSH and NAC, respectively, whereas elimination from either 22 (E2-like) or 25 (E1like) provides an alternative pathway to adducts 16 and 17 (Scheme 3). The formation of the cationic intermediate 25 also explains, at least in part, the participation of water as an oxygen source. Moreover, the nucleophilic addition of NAC or GSH to this cationic species may also partially explain the formation of the bis adducts 20 and 21. One additional evidence for the formation of 25 is the identification of a brominated product (26, Scheme 3), when the reaction of CBZ with GSH was performed in the presence of KBr. Although this product was present in trace amounts, one signal displaying the bromine isotope cluster was identified by LC-HRMS, exhibiting the monoisotopic signal at m/z 622.0950 ± 2.6 ppm and an isotope cluster compatible with the protonated molecule of 26 (Figure S17). The peroxyl intermediate 22 can also be subsequently reduced to 27, similarly to what is observed during lipid peroxidation [50]. Whereas the homolytic scission of hydroperoxides is, in general, very slow, the presence of radicals and/or nucleophiles in solution is documented to catalyse this process [51]. Therefore, the formation of hydroxyl radicals and the oxygen-centred radical 28 (along with its equilibrium product 29) from 27 is a plausible event. The subsequent oxidation/reduction of 29 will lead to the NAC adducts, 12 and 13, and the GSH adducts, 11 and 14 (Scheme 3). Overall the formation of syn and anti adducts (11 and 12) from CBZ is consistent with the proposed radical-based mechanisms, with the predominance of anti adducts presumably related to their greater stability, due to steric restraints. The formation of the hydroxyl radical from 27 allows additional reactions with CBZ and water. The direct addition of this radical to the olefinic bond of CBZ will lead to the carbon-centred radical 30 that upon oxidative/reductive processes will afford products 31 (represented as the enolic product) and 32, also detected and characterized by LC-HRMS (vide infra). The reaction of 30 with O2 can also be expected, similarly to what was shown for 15, ultimately leading to the cationic intermediate 25 (where R=OH). The identification of an MS signal matching the retention time and mass spectrum of the synthetic CBZ-EP standard is consistent with this pathway (not shown). However, no evidence for the formation of diol intermediates (6, Scheme 1) was obtained under the tested conditions. Nonetheless, this pathway, which is initiated upon addition of the hydroxyl radical (known as the most biologically

15

active free radical) to CBZ, affording the cationic species 25 (where R=OH), may constitute a reasonable explanation for the identification of the syn-CBZ-diol 6b (Scheme 1) in the urine of patients on CBZ [25]. Multiple products, with distinct retention times but sharing the same m/z at 253 (i.e., with a 16 Da increment compared to the protonated molecule of CBZ), were also identified in the reaction mixtures (not shown). This is consistent with the formation of oxidation products of CBZ, such as the phenolic metabolite 2-OH-CBZ (2) and oxcarbazepine (OXCBZ) that, together with CBZ-EP, were reported to arise from CBZ degradation under Fenton-like conditions [53]. This evidence supports not only the formation of the hydroxyl radical under the tested conditions but also the CBZ reactivity towards radicals. An exchange reaction between the hydroxyl radical and water, consistent with the proposed mechanism, explains the hydroxylated products when

H218O

18

O-enriched isotopic distribution of most of the

was used, as attested by LC-HRMS analysis

(Figure 3, see also Figures S7 and S19, Supporting Information). Moreover, the proposed mechanisms are supported by the LC-HRMS identification of CBZ-derived products, including the covalent adducts with GSH and NAC (cf. Supporting Information). In a parallel experiment, N-acetyl-cysteinyl radicals were generated, upon the addition of the free radical generator tert-butyl hydroperoxide to NAC, and then reacted with CBZ. The LC-HRMS profile was identical to the one obtained in the absence of the radical initiator, which is fully consistent with the involvement of thiyl radicals in the reaction of CBZ with NAC. To investigate if similar reactions could occur in vivo, we analysed by LC-HRMS the metabolites and adducts extracted from the livers of male Wistar rats treated orally with CBZ. Multiple products compatible with the in vivo occurrence of CBZ reactions with the biologically relevant glutathionyl and hydroxyl radicals were identified by LCHRMS (Table 2). Among these were the mercapturates, 12a, 12b, 13 and 16, which are catabolic products of the corresponding GSH conjugates. Importantly, these products were not detected when liver extracts from control rats were spiked with CBZ immediately prior to work-up, thereby demonstrating that they were not artefacts of the work-up process. Taken together, these results represent unequivocal evidence for the occurrence of non-enzymatically-mediated reactions of CBZ with GSH in vivo.

16

Table 2. Target screening results, generated by TargetAnalysis software, of liver extracts from male Wistar (n=3) rats treated by gavage with five daily 50 mg/kg CBZ doses. Metabolite/adduct

Molecular formula

Adduct

31 32

C15H12N2O2 C15H14N2O2

[M+H]+ [M+H]+

11a & 11b

C25H29N5O8S

[M+H]+ [M-H]

-

12a &12b

C20H21N3O5S

[M+H]+

13

C20H19N3O5S

16

C20H19N3O4S

[M+H]+ [M+H]+ [M-H]-

Tr mSigma (min) 0.17 0.0 0.0 0.0 0.06 0.03 0.10 0.10 0.13 0.10 0.09

6.1 2.9 18.4 14.7 34.5 19.2 17.9 36.2 54.1 8.3 27.2

m/z (measured)

m/z (calculated)

253.0972 255.1121 560.1800 560.1812 558.1664 558.1664 416.1271 416.1269 414.1121 398.1156 396.1032

253.0971 255.1128 560.1810 558.1680 416.1275 414.1118 398.1169 396.1023

Error (ppm) 0.4 -2.7 -1.8 0.3 -2.8 -2.8 -1.0 -1.4 0.4 -3.2 2.3

In summary, we demonstrated herein that glutathionyl radicals react promptly with CBZ, with no need for bioactivation. The occurrence of this reaction implies the production of reactive oxygen species (ROS) that can cause oxidative stress and its associated oxidative damage/toxicity. Taking into consideration the key role of ROS in the formation of thiyl radicals, this non-enzymatic pathway is auto-induced. We have also shown that O2 supply contributes very significantly to the extent of CBZ consumption and, therefore, to the CBZ-induced production of ROS and CBZ-derived adducts. GSH conjugation is, in general, a detoxification mechanism of xenobiotics. Actually, the catabolic products of GSH conjugates, mercapturates, are usually excreted in urine due to their hydrophilicity. Therefore, hyperoxia/oxidative stress conditions that favour the non-enzymatic pathway demonstrated in this study are expected to decrease CBZ bioavailability. Additionally, under radical or O 2 overload, this pathway can also be expected to play a role in the onset of CBZ-induced adverse events. Namely, a similar effect to what is observed with bleomicyn [54] can be anticipated for CBZ-induced pulmonary toxicity. This antineoplastic agent induces pulmonary injury, attributed in part to disruption of the thiol redox status in the lungs (lung epithelial cells), that is increased with the administration of O 2. Coherently, a clinical case of CBZ-induced interstitial pneumonitis was reported in a 19-year old lung transplant recipient, which may, at least in part, be explained on the basis of the O2 supplementation [55]. In this context, it is noteworthy that CBZ therapy has been associated with depleted activities of superoxide dismutase and glutathione peroxidase in erythrocytes and with increased levels of a disulfide GSH-hemoglobin adduct [56], which is consistent with systemic disturbance of the thiol/disulfide redox balance. The reaction of CBZ with protein thiyl radicals [57] may thus represent one additional, and previously unrecognized, pathway to the onset of CBZ-induced 17

adverse events. For instance, this mechanism is compatible with the reported identification of a covalent adduct, assumed to be formed upon reaction of microsomally-generated CBZ-EP with the Cys47 residue of histidine-tagged glutathione-S-transferase pi [36]. Taking into consideration that this residue is a site for S-glutathionylation [58], the formation of a thiyl radical at this position is anticipated. Such radical could plausibly undergo addition to CBZ, thereby leading to the formation of adducts structurally identical to those expected from reaction with CBZ-EP. Moreover, while CBZ did not react with N-based nucleophiles under our experimental conditions, the formation of cationic species such as 25 (Scheme 3) upon reaction with glutathionyl or hydroxyl radical s, is plausible in vivo. Once formed, reaction of 25 with nucleophiles, namely the nucleophilic residues of proteins (e.g., lysines, cysteines, histidines), can be expected. The in vivo formation of HSA histidine adducts presumed to derive from CBZ-EP, recently reported by Yip et al. [36], could also be explained on the basis of histidine reaction with the OH-derived cationic species 25. It should be emphasized that the dysregulation of antioxidant enzyme systems [56] and the increased oxidative stress [59] associated with CBZ use are consistent with the in vivo occurrence of the radicalbased mechanisms demonstrated in this work.

4. Conclusions The formation of multiple products by direct reaction of glutathionyl, N-acetylcysteinyl and hydroxyl radicals with the widely used antiepileptic drug CBZ was demonstrated herein for the first time. Interestingly, the same products formed upon ring-opening of the major Phase I CBZ metabolite, CBZ-EP, were also obtained in the reaction of CBZ with NAC and GSH. The product profile is complex and compatible with radical-mediated mechanisms and we have shown that O2 supply contributes very significantly to the extent of CBZ consumption and, therefore, to the production of free radicals and CBZderived adducts. The involvement of a radical-based mechanism in the reaction of CBZ with GSH and NAC is supported by several lines of evidence: 1) a mixture of anti and syn products (11 and 12) are formed with CBZ, which contrasts with the selective formation of the anti products upon nucleophilic ring-opening of CBZ-EP by the same thiols; 2) the electrophilic CBZ-EP reacts with amines and thiols; however CBZ reacts

18

solely with thiols and this reaction is more extensive under an O2 stream, i.e., under experimental conditions known to generate thiyl radicals; 3) the identification of TOCO products (23 and 24), represents a strong evidence for the involvement of the carboncentred radical intermediates 15, as it can only be explained on basis of addition of thiyl radicals to the double bond of CBZ; 4) multiple products similar to the ones reported to arise from CBZ degradation under Fenton-like conditions were identified, which demonstrates not only the formation of the hydroxyl radical under the tested conditions but also the CBZ reactivity towards radicals; and 5) the same product profile was obtained when NAC was reacted with CBZ in the presence of the free radical initiator tert-butyl hydroperoxide. The identification of CBZ-derived products and adducts in the livers of CBZ-treated rats, with retention times and mass spectra matching those obtained in vitro, attested the occurrence of these non-enzymatic pathways in vivo. Taking into consideration the susceptibility of CBZ to react with biologically relevant radical species (e.g., hydroxyl and thiyl radicals), the effects of these nonenzymatic reactions in CBZ bioavailability and/or their role in the onset adverse events is worth investigating. Actually, our results suggest that, in addition to CBZ bioactivation, non-enzymatic radical-based reactions may also be key players in CBZ biotransformation, especially under oxidative stress and hyperoxia conditions. Taken together, the results reported herein demonstrate that the search for causal relationships between the formation of non-enzymatically-driven CBZ products and the occurrence of CBZ-induced adverse events in human patients is worth pursuing further, aiming the translation of basic mechanistic findings into a clinical context, that may ultimately lead to a safer CBZ prescription.

Funding This work was supported in part by Fundação para a Ciência e a Tecnologia (FCT),

Portugal,

through

research

grants

UID/QUI/00100/2013,

RECI/QEQ-

MED/0330/2012, and IF/01091/2013/CP1163/CT0001. ILM and CC also thank FCT for doctoral fellowships (SFRH/BD/80690/2010 and SFRH/BD/102846/2014, respectively). AMMA would like to acknowledge FCT, “Programa Operacional Potencial Humano” and the European Social Fund for the IF Program (IF/01091/2013). We also acknowledge the financial support from FCT and Portugal 2020 to the RNEM (LISBOA01-0145-FEDER-402-022125) IST Node (MS facility). JN thanks Colégio de Química, Ulisboa, for fellowship 16/BAD/2017. Aknowledgements 19

We thank Dr. Nádia Grilo, CEDOC, Chronic Diseases Research Centre, NOVA Medical School, Lisboa, Portugal, for the animal procurement and handling.

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Figure 1: Superposition of the HPLC-DAD chromatograms, monitored at 254 nm, of a reaction mixture containing CBZ-EP and CBZ, in a 2:1 molar ratio, and 9.2 eq of NAC in 50 mM phosphate buffer pH 7.5/ THF (4:1) at 37 ºC, recorded after 1 (black) and 72 h (orange) incubation. The elution conditions are outlined in the Experimental section. Figure 2: LC-MS analysis of a mixture containing CBZ-EP and a 50-fold molar excess of NAC: (A1) total ion chromatogram; (A2) extracted ion chromatogram at m/z 414; (A3) extracted ion chromatogram at m/z 416, corresponding to the protonated molecule of the anti adduct 12a. LC-MS analysis of a mixture containing CBZ and a 50fold molar excess of NAC: (B1) total ion chromatogram; (B2) extracted ion chromatogram at m/z 414, corresponding to the protonated molecule of adduct 13; (B3) extracted ion chromatogram at m/z 416, corresponding to the protonated molecules of the syn and anti adducts, 12b and 12a, respectively. Also shown are the tandem mass spectra of each species. Figure 3: Isotopic pattern obtained by LC-HRMS analysis of the anti adducts 11a and adduct 14, following reaction of CBZ with GSH in: (A) water with no atmospheric control; (B) H218O with no atmospheric control; (C) H218O under O2; and (D) H218O under N2.

24

Scheme 1: Carbamazepine (CBZ), its major metabolic pathways and potentially reactive metabolites. Also shown is one of the diastereomers of the trans adduct 11a, reported to be formed in vitro. Scheme

2:

Proposed

fragmentation

mechanisms

upon

tandem

mass

spectrometry of adducts: A. 12a and 12b; and B. 13. Scheme

3:

Proposed

radical-based

mechanisms

for

the

reaction

of

carbamazepine (CBZ) with glutathione (GSH) and N-acetyl-L-cysteine (NAC). Highlighted in red are the reaction products identified by LC-MS and LC-HRMS. TOCO, thiol-olefin cooxidation process.

25

3

C

C

.0

CBZ-

BZ

BZ

mAU

EP CBZ-

1 .5

18

23

EP

0

10

20

time (min)

30

40

1h

72h

Fig. 1.

26

Fig. 2.

27

Fig. 3.

28

Scheme 1.

29

A

B

Scheme 2 . 30

Scheme 3.

31

Highlights

    

CBZ reacts with biologically relevant thiols with no need for bioactivation The product profile is complex and consistent with radical-mediated mechanisms The reaction is more extensive in the presence of oxygen Products compatible with radical-mediated mechanisms were identified in liver extracts from CBZ-treated rats The concomitant generation of reactive oxygen species can have a role on CBZinduced adverse effects

32