Article pubs.acs.org/crt
Cytochrome P450 Mediated Bioactivation of Saracatinib Jiaming Chen,† Ying Peng,*,† and Jiang Zheng*,‡,§ †
School of Pharmacy, ‡Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, P.O. Box 21, 103 Wenhua Road, Shenyang, Liaoning 110016, P. R. China § Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou 550004, P. R. China S Supporting Information *
ABSTRACT: Saracatinib is a highly selective Src kinase inhibitor against all Src kinase family members and has demonstrated anticancer effects in preclinical models. Unfortunately, it has shown multiple adverse effects during its clinical trials, along with time-dependent inhibition of P450 enzymes. The major objective of this study was to identify reactive metabolites of saracatinib in vitro and in vivo. Four oxidative metabolites (M1−M4) were detected in rat and human liver microsomal incubation systems after exposure to saracatinib. An ortho-quinone-derived reactive metabolite existing as a GSH conjugate (M5) was found in microsomes fortified with GSH as a trapping agent. The formation of the metabolites detected was NADPH dependent. Metabolites M2−M4 were also observed in bile and urine of rats given saracatinib, and M5 was only detected in bile. Inhibition and recombinant P450 enzyme incubation studies demonstrated that P450 3A4 was the primary enzyme responsible for the metabolic activation of saracatinib. The metabolism study facilitates the understanding of correlation between saracatinib-induced hepatotoxicity and bioactivation.
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INTRODUCTION Saracatinib (AZD0530), N-(5-chloro-1,3-benzodioxol-4-yl)-7[2-(4-methylpiperazin-1-yl)ethoxy]-5-(oxan-4-yloxy) quinazolin-4-amine, is an orally bioavailable and highly selective Src kinase inhibitor with potency against all Src kinase family members.1 Src kinase is a nonreceptor tyrosine kinase that is downstream of a number of growth factor receptors involved in the invasion, migration, proliferation, and survival of cells.2 Saracatinib is currently being tested in a number of Phase II clinical trials for the treatment of different solid tumors, usually advanced and metastatic, including breast, prostate, ovarian, endometrial, skin (melanoma), pancreatic, gastric, colorectal, head and neck, nonsmall-cell lung cancer, and osteosarcoma, alone or in combination with conventional chemotherapeutic agents.3,4 Additionally, saracatinib is in clinical trials for the treatment of Alzheimer’s disease.5 Adverse events were found in patients after treatment, including increased aspartate aminotransferase, alanine transaminase levels, along with severe pulmonary toxicity.6,7 Saracatinib was also reported to induce time-dependent P450 enzyme inhibition.8 Saracatinib is a 1,3-benzodioxole group-containing compound. Many compounds with 1,3-benzodioxole functional groups have been reported to induce P450-dependent toxicities and P450 inactivation. The P450-mediated metabolic activation process is generally initiated by demethylenation in those 1,3benzodioxole moieties, which leads to ring-opening and yields catechols.9 Catechols are air-labile and readily oxidized to orthoquinones.10 Quinones are highly reactive compounds that exert cytotoxicity through binding with nucleophiles of macro© 2016 American Chemical Society
molecules, such as protein and nucleic acids, and producing reactive oxygen species.11,12 The toxic effects elicited by these 1,3-benzodioxole-containing compounds are thought to be attributed to their ortho-quinone metabolites. For example, sitaxentan induced hepatotoxicity in susceptible individuals was proposed to be associated with its ortho-quinone metabolites.13,14 This led us to hypothesize that saracatinib is metabolized to an ortho-quinone, an electrophilic species, which may play an important role in the reported adverse effects of saracatinib. The objectives of the present study were to characterize reactive metabolites of saracatinib, to identify the essential cytochrome P450 enzymes responsible for the bioactivation of saracatinib, and to elucidate the bioactivation pathways of saracatinib.
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EXPERIMENTAL PROCEDURES
Chemicals and Materials. Saracatinib with purity of 98% was obtained from Dalian Meilun Biotech Co., Ltd. (Dalian, China). Glutathione (GSH), S-hexylglutathione, α-naphthoflavone, sulfaphenazole, ticlopidine, quinidine, disulfiram, methoxsalen, pilocarpine, ketoconazole, and NADPH were purchased from Sigma-Aldrich (St. Louis, MO). Human liver microsomes and recombinant human P450 enzymes were purchased from BD Gentest (Woburn, MA). Rat liver microsomes (Sprague−Dawley, male) were prepared in our laboratory according to a previously published method.15 All organic solvents were from Fisher Scientific (Springfield, NJ). All reagents and solvents were of either analytical or high-performance LC grade. Received: July 12, 2016 Published: October 21, 2016 1835
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Chemical Research in Toxicology Microsomal Incubations. Saracatinib (100 μM) and human or rat liver microsomes (1.0 mg protein/mL) were mixed in potassium phosphate buffer (pH 7.4), followed by the addition of GSH (10 mM) as a trapping agent. The total volume of the incubation mixtures was 250 μL. The reactions were started by the addition of NADPH (1.0 mM). The control samples without NADPH were included. After 1 h of incubation at 37 °C, the reactions were quenched with an equal volume of ice-cold acetonitrile. The reaction mixtures were vortexmixed and centrifuged at 16,000g for 10 min to remove protein, and the supernatants were analyzed by an LC-MS/MS system. All incubations were carried out in duplicate. Recombinant Human P450 Incubations. Incubations were carried out under the conditions described above, except that microsomes were replaced by individual human recombinant P450 enzymes (20 pmol), including P450s 1A2, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. The incubation time was reduced to 30 min. Reactions were terminated by mixing with an equal volume of ice-cold acetonitrile which contained S-hexylglutathione (10 ng/mL) as the internal standard. The resulting samples were vortexed for 2 min and then centrifuged at 16,000g for 10 min. The supernatants (10 μL) were injected into the LC-MS/MS for analysis. The amount of M5 was determined by peak area integration compared to a standard curve. The rates of metabolite formation were acquired by the amount of M5 obtained in the experiment group compared to the control group. A total normalized rate method was applied. The rates of metabolite formed in individual incubations with recombinant P450 enzymes were multiplied by the mean specific content of the corresponding P450 enzymes in human liver microsomes to obtain the normalized reaction rates of each enzyme.16 Metabolizing Enzyme Inhibition Studies. Conditions were equivalent to those of the microsomal incubations but with the addition of selective P450 inhibitors [α-naphthoflavone (1.0 μM for P450 1A2), sulfaphenazole (20 μM for P450 2C9), ticlopidine (100 μM for P450s 2B6 and 2C19), quinidine (5.0 μM for P450 2D6), disulfiram (100 μM for P450 2E1), methoxsalen (20 μM for P450s 2A13 and 2A6), pilocarpine (100 μM for P450 2A1), and ketoconazole (1.0 μM for P450 3A)] to the mixtures. Inhibitor concentrations applied were referred to the FDA Guidance for Industry on Drug Interaction Studies.17 The inhibitors were individually dissolved in dimethyl sulfoxide, and the total volume applied in the incubations did not exceed 1% of the total incubation volume.18 The microsomal reactions were initiated by the addition of NADPH. After 30 min of incubation at 37 °C, the reactions were terminated by the addition of an equal volume of ice-cold acetonitrile containing internal standard S-hexylglutathione (10 ng/mL). The mixtures were processed for sample preparation as described above and subjected to LC-MS/MS for analysis. Incubations were conducted in triplicate for each inhibitor as well as for a no inhibitor positive control, and P450 activity was expressed as the percentage of the control activity. Animal Experiments. All animal manipulations met the requirements approved by the Ethics Review Committee for Animal Experimentation of Shenyang Pharmaceutical University (Shenyang, China). Male Sprague−Dawley rats (200 ± 20 g) were obtained from the Animal Center of Shenyang Pharmaceutical University. All rats were kept in a controlled environment (temperature of 25 °C and 12 h dark/light cycle) and maintained on standard rat chow. Before administration, the experimental animals were deprived of food for 12 h. The animals were anesthetized with 10% chloral hydrate (3.0 mL/ kg), and their bile ducts were cannulated with PE-10 tubing. Saracatinib dissolved in corn oil was administered intraperitoneally at 50 mg/kg, and bile was collected for 2 h following dosing. Control bile was also collected. The other group of rats administered with the same dose of saracatinib was placed in metabolism cages. Urine samples were collected during the time periods of 0−12 and 12−24 h after the procedure. Control urine was collected prior to the treatment. During the experiment, the rats were allowed free access to food and water. Sample Preparation for LC-MS/MS Analysis. Triple volumes of acetonitrile were added to the bile or urine samples. After
centrifugation at 16,000g for 10 min, the supernatants were harvested and concentrated to dryness under a stream of nitrogen gas. The resulting concentrates were reconstituted with 100 μL of 5% acetonitrile in water and then centrifuged at 16,000g for 10 min. A 3.0 μL aliquot of the reconstituted solution was analyzed by an LCMS/MS system. Chemical Synthesis of GSH Conjugates. Saracatinib (5.0 mg, 9.24 mmol) was dissolved in 48% HBr aqueous solution (2.5 mL, 22.2 mmol). The reaction mixtures were refluxed for 14 h at 80 °C. After evaporated to dryness under a stream of nitrogen, the resulting solid was dissolved in chloroform and mixed with 2,3-dichloro-5,6-dicyano1,4-benzoquinone (DDQ) (8.5 mg, 37.0 mmol). The mixtures were stirred for 20 min at room temperature, followed by evaporation of the organic solvent under vacuum. The crude products were dissolved in 2.0 mL of tetrahydrofuran and mixed with GSH (5.7 mg, 18.5 mmol) aqueous solution (2.0 mL). The reaction mixtures were further stirred at room temperature for 5 h and then centrifuged at 16,000g for 10 min. The supernatants (5.0 μL) were subjected to LC-MS/MS for analysis. Enzyme Inactivation Study. Incubation mixtures contained saracatinib or M1 (33 μM), human liver microsomes (1.0 mg protein/mL) in potassium phosphate buffer (pH 7.4). Testosterone (200 μM) was used as a marker reaction for P450 3A activity. The total volume of the incubation mixtures was 200 μL. The reactions were initiated by the addition of NADPH (1.0 mM). Control samples without substrate were included. After incubation at 37 °C for 10 min, the reactions were stopped by adding equal volumes of ice-cold acetonitrile containing S-hexylglutathione (10 ng/mL) as the internal standard, followed by centrifugation at 16,000g for 10 min. The supernatants (10 μL) were then collected and analyzed by LC-MS/MS as described below. LC-MS/MS Method. All samples were analyzed on an AB SCIEX Instruments 5500 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) interfaced online with a 1260 infinity system (Agilent Technologies, Santa Clara, CA). Chromatographic separations were performed on an Accuore C18 column (4.6 × 150 mm, 5 μm; Thermo Fisher, Pittsburgh, PA). Column temperature was maintained at 25 °C, and the flow rate was kept at 0.8 mL/min. The mobile phase consisted of acetonitrile with 0.1% formic acid (solvent A) and 0.1% formic acid in water (solvent B). The mobile phase was initially composed of 5% solvent A and held for 2 min, followed by 5%−60% A for 6 min, 60%−100% A for 4 min, and 100%−5% A for 2 min, then maintained at 5% A for 1 min. Samples were analyzed by multiple-reaction monitoring (MRM) scanning in positive ion mode. The operating parameters were optimized and set at the following values: ion spray voltage (IS) and entrance potential (EP) were 5,500 and 10 V, and ion source temperature (TEM) was at 650 °C. Curtain gas, gas 1, and gas 2 were 35, 50, and 50 psi, respectively. The characteristics of precursor/product ion pairs (corresponding to declustering potential DP, collision energy CE, and collision cell exit potential CXP) were m/z 542 → 127 (180, 13, 10) for saracatinib; m/z 530 → 127 (110, 40, 10), 528 → 113 (110, 40, 10), 558 → 127 (110, 40, 10), and 558 → 332 (110, 40, 10) for oxidative metabolites; m/z 835 → 706 (110, 40, 10) for GSH conjugates, and m/z 392 → 246 (86, 24, 5) for S-hexylglutathione (internal standard). Furthermore, AB SCIEX Instruments 4000 Q-Trap (Applied Biosystems, Foster City, CA, USA) interfaced online with an ekspert ultra LC 100 system (Applied Biosystems) was used to analyze the metabolites/products. The information-dependent acquisition (IDA) method was employed to trigger the enhanced product ion (EPI) scans by analyzing multiple reaction monitoring (MRM). IDA was used to trigger acquisition of EPI spectra for ions exceeding 5,000 cps, with exclusion of former target ions after three occurrences for 10 s. The EPI scan was run in positive mode at a scan range for product ions from m/z 100 to 900, respectively. The collision energy (CE) was set at 50 eV with a spread of 15 eV. All data were processed using the AB SCIEX Analyst 1.6.2 software (Applied Biosystems). Metabolite analysis was also conducted on an Agilent 1200 Series Rapid Resolution LC system equipped with a hybrid quadrupole-time1836
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Chemical Research in Toxicology of-flight (Q-TOF) mass system (micro Q-TOF; Bruker Corporation, Billerica, MA). The mass spectrum data were acquired in positive ion mode. The mass spectrometric parameters were optimized as follows: end plate offset (2,500 V), capillary voltage (−4,500 V), nebulizer gas pressure (1.2 bar), the dry gas flow rate (8.0 L/min), and temperature (180 °C). The spectra were acquired at 2 s per spectrum in the range of m/z 50 to 1,500. LC conditions were similar to those described above. The data were analyzed by Bruker Daltonics Data Analysis 3.4 software.
less than that of the parent compound, indicating that the 1,3benzodioxole ring of saracatinib underwent a process of demethylenation. M2, which eluted at 8.32 min, was detected by monitoring precursor/product ion pair m/z 528/113. The value of its [M + H]+ (m/z 528) was 14 Da lower than that of saracatinib (Figure 3B), suggesting that saracatinib experienced a loss of methyl group during the microsomal incubations. The fragment ions at m/z 113 (C6H13N2), 332 (C15H11ClN3O4), and 416 (C20H19ClN3O5) were associated with the fragmentation of parent saracatinib (Figure 3D). M3/M4 (Rt = 7.91 and 8.58 min) displayed a protonated molecule at m/z 558, which was proposed to be consistent with the introduction of a hydroxyl group to saracatinib (Figure 3C). Product ions m/z 123, 348, and 432 observed in the mass spectrum of M3 were possibly responsible for C7H2O2, C15H11ClN3O5, and C20H19ClN3O6, respectively (Figure 3E). This led us to propose that the hydroxylation occurred at the pyrimidine/phenyl ring. Fragment ions m/z 143, 332, and 416 of M4 may be associated with C7H15N2O, C15H11ClN3O4, and C 20H 19 ClN 3 O5 , respectively (Figure 3F), implying the introduction of a hydroxyl group on the piperazine ring. The MS/MS spectrum of M5 was obtained by MRM-EPI scanning (ion transition m/z 835 → 706) with the retention time at 7.46 min (Figures 4B and S1), and the spectrum showed the indicative characteristic fragment ions associated with the cleavage of GSH moiety (Figure 4D). The product ions at m/z 562 and 706 were derived from the cleavage of the C−S bond of the GSH moiety (−273 Da) and the neutral loss (NL) of the γ-glutamyl portion (−129 Da) from m/z 835 (Figure 4D), respectively. The fragment ion at m/z 127 was considered to originate from the saracatinib portion. It is worth mentioning that the same technique was applied to screen other potential metabolites, and we failed to find any others. Chemical synthesis was performed to further characterize metabolites M1 and M5 detected in microsomal incubations. Demethylenation was achieved by refluxing saracatinib in 48% HBr aqueous solution. The resulting product showed the same chromatographic and mass spectrometric identities as M1 generated in the microsomal incubations (Figure 2C and E). The demethylenated product was sequentially oxidized with DDQ, followed by reaction with GSH. The GSH conjugate formed in the reaction was found to correspond to M5 observed in the microsomal incubations (Figure 4C and E), based on their chromatographic and mass spectrometric identities. Synthetic M1 was also analyzed by LC/Q-TOF mass spectrometry. The protonated molecular ion was m/z 530.2179 (Table 1) with a clear 3:1 ratio chlorine isotope pattern (Figure 6), consisting of the molecular weight of the elemental formula C26H33Cl1N5O5. The accurately measured mass in the high-resolution MS system was in line with the corresponding theoretical mass within 5 ppm based on the predicted formula (Table 1). Furthermore, synthetic M1 was purified and incubated with human liver microsomes containing GSH and NADPH. The produced GSH conjugate showed the same chromatographic and mass spectrometric identities as that generated in microsomal incubations with saracatinib (Figure 4F). Unfortunately, we were unable to obtain enough amounts of the metabolites/products for nuclear magnetic resonance (NMR) characterization, due to the high instability of the demethylenated product (M1). Biliary and Urinary Metabolites of Saracatinib. To investigate the bioactivation of saracatinib in vivo, bile and urine
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RESULTS Mass Spectrometric Behavior of Saracatinib. To facilitate metabolite identification, we started with examining the chromatographic and mass spectrometric properties of saracatinib. The parent compound eluted at 8.26 min under the employed chromatographic conditions (Figure 1A), and the
Figure 1. Chromatographic and mass spectrometric behavior of saracatinib. (A) Extracted ion (m/z 542 → 127) chromatogram obtained from LC-MS/MS analysis. (B) MS/MS spectrum of saracatinib.
protonated molecule [M + H]+ was m/z 542 in positive ion mode. The mass spectrum showed major fragment ions at m/z 127 (C7H16N2) and 332 (C15H11Cl1N3O4) formed by the cleavage of the alkyl C−O bond (Figure 1B). In Vitro Metabolic Activation of Saracatinib. Saracatinib was incubated in human or rat liver microsomes supplemented with NADPH and GSH as a trapping agent. Four oxidative metabolites (M1−M4) and a GSH conjugate (M5) were detected in both human and rat liver microsomes. No such metabolites were observed in the control group (Figures 2A and 3A), which indicates that the formation of these metabolites was NADPH-dependent and that P450 enzymes played an important role in the formation of these metabolites. M1 with a retention time of 7.89 min was detected by acquiring precursor/product ion pair m/z 530 → 127 in positive mode (Figure 2B). The tandem mass spectrometry (MS/MS) spectrum of M1 was obtained by MRM-EPI scanning displaying three indicative characteristic fragment ions at m/z 127 (C7H16N2), 320 (C14H11ClN3O4), and 416 (C20H19ClN3O5) (Figure 2E). The m/z value of M1 was 12 Da 1837
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Figure 2. Characterization of oxidative metabolite M1. Extracted ion (m/z 530 → 127) chromatograms obtained from LC-MS/MS analysis of human liver microsome incubations containing saracatinib and GSH in the absence (A) or presence (B) of NADPH. (C) Extracted ion (m/z 530 → 127) chromatogram of synthetic M1. MS/MS spectra of M1 generated in human liver microsome incubations (D) and chemical synthesis (E).
Figure 3. Characterization of metabolites M2−M4. Extracted ion chromatogram of M2−M4 obtained from human liver microsome incubations in the absence of NADPH (A). Extracted ion (m/z 528 → 113) chromatogram of M2 obtained from incubations in the presence of NADPH (B). Extracted ion (m/z 558 → 127, m/z 558 → 332) chromatogram of M3/M4 obtained from microsomal incubations (C). (D) MS/MS spectra of M2 and (E/F) M3/M4 generated from microsomal incubations.
samples were collected in saracatinib-exposed rats and analyzed by LC-MS/MS. Metabolites M2−M4 were detected in the bile and urine, but no such metabolites were observed in the control group. Unfortunately, M5 was only found in the bile of rats. The metabolites found in vivo studies showed the same
chromatographic (Figure 5A−E) and mass spectrometric behaviors (Figure 5F−I) as those formed in the microsomal incubations. However, we failed to detect M1 in the bile and urine samples. 1838
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Figure 4. Characterization of GSH conjugate M5. Extracted ion (m/z 835 → 706) chromatogram obtained from LC-MS/MS analysis of human liver microsome incubations containing saracatinib and GSH in the absence (A) or presence (B) of NADPH. (C) Extracted ion (m/z 835 → 706) chromatogram of synthetic M5. MS/MS spectra of M5 obtained from human liver microsome incubations (D) and chemical synthesis (E). (F) Extracted ion (m/z 835 → 706) chromatograms obtained from LC-MS/MS analysis of microsomal incubations of synthetic M1 with human liver microsomes, NADPH, and GSH. Neutral loss spectra of M5 obtained from human liver microsome incubations (G) and chemical synthesis (H).
Table 1. Mass Spectrometric Profiling Data Obtained from LC-Q-TOF MS Analysis of Synthetic M1 [M + H]+
[M + H]+
error
compound
formula
calulated
detected
ppm
mDa
sigma
M1
C26H33ClN5O5
530.2165
530.2179
1.68
1.43
0.009
Figure 5. Extracted ion (m/z 528 → 113, M2) chromatograms obtained from bile (A) and urine (B) of rats given saracatinib. Extracted ion (m/z 558 → 127, m/z 558 → 332, M3 and M4) chromatograms obtained from bile (C) and urine (D) samples. Extracted ion (m/z 835 → 706, M5) chromatogram obtained from bile (E) samples of rats treated with saracatinib. (F) MS/MS spectra of M2 and G/H M3/M4 and (I) M5 generated from in vivo experiments.
P450 Enzymes Involved in Saracatinib Biotransformation. Recombinant human P450 incubation experiments were performed to determine which P450 enzymes were responsible for the metabolism of saracatinib. Eight P450 enzymes were individually incubated with saracatinib in the
presence of NADPH and GSH, followed by monitoring the formation of M5. P450 3A4 showed the most catalytic activity, followed by P450s 2D6 and 1A2 (Figure 7A). A selection of P450 inhibitors, including α-naphthoflavone (P450 1A2 inhibitor), sulfaphenazole (P450 2C9 inhibitor), 1839
DOI: 10.1021/acs.chemrestox.6b00242 Chem. Res. Toxicol. 2016, 29, 1835−1842
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Chemical Research in Toxicology
Figure 6. High-resolution mass spectrum of synthetic M1.
P450 3A4 was the primary enzyme involved in the oxidative metabolism of saracatinib. P450 3A4 Inactivation. To determine whether orthoquinone is responsible for P450 time-dependent inhibition, we carried out inactivation experiments in human liver microsomes with saracatinib or M1 as the substrate. The remaining P450 3A4 activity was monitored by measuring the amount of 6βhydroxyl-testosterone produced. Both saracatinib and M1 showed an inactivation effect to P450 3A4 (about 40.9% and 98.2%), suggesting that ortho-quinone was involved in the P450 inhibition.
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DISCUSSION Several adverse events were found in patients given saracatinib, including pulmonary toxicity and potential liver injury. The mechanisms of toxicities induced by saracatinib remain unknown. The present study was designed and performed to elucidate the metabolism pathways of saracatinib to better understand the mechanisms of the toxic action. As an initial step, we performed microsomal incubations with saracatinib supplemented with GSH and NADPH. Four oxidative metabolites (M1−M4) and a GSH conjugate (M5) were detected by LC-MS/MS, and the formation of these metabolites was NADPH-dependent. On the basis of the obtained mass spectrometric data, we proposed the routes of saracatinib biotransformation including O-dealkylation (M1), N-dealkylation (M2), and hydroxylation (M3/M4). The identified putative activation processes of saracatinib are shown in Scheme 1. M5 showed the characteristic neutral loss of 129 Da (γ-glutamyl portion) derived from the GSH moiety (Figure 4G) and the characteristic precursor ion at m/z 127 resulting from the saracatinib moiety. This indicates that M5 was a saracatinib-GSH conjugate. DDQ-mediated oxidation of M1 followed by reaction with GSH produced M5 (Figure 4F). This further supports that M1 was the metabolite resulting from oxidative demethylenation. As expected, the treatment of M2−M4 by the same procedures failed to offer any GSH conjugates since it appears that no immediate electrophilic species could be produced by a single step of oxidation of M2− M4 with DDQ. Chemical synthesis was executed to verify the metabolite identification work. The resulting products showed the same chromatographic and mass spectrometric identities as M1 and M5 detected in the microsomal incubations. The synthetic work further characterized M1 and M5. Four metabolites, including M2−M5, were detected in bile of animals given saracatinib as observed in microsomal incubations. Additionally, metabolites M2−M4 were also observed in
Figure 7. Formation of M5 in incubations of saracatinib with recombinant human P450 enzymes. (A) Rates of M5 formation in incubations of saracatinib with individual recombinant P450 enzymes after normalization based on the relative content of the corresponding P450 enzyme in human liver microsomes. (B) Effects of selective P450 inhibitors on the formation of M5. Saracatinib was incubated with human liver microsomes, GSH, and NADPH in the presence of individual P450 enzyme inhibitors. Data represent the mean ± SD (n = 3). *p < 0.05 and **p < 0.01 were considered significantly different.
ticlopidine (P450 2B6 and P450 2C19 inhibitor), quinidine (P450 2D6 inhibitor), disulfiram (P450 2E1 inhibitor), methoxsalen (P450 2A13 and P450 2A6 inhibitor), pilocarpine (P450 2A1 inhibitor), and ketoconazole (P450 3A inhibitor) were used to probe the contributions of P450s in saracatinib bioactivation in human liver microsomes. Incubation with ketoconazole elicited a significant inhibitory effect (86.2%) on the formation of M5 (p < 0.01), followed by α-naphthoflavone (50.1%), pilocarpine (36.4%), quinidine (35.0%), ticlopidine (20.7%), sulfaphenazole (13.6%), methoxsalen (4.9%), and disulfiram (1.8%) (Figure 7B). In addition, a similar observation was made in rat liver microsome incubations, indicating that there was no species difference in the bioactivation of saracatinib. The above results verified that 1840
DOI: 10.1021/acs.chemrestox.6b00242 Chem. Res. Toxicol. 2016, 29, 1835−1842
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Chemical Research in Toxicology Scheme 1. Proposed Metabolism Pathways of Saracatinib
responsible for the metabolic activation of saracatinib. This study enables us to better understand the mechanisms of saracatinib-induced toxicities and P450 enzyme inactivation.
urine. Interestingly, we failed to detect biliary and urinary M1, the demethylenation metabolite. This may be explained by the instant and spontaneous/enzymatic oxidation of M1 and sequential GSH conjugation to M5. The formation of the GSH conjugate in vitro and in vivo implies that the reactive metabolite might covalently bind to proteins or other biologic macromolecules. Protein modification could initiate the toxicities of saracatinib. P450 inactivation (irreversible inhibition) is generally triggered by a covalent modification of the P450 apoprotein and/or alkylation or arylation of the prosthetic heme moiety, or destruction of the prosthetic heme groups via the reactive intermediate complex.19,20 Filppula and co-workers speculated that the piperazine ring of saracatinib was responsible for the inactivation of P450 3A, following bioactivation.8 However, our study demonstrated that the inactivation of P450 3A possibly attributed to the 1,3-benzodioxole moiety rather than the piperazine ring. Enzyme inactivation experiments further verified that ortho-quinone played a role in P450 inhibition. Bioactivation studies with individual recombinant enzymes demonstrated that P450 3A4 was the major enzyme involved in the bioactivation of saracatinib, followed by P450 2D6 and P450 1A2 (Figure 7A). Furthermore, coincubation with ketoconazole in microsomes significantly attenuated the formation of the GSH conjugate (Figure 7B). α-Naphthoflavone, quinidine, and pilocarpine also elicited certain inhibitory effects, and no or minor inhibition was observed in the presence of the other P450 inhibitors applied. The individual recombinant enzyme and enzyme inhibition studies demonstrated that P450 3A4 dominated the bioactivation of saracatinib. Any factors which cause P450 3A4 induction and activation may accelerate the metabolic activation of saracatinib and possibly intensify its toxicities. In conclusion, P450-mediated O-demethylenation of saracatinib generated electrophilic intermediate ortho-quinone both in vitro and in vivo. P450 3A4 was the primary enzyme
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.6b00242. Extracted ion (m/z 835 → 706, M5) chromatogram obtained from LC-MS/MS analysis of human liver microsome incubations containing saracatinib (0.8 μM) and GSH (10 mM) in the presence of NADPH (1.0 mM) (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*(Y.P.) Tel: +86-24-23986361. Fax: +86-24-23986510. E-mail:
[email protected]. *(J.Z.) Tel: +86-24-23986361. Fax: +86-24-23986510. E-mail:
[email protected]. Funding
This work was supported in part by the National Natural Science Foundation of China (No. 81430086 and 81373471). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS GSH, glutathione; NADPH, β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt; LC-MS/MS, liquid chromatography−tandem mass spectrometry; DDQ, 2,3dichloro-5,6-dicyano-1,4-benzoquinone; MRM, multiple-reaction monitoring; IS, ion spray voltage; EP, entrance potential; CE, collision energy; DP, declustering potential; CXP, cell exit potential; IDA, information-dependent acquisition; EPI, 1841
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Article
Chemical Research in Toxicology
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enhanced product ion; NMR, nuclear magnetic resonance; NL, neutral loss
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DOI: 10.1021/acs.chemrestox.6b00242 Chem. Res. Toxicol. 2016, 29, 1835−1842
