Article pubs.acs.org/crt
In Vitro Bioactivation of a Selective Estrogen Receptor Modulator (2S,3R)‑(+)-3-(3-Hydroxyphenyl)-2-[4-(2-pyrrolidin-1ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (I) in Liver Microsomes: Formation of Adenine Adducts Ying Li, George A. Doss, Yan Li,† Qing Chen, Wei Tang,‡ and Zhoupeng Zhang* Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey 07065, United States
ABSTRACT: As part of our efforts to develop safer selective estrogen receptor modulators (SERMs), compound I {(2S,3R)(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)-phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol} was previously identified as a lead for further development. Subsequent studies showed that compound I is genotoxic in both in vitro Chinese hamster ovary (CHO) cells and in vivo mouse studies. To better understand the possible mechanisms for the observed genetoxicity effects, in vitro incubations of I with liver microsomes of human, monkey, and mouse in the presence of adenine were performed, which led to the detection of five adenine adducts. The formation of these adducts was NADPH-dependent, suggesting the involvement of oxidative bioactivation catalyzed by cytochrome P450 enzymes. The mechanism for the formation of the major adenine adduct (A1) involves the formation of a reactive ring-opened para-quinone intermediate. The formation of four other adenine adducts may involve the formation of a reactive epoxide or ortho-quinone intermediate. Furthermore, incubations of compound I with human hepatocytes showed dose-dependent DNA damages in Comet assays. All of the above suggest that some reactive metabolites of compound I, formed through bioactivation mechanisms, have a potential to interact with DNA molecules in vitro and in vivo. This may be one of the causes of the genotoxicity observed preclinically both in vitro and in vivo. This case study demonstrated an approach using in vitro DNA trapping assays for assessing the genotoxicity potential of drug candidates.
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like effects on bone tissue and the cardiovascular system.13,14 However, some SERM drugs, that is, tamoxifen and raloxifene, are bioactivated to form species, which react with protein or DNA molecules to form corresponding adducts in biological systems.15−22 Because reactive metabolites formed through bioactivation have been hypothetically associated with organ toxicity in some cases, there is a high possibility that the formation of hepatocellular carcinoma in rats and the increased clinical incidences of endometrial cancer in patients might be related to the bioactivation of tamoxifen.14,23 In an effort to develop a safer SERM drug, we previously identified (2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (compound I, Figure 1) as a lead compound with a low propensity to form reactive intermediates.24 It was demonstrated that compound I had a covalent protein binding value of 461 pmol equiv/mg protein in human liver microsomes. However, its covalent protein binding in human hepatocytes was significantly reduced to 48 pmol equiv/mg protein. In addition, the in vivo studies demonstrated that the protein binding of compound I
INTRODUCTION Bioactivation or metabolic activation of drugs or drug candidates is a biological process where drugs or drug candidates are metabolized to form reactive species, which have a potential to react with protein or DNA in biological systems.1,2 Even though the direct correlation between bioactivation and potential clinical toxicity is not always readily available, the efforts to try to better understand such a correlation have been a research subject in both academic institutions and the pharmaceutical industry for many years.3−11 The current biggest challenge is how to use the safety-related preclinical data to confidently predict the potential clinical risk to assist making a critical decision at drug discovery and development stages. Estrogens are a class of estrogen receptor agonists commonly used in hormone replacement therapy to alleviate symptoms and urogential atrophy in menopausal women.12 However, the potential risk to promote development of breast and endometrial tumors limits the long-term usage of estrogens in breast cancer patients.12 Selective estrogen receptor modulators (SERMs) are a class of nonsteroidal compounds that are targeted to antagonize the adverse effects of estrogens on uterine and breast tissues while producing beneficial estrogen© 2012 American Chemical Society
Received: June 4, 2012 Published: September 21, 2012 2368
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Chemical Research in Toxicology
Article
The collision energy ramp of 30−50 V was used for MS/MS analysis of targeted ions. A solution of aqueous leucine−enkephalin (200 ng/ mL in water−methanol 80:20) was used as a lock spray solution, and two ions of m/z 278.1141 and 556.2772 were selected as the lock masses. The liquid chromatography separations were performed on an Acquity UPLC system (Waters) consisting of a binary pump and an autosampler. For radiochromatographic analysis of a sample from incubation in recombinant human cytochrome P450 3A4 and for isolation of the adenine adduct A1 in monkey liver microsomes, a Finnigan LCQDeca XP Plus mass spectrometer (San Jose, CA) interfaced to a HPLC system consisting of two Shimadzu LC-10AD pumps (Kyoto, Japan), a Shimadzu SIL-10AD auto injector, and a Finnigan UV6000LP photodiode array detector was used. The LCQDeca XP Plus mass spectrometer employed ESI in the positive ion mode. The heated capillary temperature was set at 200 °C, the normalized collision energy was 40%, the sheath gas flow rate was 60 units, and the auxiliary gas flow rate was 20 units. The ion spray voltage, the capillary voltage, and the tube lens offset were adjusted to achieve maximum sensitivity using compound I. The collision gas was helium. For liquid chromatography−tandem mass spectrometry (LC/MS/ MS) analysis using QTof G2 mass spectrometer, samples (10 μL) were loaded onto an Acquity UPLC BEH C18 column (1.7 μm, 2.1 mm × 30 mm; Waters). The flow rate was set at 0.5 mL/min with a 1:5 split to the mass spectrometer ion source and a waste container, respectively. The mobile phase consisted of solvent A (100% water, 0.1% formic acid, v/v) and solvent B (100% acetonitrile, 0.1% formic acid, v/v). The UPLC runs were programmed by a linear increase from 5 to 90% of solvent B during a 10 min period. For LC/MS/MS analysis using Finnigan LCQDeca XP Plus mass spectrometer interfaced to a HPLC system consisting of two Shimadzu LC-10AD pumps, a Shimadzu SIL-10AD autoinjector for radiochromatographic analysis, a sample (50 μL) was loaded onto an Agilent Zorbax Rx-C8 column (4.6 mm × 250 mm, 5 μm, Wilmington, DE). The flow rate was set at 1 mL/min with a 1:5 split to the mass spectrometer ion source and a Packard flow scintillation analyzer (model 500TR, Boston, MA), respectively. The mobile phase consisted of solvent A (5 mM ammonium acetate in water− acetonitrile−acetic acid, 95:5:0.05, v/v/v) and solvent B (5 mM ammonium acetate in acetonitrile−water−acetic acid, 95:5:0.05, v/v/ v). The HPLC runs were programmed by a linear increase from 20 to 50% of solvent B during a 30 min period. The MS/MS spectra were recorded by collision-induced dissociation (CID) of MH+ species. Incubations of Compound I in Liver Microsomes or Human Cytochrome P450 3A4 in the Presence of DNA Bases. Liver microsomes of mouse, monkey, and human (1 mg protein/mL) or recombinant human P450 3A4 (250 pmol/mL) were suspended in potassium phosphate buffer (100 mM, pH 7.4) containing EDTA (1 mM) and MgCl2 (0.1 mM) in a total volume of 1 mL. Compound I in methanol was added to a final concentration of 50 μM, such that the concentration of methanol in the incubation mixture did not exceed 0.5%. Incubations were performed in the presence of individual DNA bases (adenine, guanine, thymine, or cytosine, 5 mM) and NADPH (1.2 mM) at 37 °C for 60 min. The reactions were quenched by adding 100 μL of a 10% TFA solution. The suspensions were centrifuged at 3000g for 10 min. The supernatants were loaded onto Oasis HLB extraction cartridges (60 mg, 3 mL), which were preconditioned with methanol and water. The pellets were extracted with 3 mL of methanol by sonicating for 10 min and vortexing for 10 min. After centrifugation at 3000g for 10 min, the methanolic supernatants were used to elute the above cartridges, followed by the addition of 2 mL of methanol. The eluted samples were evaporated to dryness under nitrogen in a Zymark TurboVap LV evaporator at room temperature. The dried samples were reconstituted in 300 μL of solvent B, and an aliquot was loaded onto an UPLC column for LC/ MS/MS analysis. A separate incubation of [3H]I with recombinant human P450 3A4 (250 pmol/mL) in the presence of adenine was performed to obtain a radiochromatographic profile. The final specific activity of [3H]I was 5 μCi/mg. The incubation and processing procedures were the same as described above in incubations with liver
Figure 1. Structures of compound I, five adenine adducts A1−A5, and the hydroquinone metabolite M12. Lowercase letters denote specific hydrogen atoms cited in the descriptions of NMR spectra.
was less than 10 pmol equiv/mg protein in rat liver and plasma. These low levels of in vivo protein binding might be explained by the fact that the major clearance pathway of compound I in rats is glucuronidation.24 All of these findings supported the selection of compound I as a lead compound for further development in the SERM program. Subsequent in vitro chromosomal aberration assay of compound I in Chinese hamster ovary (CHO) cells was positive for both structural aberrations and polyploidy (unpublished results). In addition, the in vivo micronucleus induction assay in mouse bone marrow was also positive (unpublished results). These new data suggest that compound I is genotoxic under the conditions of these assays. To better understand possible mechanisms for the observed in vitro and in vivo genotoxicity, we performed in vitro trapping studies of compound I using DNA bases, a Comet assay in human hepatocytes, as well as an assay to measure intracellular GSH/GSSG ratio in freshly isolated rat hepatocytes.
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MATERIALS AND METHODS
Materials. NADPH and menadione were purchased from SigmaAldrich Co. (St. Louis, MO). All other reagents and solvents were obtained from Fisher Scientific (Fair Lawn, NJ). Human liver samples were obtained from Pennsylvania Regional Tissue Bank (Exton, PA). Liver microsomes were prepared from individual livers by differential centrifugation, and aliquots of each preparation were pooled on the basis of equivalent protein concentrations.25,26 Microsomes from baculovirus-infected cells coexpressing human P450 3A4 and NADPHP450 oxidoreductase were prepared at Merck Research Laboratories.24 The synthesis of compound I and isolation of the hydroquinone metabolite M12 from incubations of compound I with recombinant cytochrome P450 3A4 were previously reported.24 Tritium-labeled compound I tracer ([3H]I) was synthesized at Merck Research Laboratories with a radiochemical purity of >98.5% in HPLC analysis. The specific activity of [3H]I was 29.0 mCi/mg. The Comet assay reagent kit was purchased from Trevigen Inc. (Gaithersburg, MD). Cryopreserved human hepatocytes from three male and two female donors (lot numbers 70, 78, 88, 91, and 95) were obtained from In Vitro Technologies (Baltimore, MD), and their testosterone 6βhydroxylation activity ranged from 18 to 105 pmol/min/106 cells. Instrumentation. Waters QTof Xevo G2 mass spectrometer (Waters, Milford, MA) was used to perform full scan mass spectrometry (MS) and MS/MS analysis of compound I and its metabolites. An electrospray ionization (ESI) source was used in the positive ion mode. The source temperature was set at 100 °C, the capillary voltage was set at 2.5 kV, the sample cone voltage was set at 25 V, and the desolvation temperature was set at 450 °C. The scan times were set at 0.2 s for full scan MS and 0.5 s for MS/MS analysis. 2369
dx.doi.org/10.1021/tx3002466 | Chem. Res. Toxicol. 2012, 25, 2368−2377
Chemical Research in Toxicology
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Figure 2. Extracted ion chromatograms of compound I incubated with liver microsomes of mouse, monkey, and human and human cytochrome P450 3A4 in the presence of adenine. Incubations were performed, as described in the Materials and Methods, for a period of 60 min. Ions were extracted at m/z 583.213 ± 0.010 Da. Five adenine adducts were labeled as A1−A5. Comet Assays. Cryoperserved human hepatocytes were thawed according to the manufacturer's instructions and were diluted to 1 × 106 live cells/mL with cell viability of >80% (based on the trypan blue exclusion test). Incubations were performed by suspending pooled hepatocytes in Krebs−biocarbonate buffer followed by addition of compound I in DMSO with a final DMSO concentration of 0.1% of total incubation volume. The final substrate concentrations in the suspension were 3.1, 6.2, 12.5, or 25 μM in a final volume of 1 mL (1 × 106 cells/mL) under CO2/O2 (95%/5%) at 37 °C for 90 min. Clofibric acid (2 mM) and DMSO (0.1%) were used as a positive control and a solvent control, respectively. Cells were harvested after centrifugation (3000 rpm for 5 min) and were then resuspended in ice-cold PBS buffer (1 × 106 cells/mL). Fifty microliters of the above suspension was mixed with 500 μL of low melting point agarose at 37 °C. Thirty microliters of the above agarose mixture was spread over a well on a 20-well CometSlide (Trevigen Inc.). The gels were allowed to set at 4 °C for 5 min, and the slides were placed into a prechilled lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Trizma base, and 1% Triton X-100) at 4 °C for 30 min and then in an alkaline solution (pH > 13) at room temperature for 20 min. Electrophoresis was performed in a prechilled alkaline electrophoresis solution (24 V, 300 mA for about 30 min). The slides were washed twice with distilled water and 70% of ethanol once and dried at room temperature for 20 min. To each well was added 50 μL of SYBR Green dye to stain nucleoids for 30 min. After the excess SYBR Green dye was removed, the slides were allowed to dry completely at room temperature. Stained nucleoids were viewed under a fluorescence microscope at excitation and emission wavelengths of 425 and 521 nm, respectively. Measurement of Intracellular GSH/GSSG Ratio in Freshly Isolated Rat Hepatocytes. Hepatocytes were freshly isolated by perfusion of a liver from a male Sprague−Dawley rat and were diluted with Krebs−bicarbonate buffer (pH 7.4) to 1 × 106 cells/mL. The cell viability (>85%) was analyzed by the trypan blue exclusion method. Stock solutions of menadione, compound I, and its hydroquinone metabolite M12 were added to rat hepatocytes at the concentrations of 30 μM for menadione, 10, 50, 100, and 300 μM for compound I, and 10, 50, and 100 μM for M12, respectively. A methanol solution was
microsomes. Control experiments were also performed in the absence of either a DNA base or NADPH. Isolation of the Adenine Adducts from Monkey Liver Microsomal Incubation. Monkey liver microsomes (1 mg protein/mL) were suspended in potassium phosphate buffer (100 mM, pH 7.4) containing EDTA (1 mM) and MgCl2 (0.1 mM) in the presence of adenine (5 mM) in a total volume of 300 mL. [3H]I in methanol was added to a final concentration of 50 μM at a final specific activity of 10 μCi/mg, such that the concentration of methanol in the incubation mixture did not exceed 0.2%. Incubations were initiated by adding NADPH (1.2 mM) and were incubated at 37 °C for 60 min. The reactions were quenched by adding 2 volumes of acetonitrile. The sample extractions were performed as described above. The eluted samples in methanol were evaporated to dryness under nitrogen, and the residues were dissolved in solvent A−solvent B (1:3) (solvent A: 5 mM ammonium acetate in water−acetonitrile− acetic acid, 95:5:0.05, v/v/v; solvent B: 5 mM ammonium acetate in acetonitrile−water−acetic acid, 95:5:0.05, v/v/v). Aliquots were loaded onto a semipreparative Waters YMC-AQ C18 column (10 mm × 250 mm, 5 μm) and eluted at a flow rate of 3 mL/min with a 1:25 split to a Finnigan LCQDeca XP Plus mass spectrometer ion source and a fraction collector. The HPLC mobile phase consisted of solvent A (5 mM ammonium acetate in water−acetonitrile−acetic acid, 95:5:0.05, v/v/v) and solvent B (5 mM ammonium acetate in acetonitrile−water−acetic acid, 95:5:0.05, v/v/v). The HPLC runs were programmed by a linear increase from 0 to 80% of solvent B during a 30 min period. Fractions containing radioactivity were collected, and the fractions corresponding to the adenine adducts at m/z 583.2 at the same retention times were pooled and evaporated to dryness under nitrogen at room temperature. Purified samples were used for nuclear magnetic resonance (NMR) analysis. NMR Analysis. NMR spectra of isolated metabolites dissolved in CD3CN:D2O (4:1) were recorded with a Varian Inova 600 spectrometer operating at 600 MHz. Chemical shifts (δ) are expressed as parts per million (ppm) downfield relative to tetramethylsilane, and coupling constants (J) are expressed in Hertz (Hz). 2370
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Figure 3. Full MS spectra of adducts A1−A5. A range of m/z 490−670 is shown here.
Figure 4. Radiochromatogram of [3H]I incubated with human cytochrome P450 3A4 in the presence of adenine. Five adenine adducts were labeled as A1−A5. used as a solvent control (negative control). Aliquots (100 μL) of incubation samples at 0 and 5 min were added to amber test tubes containing iodoacetamide (IAM) for derivatization of GSH. The detailed procedures for preparation of standard curves, derivatization of GSH, and LC/MS analysis were previously reported.2
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the presence of an individual DNA base (adenine, guanine, thymine, or cytosine) and NADPH. Five isomeric adenine adducts (A1−A5) with m/z of 583.2127 in liver microsomal incubations were detected in LC/MS analysis, which corresponded to the molecular ion of parent + adenine − 2H with a mass error of
