Sorafenib (free base, >99%) was obtained from Chemie Tek (Indianapolis, IN), and

Transcription

1 Supplemental Methods Chemicals and Reagents Sorafenib (free base, >99%) was obtained from Chemie Tek (Indianapolis, IN), and isotopically-labeled 13 C- 2 H 3 -sorafenib (labeled atoms on N-methyl position) was purchased from Alsachim (Strasbourg, France). Sorafenib N-oxide (>98%) was purchased from Toronto Research Chemicals Inc. (North York, Canada). HPLC-grade methanol and acetonitrile were obtained from Burdick & Jackson (Muskegon, MI). Dimethyl sulfoxide (>99.5%, gas chromatography grade) was purchased from Sigma (St. Louis, MO). Analytical-grade formic acid (98%) was obtained from EMD Chemicals (Gibbstown, NJ). Potassium phosphate (0.5 M; ph 7.4) buffer solution, NADPH Regenerating System Solutions A (31 mm NADP +, 66 mm glucose 6-phosphate, and 66 mm MgCl 2 ) and B (40 U/mL Glucose 6-phosphate dehydrogenase in 5 mm sodium citrate), and UGT Reaction Mixture Solution A (25 mm UDP glucuronic acid) and B (containing alamethicin; ph 7.5) were purchased from BD Biosciences (Woburn, MA). Ultrapool HLM 150 microsomes (S9 fraction) and insect cell control were purchased from BD Biosciences. Purified human UDPglucuronosyltransferase (UGT) enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, and UGT2B15) and cytochrome P450 (CYP) enzymes (CYP1A1, CYP1A2, CYP3A4, CYP3A5, CYP2C8, CYP2C9, CYP2C19, and CYP2D6), which were prepared from baculovirus-infected insect cells, were purchased from BD Bioscience. RNeasy kits were purchased from Qiagen (Valencia, CA). SuperScript III First-Stand Synthesis Kits were purchased from Invitrogen (Grand Island, NY). Taqman Gene Expression Assays for human CYP3A4 (ID: Hs _sH), UGT1A9 (ID: Hs _m1), and GAPDH (ID: Hs _m1) were purchased from Applied Biosystems (Carlsbad, CA). Identification of UGT enzymes responsible for metabolism of sorafenib

2 An initial screen was performed to determine the UGT enzymes responsible for sorafenib glucuronidation including: UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15. Reactions were performed in triplicate, except for UGT1A9 (6 reactions total). The reaction mixture (final volume 500 µl) was composed of UGT Reaction Mixture Solution A and B, sorafenib (final concentration 10 µm) and deionized water. The mixture was incubated at 37 C for five minutes, and the metabolic reaction was initiated following the addition of UGT enzyme or UGT control (final concentration 0.2 mg/ml). After 60 min incubation at 37 C, 100 µl was transferred to a microcentrifuge tube and the reaction was terminated with the addition of 100 µl of cold methanol/acetonitrile (50:50 v/v) containing internal standard solution. Kinetics of sorafenib glucuronidation by UGT1A9 Optimal conditions (incubation time and protein concentration) for sorafenib glucuronide formation by UGT1A9 were determined by performing a time course for up to 60 min and evaluating protein concentrations up to 1 mg/ml. The reaction mixture (final volume 200 µl) consisted of UGT1A9 0.5 mg/l, varying sorafenib concentrations (0.1 to 50 µm), and an incubation time of 60 min. Reactions were performed in triplicate following the procedure described for UGT enzyme screen. Reaction rate (velocity) was calculated as peak area of glucuronide per minute per milligram UGT1A9. Substrate concentration (x-axis) was plotted against UGT1A9 velocity (y-axis) and the kinetic parameters maximum metabolite formation (Vmax) and substrate concentration at which 50% of Vmax is obtained (Km) were estimated by fitting the Michaelis-Menten equation to the data by nonlinear regression analysis as implemented in GraphPad Prism (GraphPad Software, Inc., La Jolla, CA USA). Identification of CYP enzymes responsible for metabolism of sorafenib An initial screen was performed to determine the CYP enzymes responsible for metabolism of sorafenib to sorafenib N-oxide including: CYP1A1, CYP1A2, CYP3A4, CYP3A5, CYP2C8, CYP2C9,

3 CYP2C19, and CYP2D6. Reactions were performed in triplicate. The reaction mixture (final volume 500 µl) was composed of 0.5 M potassium phosphate buffer (final concentration 100 mm), NADPH Regenerating System Solution A and B, and sorafenib (final concentration 10 µm). The mixture was incubated at 37 C for five minutes, and the metabolic reaction was initiated with the addition of the desired CYP enzyme or control (final concentrations of 20, 40, and 80 pmol/ml). After a 60 min incubation at 37 C, 100 µl of the reaction mixture was transferred to a microcentrifuge tube and the reaction was terminated with the addition of 100 µl of cold internal standard solution. Kinetics of sorafenib N-oxide formation by CYP3A4 Incubation time and protein concentration was optimized for sorafenib N-oxide formation by CYP3A4 by performing a time course up to 60 min and evaluating protein concentrations of 20, 40, and 80 pmol/ml. Reactions (final volume of 200 µl) consisted of CYP3A4 40 pmol/ml, varying concentrations of sorafenib ( µm), and an incubation time of 30 min. Reactions were performed in triplicate following the procedure described for the CYP enzyme screen. Reaction rate (velocity) was calculated as nmol sorafenib N-oxide per minute per nmol CYP3A4. Substrate concentration (x-axis) was plotted against CYP3A4 velocity (y-axis) and the apparent kinetic parameters Vmax and Km were estimated by fitting the Michaelis-Menten equation to the data by nonlinear regression analysis as implemented in GraphPad Prism. Inhibition of UGT1A9-mediated sorafenib glucuronidation and CYP3A4-mediated sorafenib N- oxide formation by azole antifungals The effect of the azole antifungal drugs ketoconazole, voriconazole and posaconazole on sorafenib glucuronidation by UGT1A9 and sorafenib N-oxide formation by CYP3A4 was investigated using optimized conditions and reaction mixtures as described above. An initial screen was performed with sorafenib at 10 µm and azole antifungal at 100 µm and 1,000 µm. Reactions were performed in triplicate. For reactions showing strong inhibition of sorafenib metabolism (e.g., to values of <50% of control), inhibition studies were repeated at 3 to 5 sorafenib concentrations (2-100 µm)

4 and at varying concentrations of azole antifungal ( µm). To obtain a graphical estimate of apparent inhibition constant (Ki), which is the concentration of inhibitor required to produce half maximal inhibition, Dixon plots were constructed by graphing substrate concentration (x-axis) versus 1/velocity (y-axis). Substrate concentration (x-axis) was plotted against velocity (y-axis) and Ki was estimated by fitting the corresponding equation for the appropriate inhibitor type to the data using nonlinear regression analysis. Construction of plots and data fitting were performed using GraphPad Prism. Metabolism of Sorafenib in Human Liver Samples Human liver microsomes (HLMs) were extracted from liver samples (~100 mg) using the method of Wang et al (1). Briefly, liver tissue was homogenized in buffer (100 mm Tris, 100 mm KCl, 1 mm EDTA, and 20 µm butylated hydroxytolulene (BHT); ph 7.4) containing protease inhibitor cocktail (Roche; Nutley, NJ) and 100 µm PMSF using a homogenizer (Next Advance; Averill Park, NY). Homogenate was then centrifuged at 12,000 RPM for 15 min at 4 C. The supernatant was then centrifuged at 34,000 RPM for 1 h at 4 C. The pellet was then resuspended in buffer containing 100 mm potassium phosphate, 1 mm EDTA, 20% glycerol, 1 mm DTT, and 20 µm BHT (ph 7.25) supplemented with protease inhibitor cocktail and 100 µm PMSF. Assessment of UGT- and CYPdependent sorafenib metabolism was performed using the BD Gentest CYP and UGT reaction mix, respectively, according to the manufacturer s instructions. Briefly, human liver microsomes (1 mg/ml) or Ultrapool HLM 150 microsomes (S9 fraction; 1 mg/ml), as a positive control, were combined with sorafenib (10.0 µm) and the appropriate reaction buffers. The total reaction volume was 200 µl. The reaction was incubated at 37 C with constant agitation for 60 min for both the UGT and CYP reactions, and the reaction was stopped by the addition of 100 µl of cold internal standard solution to 100 µl reaction mixture. A total of 61 liver samples were initially evaluated. Criteria for inclusion of liver samples included: 1) 0-20 years of age, 2) Caucasian ethnicity, and 3) no history of disease. However, 9

5 samples had unevaluable enzymatic activity (sorafenib N-oxide or sorafenib glucuronide concentrations were below the assay lower limit of quantitation). Because we could not determine if this was due to poor sample quality, we excluded these samples from the study. RNA extraction, reverse transcription, and quantitative (q)-pcr Analysis Frozen human liver samples (20 mg) were homogenized and RNA was extracted using the RNAeasy kit according to the manufacturer s instructions. RNA quality was determined spectrophotometrically. Reverse transcription of RNA (1 µg) was then performed using the SuperScript III First-Stand Synthesis Kit for qrt-pcr according to the manufacturer s instructions. qpcr reactions containing 50-ng cdna were performed on an Applied Biosystems 7900 HT Sequence Detection System, and gene-specific amplification was performed using Taqman Gene Expression Assays for CYP3A4, UGT1A9, and GAPDH according to the manufacturer s instructions. Standard curves for each gene were generated using serial dilutions of plasmids containing human cdna constructs or cdna amplicons ranging from copies. mrna transcript numbers were calculated from linear regression analysis of the respective standard curves and normalized to the expression of GAPDH mrna. Sorafenib Glucuronide Isolation and Identification Sorafenib glucuronide was isolated from patient urine using 4 volumes of ethyl acetate. The extract was evaporated in a rotary evaporator at 37 C, and the residue was reconstituted in methanol at 20 mg/ml. Sorafenib glucuronide was then isolated from the sample solution using a Waters Prep LC controller and Photodiode Array Detector (Milford, MA). A Phenomenex Prep column (Gemini 5 µ C 18, 110 A, 100x10 mm; Torrance, CA) was used for semi-preparative isolation. A HPLC gradient method consisting of 0.1% formic acid of H 2 O and acetonitrile as the mobile phase at a flow rate of 10 ml/min was used with an injection volume of 0.5 ml and detection at 270 nm. Fractions between retention time 13.5 and 14.0 min were pooled and dried under nitrogen gas at room temperature.

6 Sorafenib glucuronide was identified on a Waters UPLC system coupled with a Waters Xevo G2 QTOF spectrometer operating in positive electrospray ionization and full-scan mode (Supplemental Figure 6). Sorafenib glucuronide formed a protonated molecule [M + H]+ at m/z Its fragments were formed predominantly by the loss of glucuronic acid and the cleavage of the N-C=O bond. The correspondent product ion at m/z (sorafenib) and m/z (cleavage between the 4-amino phenoxy nitrogen and the phenyl carbamoyl carbon) were observed. The formation of the crucial daughter ion at m/z confirmed the nitrogen binding positions for the glucuronide group. This ion could only be formed if the glucuronide moiety is bound to the nitrogen atom. The purity of the collected sorafenib glucuronide was determined by HPLC-UV-MS (>98% purity). Generation of LC-MS/MS Internal Standard Solution The internal standard 13 C- 2 H 3 -sorafenib glucuronide was prepared by incubation of 13 C- 2 H 3 - sorafenib with purified UGT1A9 to obtain 13 C- 2 H 3 -sorafenib glucuronide. Briefly, 13 C- 2 H 3 -sorafenib (0.1 mg/ml in MeOH) was added to UGT1A9 (0.2 mg/ml) and UGT reaction buffers as described by the manufacturer. The mixture was incubated for 24 h at 37 C, and the reaction was terminated by the addition of acetonitrile. After centrifuging at 10,000 g for 3 min, 4 volumes of acetonitrile were added into the supernatant. 13 C- 2 H 3 -sorafenib stock solution was added to yield a final concentration of 25 ng/ml 13 C- 2 H 3 -sorafenib in the internal standard solution. Tandem LC-MS/MS Spectrographic Analysis of Sorafenib and Metabolites in Metabolic Reaction Mixtures and human plasma Analytes were extracted from 100 µl of standard, quality control (QC), or metabolic reaction samples with 500 µl acidified ethyl acetate supplemented with 0.5% formic acid. After vortexing for 15 min and centrifugation at 10,000 g for 5 min, the sample was put on dry ice for 1 min. The upper

7 organic layer was evaporated to dryness under nitrogen at 35 C and the residue was reconstituted in 200 µl acetonitrile, which was transferred to an autosampler vial and 1 µl was injected for analysis. Sorafenib, sorafenib glucuronide, and sorafenib N-oxide were measured by HPLC with tandem mass spectrometric detection (LC-MS/MS) using a TQD triple-quadrupole system. Separation was achieved on a Waters ACQUITY BEHC 18 column (1.7 µm; 50 x 2.1 mm) using a column heater operating at 40 C with a Waters ACQUITY in-line filter. Autosampler temperature was maintained at 15 ± 5 C, and the gradient mobile phase was composed of 0.1% formic acid in acetonitrile (B) and 0.1% formic acid in H 2 O (A). After sample injection, B was increased from 30% to 65% over 1 min and then maintained for 0.7 min; after flushing for 0.8 min with 95% B, the column was equilibrated for 2 min with 30% B before the next injection. The flow rate was 0.7 ml/min with a total run time of 5 min. The mass spectrometer was operated in the positive mode using Masslynx 4.1 software. Description of peak analysis is found in the supplemental methods. Spectrographic peak analysis was performed in MRM mode, and the following mass ions (m/z) were used for detection: m/z 465.1>252.9 for sorafenib; m/z 481.0>286.0 for sorafenib N-oxide, m/z 641.2>270.1 for sorafenib glucuronide, m/z > for isotopically labeled 13 C- 2 H 3 -sorafenib, and m/z 645.1>469.1for isotopically labeled 13 C- 2 H 3 -sorafenib glucuronide. The MS/MS conditions were as follows: capillary voltage: 1.5 kv; cone voltage: 48 V for sorafenib, 45 V for sorafenib glucuronide, 54 V for sorafenib N-oxide, and 46 V for the internal standard; source temperature: 150 C; desolvation temperature: 500 C; cone gas flow: 5 L/h; desolvation gas flow: 950 L/h and collision energy: 33 for sorafenib, 27 for sorafenib N-oxide, 20 for sorafenib glucuronide and 35 for the internal standard. Sorafenib, sorafenib glucuronide and sorafenib N-oxide stock solutions were prepared by dissolving 10 mg, 5 g, and 5 mg, respectively, with methanol in a 10-ml volume flask. Stock solutions were stored at -20 C. Working solutions of sorafenib, sorafenib glucuronide and sorafenib N-oxide were prepared by diluting the stock solutions with 70% aqueous acetonitrile. The working solutions were diluted in insect control extracts to prepare calibration standards at concentrations ranging from

8 50 to 10,000 ng/ml for sorafenib and 10 to 5000 ng/ml for sorafenib glucuronide and sorafenib N- oxide. For each analytical run, QC samples were prepared independently at three different concentrations (low, medium, and high), and seven concentration points were used to generate the calibration curves. The back-calculated concentration for each standard was less than 15% of the nominal concentration except the LOQ, which was less than 20%. Sorafenib and sorafenib N-oxide were quantitated in plasma samples from the first 26 of 30 patients using a previously validated LC-MS/MS assay (2), and sorafenib glucuroinde was measured in all samples with the new analytical method described above except for a few exceptions outlined below. For the most recent 4 patients that were evaluated, all 3 analytes were measured using the new analytical method. For the new assay, a 30-µL aliquot of standard, QC or patient plasma sample was spiked with internal standard solution (described above). The tube was vortexed for 45 sec followed by centrifugation at 16,000 g for 8 min at 4 C. The supernatant was transferred to an autosampler vial and 1 µl was injected for analysis. For method validation, QC samples, containing all 3 analytes in human plasma, were prepared independently at four different concentrations (lower limit of quantitation [LOQ], low, medium and high concentrations), and were analyzed over 4 days. Average accuracies for QC samples for sorafenib, sorafenib N-oxide, and sorafenib glucuronide ranged from 92.7%-106%, %, and %, respectively; within- and between-day variability was 4.0%, 8.0%, and 12.0%, respectively. Sorafenib glucuronide was stable in plasma at room temperature up to 6 h with 6% deviation from initial concentrations. Sorafenib glucuronide was stable through 3 freeze-thaw cycles with 9% deviation from initial concentrations. Sorafenib glucuronide in reconstitution solution in the autosampler at 10ºC was stable for up to 26 h with 4% deviation from initial concentration. The last long-term stability testing we performed of sorafenib glucuronide QC samples at low (30 ng/ml) and high (2000 ng/ml) concentrations was for samples that were stored at -80 ºC for 184 days. Sorafenib glucuronide concentrations were 122.6% and 103.7% of initial concentrations, respectively, at day 184. The two different analytical assays used in the PK study were also cross-validated for sorafenib and sorafenib N-oxide by measuring both

9 analytes in quadruplicate QC samples at low and high concentrations with both methods over several days. Sorafenib and sorafenib N-oxide concentrations differed by < 15% (median 1%; range, 0% - 12%). The median time between sample collection and analysis of sorafenib and sorafenib N-oxide for the first 26 patients was 28 days (range, 1 to 379 days). By reanalyzing samples for the first 26 patients (total of 107 samples) with the new analytical method, we performed an incurred sample reanalysis and were able to assess long-term freezer stability of sorafenib and sorafenib N-oxide in human plasma. This was assessed as run-run interval (RRI), which was estimated as the time from the initial analysis to re-analysis. RRI for sorafenib and sorafenib N-oxide are illustrated in Supplemental Figure 7. Sorafenib concentrations varied by 85% to 115% for 94% of samples up to a RRI of ~ 1000 days. As RRI increased, more of the sorafenib N-oxide concentrations varied by > 115%, which resulted in a large percentage of sorafenib N-oxide metabolic ratios deviating by > 115%. This is most notable at ~ > 500 days. The data indicate that sorafenib N-oxide is not as stable as sorafenib in long-term freezer storage. The plasma samples from patients were thawed twice prior to re-analysis, which could have contributed to the observed changes in sorafenib N-oxide concentrations. However, our previously published validation data indicate that sorafenib N-oxide is stable through 3 freeze-thaw cycles with < 15% deviation from initial concentration(2). Independently of the incurred sample analysis, we evaluated long-term stability of sorafenib and sorafenib N-oxide at low and high plasma QC samples stored at -80 ºC for 213. Sorafenib concentrations were 97.4% and 103.0% of initial concentrations, respectively, at day 213; and sorafenib N-oxide concentrations were 90.8% and 96.0% of initial concentrations, respectively. References (1) Wang L, Christopher LJ, Cui D, Li W, Iyer R, Humphreys WG, et al. Identification of the human enzymes involved in the oxidative metabolism of dasatinib: an effective approach for determining metabolite formation kinetics. Drug Metab Dispos 2008;36: (2) Li L, Zhao M, Navid F, Pratz K, Smith BD, Rudek MA, et al. Quantitation of sorafenib and its active metabolite sorafenib N-oxide in human plasma by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2010;878: