AT7519

Liquid chromatography-tandem mass spectrometric assay for the light sensitive survivin suppressant sepantronium bromide (YM155) in mouse plasma

M. Emmy M. Dolmana, Ilona J.M. den Hartoga, Jan J. Molenaara, Jan H.M. Schellensb,c,
Jos H. Beijnenb,c,d, Rolf W. Sparidansb,∗
a Amsterdam Medical Center, University of Amsterdam, Department of Oncogenomics, Meibergdreef 15, PO Box 22700, 1105 AZ Amsterdam,
The Netherlands
b Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacoepidemiology & Clinical Pharmacology,
Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
c The Netherlands Cancer Institute, Department of Clinical Pharmacology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
d Slotervaart Hospital, Department of Pharmacy & Pharmacology, Louwesweg 6, 1066 EC Amsterdam, The Netherlands

Abstract

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for sepantronium bromide (YM155), an inhibitor of survivin, was developed and validated. Under reduced light exposure, plasma samples were pre-treated using protein precipitation with acetonitrile contain- ing AT7519 as internal standard. After dilution with water, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray inter- face with positive ionization and compounds detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer.

The assay was validated in a 0.5–100 ng/ml calibration range with r2 = 0.9981 ± 0.0007 using double logarithmic calibration (n = 5). Within day precisions (n = 6) were 3.6–8.8% and between day (3 days; n = 18) precisions 6.5–11.1%. Accuracies were between 92 and 111% for the whole calibration range. The light sensitive drug sepantronium was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully used to determine plasma drug levels in mice after administration of sepantronium bromide by continuous infusion from subcutaneously implanted osmotic pumps.

1. Introduction

Almost all neuroblastoma tumors have enhanced levels of sur- vivin (BIRC5), a member of the inhibitor of the apoptosis protein (IAP) family [1]. Survivin expression levels in neuroblastoma cor- relate with a more aggressive phenotype and worse prognosis [1]. Sepantronium (Fig. 1), used as bromide salt (YM155) was identified as an inhibitor of survivin expression in a compound screen using SY5Y neuroblastoma cells. The compound might be beneficial for neuroblastoma treatment. In vitro studies have shown that sepa- ntronium causes cell death of neuroblastoma cells with low levels of the ABCB1 multidrug transporter [2]. For further preclinical development of sepantronium bromide a sensitive method for the analysis of drug plasma levels is warrented, which makes it possible to evaluate if plasma sepantronium levels correlate with efficacy.

Thus far, a validated bioanalytical assay for sepantronium using LC-MS/MS was reported for dog plasma [3] while for human plasma an assay was summarized in a pharmacokinetic study [4]. Both methods use relatively large sample volumes of 250 and 500 µl respectively, and a labor intensive and expensive solid phase extraction as a pretreatment method. Sample pretreatment was performed under reduced light exposure by Minematsu et al. [3]. Therefore, we now report the development and validation of a new, alternative, bio-analytical assay for sepantronium bromide in mouse plasma to support our preclinical mouse studies with the agent, using small sample volumes (20 µl), LC-MS/MS and protein precipitation as a simple pre-treatment procedure. Light sensitivity of the drug was given special attention.

2. Experimental
2.1. Chemicals

Sepantronium bromide (>96.5%) was purchased from Selleck Chemicals (Houston, TX, USA) and the internal standard AT7519 dwell time and a −5 V skimmer voltage. AT7519 was monitored at m/z 382.1→281.9; 135.9 at −25 and −40 V, respectively, with 0.05 s dwell times and a −8 V skimmer voltage. Mass resolutions were set at 0.7 full width at half height (unit resolution) for both separating quadrupoles.

2.4. Sample pre-treatment

To a volume of 20 µl of mouse plasma, pipetted into a poly- propylene reaction tube, 30 µl of 250 ng/ml AT7519 in acetonitrile were added. Tubes were closed and shaken by vortex mixing for 5–10 s. After centrifugation of the sample at 10,000 × g at 20 ◦C for 1 min, 40 µl of the supernatant was transferred into a 250 µl glass insert placed in an autoinjector vial. Before closing the vial, 100 µl of water was added and finally, 5 µl of the mixture was injected onto the column.(>98%) was kindly supplied by Astex (Cambridge, UK). Water (LC- MS grade), methanol (HPLC grade) and acetonitrile (HPLC-S grade) were obtained from Biosolve (Valkenswaard, The Netherlands). Water, not used as eluent, was home purified by reversed osmo- sis on a multi-laboratory scale. Formic acid was of analytical grade originating from Merck (Darmstadt, Germany). Pooled female mouse tripotassium EDTA plasma was supplied by Seralab Labo- ratories (Haywards Heath, UK).

Fig. 1. Chemical structure and product spectrum, formed by collision induced dis- sociation of sepantronium, m/z 363.16 at −24 V.

2.2. Equipment

The LC-MS/MS equipment consisted of a DGU-14A degasser, a CTO-10Avp column oven, a Sil-HTc autosampler and two LC10- ADvp-µ pumps (all from Shimadzu, Kyoto, Japan) and a Finnigan TSQ Quantum Discovery Max triple quadrupole mass spectrome- ter with electrospray ionization (Thermo Electron, Waltham, MA, USA). Data were recorded on and the system was controlled by the Finnigan Xcalibur software (version 1.4, Thermo Electron).

2.3. LC-MS/MS conditions

Partial-loop injections (5 µl) were made on a Polaris 3 C18-A col- umn (50 × 2 mm, dp = 3 µm, average pore diameter = 10 nm, Varian, Middelburg, The Netherlands) with a corresponding pre-column (10 × 2 mm, Agilent Technologies, Amstelveen, The Netherlands). The column temperature was maintained at 40 ◦C and the sam- ple rack compartment at 4 ◦C. A gradient (0.5 ml/min) using 0.02% (v/v) formic acid (A) and methanol (B) was used. After injection, the percentage of methanol was increased linearly from 20 to 40% (v/v) during 1.33 min. Next, the column was flushed with 100% (v/v) methanol for 0.67 min and finally, the column was reconditioned at the starting conditions (20% (v/v) B) for 1 min resulting in a total run time of 3 min per sample. The whole eluate was transferred into the electrospray probe, starting at 0.6 min after injection by switching the MS divert valve until 2.0 min after injection. The elec- trospray was tuned in the positive ionization mode by introducing 0.5 ml/min of a solvent mixture containing 50% (v/v) of 0.1% (v/v) formic acid and 50% (v/v) methanol and 5 µl/min of 10 µg/ml of sepantronium bromide. Electrospray settings of the assay were a 500 V spray voltage, a 382 ◦C capillary temperature and the nitrogen sheath, ion sweep and auxiliary gasses were set at 42, 24 and 0 arbitrary units, respectively. The SRM mode was used with argon as the collision gas at 1.6 mTorr. The tube lens off set was 101 V for sepantronium and 130 V for AT7519. Sepantronium was mon- itored at m/z 363.15→305.0 at −24 V collision energy with a 0.1 s

2.5. Validation

A laboratory scheme based on international guidelines was used for the validation procedures [5–7].

2.5.1. Calibration

Stock solutions of 1 mg/ml sepantronium bromide were prepared in water. AT7519 was prepared at 1 mg/ml in methanol. Stock solutions were stored in 1.5 ml polypropylene tubes at −30 ◦C, for sepantronium amber colored containers were used. Sepantronium bromide stock solutions were diluted to 2000 ng/ml working solutions with water and one working solution was further diluted to a 100 ng/ml calibration solution in pooled female tripotassium EDTA mouse plasma. Working and calibration solutions were stored in amber 1.5-ml polypropylene tubes at −80 ◦C. Additional calibration samples were prepared daily at 25, 10, 2.5, 1 and 0.5 ng/ml by dilution of the 100 ng/ml calibration solution with blank mouse plasma. The highest and two lowest calibration samples were processed in duplicate for each daily calibration, whereas the levels in between were processed only once. Least-squares double logarithmic linear regression was employed to define the calibration curves using the ratios of the peak area of the analyte and the IS.

2.5.2. Precision and accuracy

A second stock solution of sepantronium bromide was used to obtain validation (quality control; QC) samples in pooled mouse female tripotassium EDTA mouse plasma at 80 (QC-high), 10 (QC-med), 1.6 (QC-low) and 0.5 ng/ml (QC-LLOQ). Samples were stored in amber polypropylene tubes at −80 ◦C. Precisions and accuracies were determined by sextuple analysis of each QC in three analyt- ical runs on three separate days for all QCs (total: n = 18 per QC). Relative standard deviations were calculated for both, the within and between day precisions.

2.5.3. Selectivity

Six individual mouse plasma samples were processed to test the selectivity of the assay. Samples were processed without sepa- ntronium and IS and with sepantronium bromide at the LLOQ level (0.5 ng/ml), supplemented with the IS.

2.5.4. Recovery and matrix effect

The recovery was determined (n = 4) by comparing processed samples (QC-high, -med, -low; the same samples as used for preci- sion and accuracy) with reference sepantronium solutions in blank pooled plasma extract at the same levels The matrix effect was assessed by comparing the reference solutions in blank plasma extracts with the same matrix-free solutions at the three validation levels. An analogous procedure was used for the internal standard.

2.5.5. Stability

The stability of sepantronium was investigated in QC-high and -low plasma samples stored in polypropylene tubes protected from light. Quadruplicate analysis of these samples from separate tubes was performed after storage at 20 ◦C (ambient temperature) for 24 h, three additional freeze–thaw cycles (thawing at 20 ◦C during ca. 2 h and freezing again at −80 ◦C for at least one day), and stor- age at −80 ◦C for 3.5 months, respectively. Additional experiments were performed at the low level during 8 h at ambient temperature and after only two freeze–thaw cycles, respectively. Drug stabil- ity was also investigated in polypropylene reaction tubes without light protection standing for 0, 2, 4 and 8 h, respectively, on the laboratory bench for the QC-high and -low levels, both in plasma and in water (n = 3 for each exposure time and sample (20 µl)). Furthermore, an analytical run was re-injected after additional storage of the extracts at 4 ◦C for five nights to test the stability at these conditions in the autoinjector.

Finally, the responses of sepantronium from the stock solutions in water after 6 h at 20 ◦C (n = 2) and after 3.5 months at −30 ◦C (n = 2) were compared to fresh stock solutions with LC-MS/MS after appropriate dilution of the samples and adding IS.

2.6. Mouse samples

Female NMRI nu/nu mice were obtained from Harlan (Zeist, The Netherlands) and experiments were performed with per- mission from and according to the standards of the Dutch animal ethics committee (DEC 102710). Mice with KCNR neu- roblastoma xenografts were treated with 0.1, 1 or 3 mg/kg/day sepantronium bromide, administered via continuous infusion from subcutaneously implanted Alzet® osmotic mini pumps (Cupertino, CA, United States). Osmotic pumps were implanted opposite the tumor side and sepantronium bromide (formulated in 25% dimethyl sulfoxide and 75% PBS in concentrations of 8.33 mg/ml/kg (dose: 0.1 mg/kg/day), 83.3 mg/ml/kg (dose: 1 mg/kg/day) or 250 mg/ml/kg (dose: 3 mg/kg/day)) was released from the pumps at a continuous rate of 0.5 µl/h. Blood samples were collected from the inferior vena cava at day 7 after start of treatment in tripotassium EDTA vials. Plasma samples were obtained by centrifugation twice at 1150 × g for 15 min and were stored at −80 ◦C until pretreatment and analysis as described above. For the comparison with previous published results, statistical analysis was performed using the two- tailed unpaired Student’s t-test, with p < 0.05 as the minimum level of significance. 3. Results and discussion 3.1. Method development Previous ESI-MS/MS settings were optimized for sepantronium to obtain maximal sensitivity. Maximized ion detection could be obtained at low electrospray voltage, probably due to the intrinsic charge of the sepantronium ion. A product spectrum of sepantron- ium is presented in Fig. 1; a spectrum of the IS had been reported previously [8]. Light protection of the drug was reported in an exist- ing assay [3] and light exposure in front of a window during the day resulted in a half-life for sepantronium of ca. 2 h (data not shown). Therefore, samples were protected in this study from light as far as possible by using reduced light intensity during sample pretreat- ment (artificial lights off in the laboratory) and sample storage in amber tubes and vials. Sample pretreatment was as simple and fast as possible by using only protein precipitation and dilution of the sample. Strongly retained plasma constituents were removed from the column using a high organic flush at the end of each analyt- ical run in order to prevent long term suppression effects of the ionization. Because a stable isotopically labeled analogue of sep- antronium was not available, AT7519, showing almost identical retention as sepantronium in the present assay (Fig. 2), was chosen as internal standard. Fig. 2. SRM chromatograms of sepantronium and the IS in plasma extracts: blank mouse plasma (A), LLOQ (0.5 ng/ml) spiked plasma (B) and wild type female mouse EDTA tripotassium plasma, containing 0.89 ng/ml of the drug (C), 7 days after starting a 0.1 mg/kg/day sepantronium bromide infusion. An artificial off set was given to the chromatograms. 3.2. Validation Because in a preliminary experiment (n = 6) plasma levels were observed up to 34 ng/ml and 0.5 ng/ml seemed to be near the low- est level to be assessed with the presented assay, a 0.5–100 ng/ml range was chosen for validation. SRM chromatograms are depicted in Fig. 2, showing chromatograms of blank and LLOQ spiked plasma samples. 3.2.1. Calibration The relative response of sepantronium showed a small but significant deviation from a linear function (p = 0.02 for a 1-tailed Student’s t-distribution of the average double-logarithmic slope (n = 5) compared to 1); therefore, and because of failing results for the selectivity experiments using linear regression, the double logarithmic linear function was used for assay calibration [5]. For 5 calibrations (60 samples) the concentrations were back-calculated from the ratio of the peak areas (analyte and IS), using the calibra- tion curves of the run in which they were included. No deviations from the average of each level higher than 1.8% were observed (data not shown), indicating an excellent suitability of the double log- arithmic regression model [5,9]. The average of the reproducible regression parameters of the double logarithmic regression functions (n = 5) were log(y) = −1.81(±0.05) + 0.967(±0.011) log(x) with a regression coefficient of 0.9981 ± 0.0007. Here, x is the sepantronium bromide concentration (ng/ml) and y is the drug response relative to the IS. 3.2.2. Precision and accuracy Assay performance data of the validation samples at four con- centrations are reported in Table 1. Within day and between day variations lower than 11.1% were observed and deviations of the accuracies were lower than 10.4%. The precision and accuracy therefore met the required ±15% variation (±20% for the LLOQ) [5–7]. 3.2.3. Selectivity The analysis of six independent blank mouse plasma samples showed no interfering peaks in the SRM traces for sepantron- ium and the IS AT7519. Blank sepantronium responses were all below 5% of the LLOQ response, meeting the required 20% [9], and blank IS responses below 0.1% of the normal response. The signals at the LLOQ level (0.5 ng/ml) were all distinguishable from blank responses with signal-to-noise ratios in the range 10–20; con- centrations found at the LLOQ level (n = 6) were 0.57 ± 0.04 ng/ml, demonstrating the applicability of the investigated LLOQ level [5–7]. 3.2.4. Recovery and matrix effect Extraction recoveries showed no losses for sepantronium and ranged from 94 to 103% (data not shown). Matrix effects were also not observed; ionization recoveries ranged from 94 to 104% for sepantronium at the investigated levels. For the IS AT7519,102.1 ± 3.2% for extraction recovery and 114.2 ± 4.5% (both n = 4) for matrix effect were found, indicating there may be only a very small matrix effect. Overall, the absence of significant extraction losses and matrix effects contributed to a successful validation of the assay [5–7]. 3.2.5. Stability The stability of sepantronium in female mouse EDTA tripotas- sium plasma after different storage procedures is presented in Table 2. Because sepantronium degradation in the QC-low sam- ple exceeded 15% during 24 h storage at ambient temperature as well after 3 freeze–thaw cycles, experiments were repeated for 8 h storage at ambient temperature and 2 freeze–thaw cycles, respec- tively. The drug showed sufficient stability under these conditions. Without light protection on the laboratory bench, drug half-life values were 12.1 h (QC-high in plasma), 9.3 h (QC-low in plasma), 4.6 h (QC-high in water) and 3.6 h (QC-low in water), respectively. These results show a limited risk of sample disintegration during short periods of light exposure. Degradation in plasma is proba- bly slower than in water because of the limited transparency of the matrix. Re-injection of calibration and QC samples after addi- tional storage at 4 ◦C for five nights resulted again in successful performances without any loss of precision and accuracy, thus QC failures remained far below a 33% frequency (data not shown) as required [5,9]. Recoveries of sepantronium bromide in stock solu- tions were excellent: 99.3% (after 6 h at 20 ◦C; n = 2) and 101.7% (after 3.5 months at −30 ◦C; n = 2), respectively. Fig. 3. Plasma sepantronium bromide concentrations at day 7 after continuous sub- cutaneous infusion of 0.1 (n = 6), 1 (n = 6) or 3 (n = 9) mg/kg/day in female NMRI nu/nu mice with KCNR neuroblastoma xenografts (268 mm3 ). Each data point (Ç) represents an individual mouse and horizontal (dark grey) lines represent the aver- age plasma levels for each dose. Plasma levels of sepantronium bromide linearly increased with dose, as shown by the dotted (dark grey) line. Linear regression was performed using GraphPad Prism 5, forcing the line through zero. 3.3. Mouse pharmacokinetics After the successful validation procedure, the new assay was used to investigate sepantronium bromide plasma levels at day 7 after administration of 0.1, 1 and 3 mg/kg/day sep- antronium bromide (equal to approximately 0.082, 0.82 and 2.5 mg/kg/day sepantronium, respectively) to mice with KCNR neuroblastoma xenografts via continuous infusion from subcuta- neously implanted osmotic pumps. Plasma levels of sepantronium bromide increased dose-dependently as shown by the average plasma concentrations of 0.91 ± 0.39 (n = 6), 6.71 ± 1.57 (n = 6) and 18.91 ± 6.48 (n = 9) ng/ml observed after administration of 0.1, 1 and 3 mg/kg/day sepantronium bromide, respectively (Fig. 3). Previous publications reported sepantronium concentrations in human plasma after continuous intravenous administration [4,10]. Conversion of these sepantronium concentrations into sepantro- nium bromide concentrations and translation of human doses to animal doses using the formula described by Reagan-Shaw et al. (human Km = 37 and mouse Km = 3) [11] shows that the average sepantronium bromide plasma levels found in the cur- rent study after continuous subcutaneous infusion in mice were slightly higher as compared to the steady-state plasma levels found after continuous intravenous infusion in human. For example, the steady-state plasma concentration at day 7 after continuous intra- venous infusion of 8 mg/m2/day sepantronium in humans (equal to approximately 3.2 mg/kg/day sepantronium bromide in mice) was 13 ± 3 ng/ml [10] sepantronium, which is equal to approximately 15.9 ± 3.7 ng/ml sepantronium bromide. This is slightly lower, but not statistically different, than the average plasma concentration of 18.91 ± 6.48 ng/ml sepantronium bromide found at day 7 after continuous subcutaneous infusion of 3 mg/kg/day in mice. Results suggest a complete biological availability after subcutaneous infusion. 4. Conclusions The current article reports the first fully validated assay for the quantification of sepantronium bromide in female mouse EDTA tripotassium plasma samples. The sensitive LC-MS/MS assay includes a fast and simple sample pre-treatment method. Results showed values of accuracy, precision, recovery and stability com- pliant to international guidelines [5–7]. The new assay was successfully used for the analysis of plasma levels after continu- ous subcutaneous infusion of the drug in mice with neuroblastoma xenografts. References [1] F. Lamers, I. van der Ploeg, L. Schild, M.E. Ebus, J. Koster, B.R. Hansen, T. Koch, R. Versteeg, H.N. Caron, J.J. 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