AZD5363

A validated LC-MS/MS method for the quantification of capivasertib in dog plasma: application to its pharmacokinetics study

Abstract

In this study, a simple and reliable liquid chromatography tandem mass spectrometric method was first developed for the determination of capivasertib in dog plasma with ipatasertib as internal standard. The plasma samples were deproteinated by using acetonitrile. An ACQUITY BEH C18 column (1.7 μm, 2.1 × 50 mm) maintained at 40 oC was used for chromatographical separation, with water containing 0.1% formic acid and acetonitrile as mobile phase. MRM transitions were m/z 429.2 > 135.1 for capivasertib and m/z 458.2 > 387.2 for ipatasertib, respectively. An excellent linearity was achieved in the concentration range of 1-1000 ng/mL with correlation coefficient more than 0.9981. The lower limit of quantification was 1 ng/mL. The extraction recovery of capivasertib from dog plasma was > 85.81% and no significant matrix effect was found. The intra- and inter-day precision was less than 9.58 % and the accuracy ranged from -10.60% to 12.50%. The validated method was further applied to the pharmacokinetic study of capivasertib in dog plasma after single oral (5 mg/kg) and intravenous (1 mg/kg) administrations. The results revealed that capivasertib was rapidly absorbed into plasma with good bioavailability (47.04%) and low clearance.

Keywords: capivasertib, pharmacokinetics, bioavailability, liquid chromatography tandem mass spectrometry

1. Introduction

Cancer is one of the life-threaten diseases that has plagued humans for thousands of years. The PI3k/AKT pathway is a growth-regulating cellular signaling pathway (Uko et al., 2020). Activation of the PI3K/AKT/mTOR pathway is one of the most common aberrations in human cancer, which is associated with tumor growth and survival (Sarker et al., 2009). Decrease of AKT phosphorylation is associated with reduction in cancer cell proliferation (Uko et al., 2020). Capivasertib, a novel pyrrolopyrimidine-derived drug, is a highly selective pan-AKT kinase inhibitor that inhibits all AKT isoforms with a potency of 10 nM or less (Davies et al., 2012).Capivasertib not only suppress cancer cell proliferation and metastasis, but may also inhibit cytokine regulation and PD-L1 expression (Jabbarzadeh et al., 2020). Capivasertib is currently in clinical trials (Banerji et al., 2018; Tamura et al., 2016).

Pharmacokinetic study plays an integral role in drug discovery and development, which is a key determinant of drug success in some cases (He and Wan, 2018; Fan and de Lannoy,2014). Information from pharmacokinetic study is of great importance in understanding the bioactivities and safety profiles of drugs. To the best of our knowledge, there is no report regarding the pharmacokinetic study. To support the pharmacokinetic study, a reliable and accurate quantitation method is necessary. In recent years, liquid chromatography combined with electrospray tandem mass spectrometry has emerged as one of the most powerful tools to quantify drugs in biological matrices due to its high sensitivity and selectivity (Solliec et al., 2015; de Paepe et al., 2013; Zhao et al., 2014; Liu et al., 2011).

In this study, a rapid and reliable ultra-high performance liquid chromatography combined electrospray ionization tandem mass spectrometric method (LC-MS/MS) developed for the determination of capivasertib in dog plasma. The developed assay was fully validated according to the guidance issued by Food and Drug Administration (Food and Drug Administration, 2018). The validated LC-MS/MS method presented high sensitivity (1 ng/mL) and short chromatographic run time (2 min), which met the requirement of pharmacokinetic study of capivasertib in dog plasma.

2. Materials and methods

2.1. Chemicals and reagents

Capivasertib (purity > 98%) and ipatasertib (purity > 98%, internal standard, ISTD) were purchased from MedChemExpress (Shanghai, China). Acetonitrile and methanol were of HPLC grade and purchased from Fisher Scientific. LC-MS grade formic acid was supplied by Sigma-Aldrich. Water for LC-MS analysis was prepared by a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals and solvents met the highest HPLC grade or quality available.

2.2. LC-MS/MS conditions

The liquid chromatography was carried out on Dionex Ultimate 3000 UHPLC system equipped with a vacuum degasser, an auto-sampler, a binary pump and a column oven. An Acquity BEH C18 column (50 × 2.1 mm, i. d. 1.7 μm, Waters Corp.) kept at 40 oC was used for chromatographical separation with water containing 0.1% formic acid and acetonitrile as mobile phase, at a flow rate of 0.4 mL/min. The gradient programs were 0-0.2 min 20% B, 0.2-1.3 min 20-80% B, 1.3-1.8 min 80% B and 1.8-2.0 min 20% B. The mixture of methanol- acetonitrile-water-isopropanol (1:1:1:1, v/v/v/v) was used for washing the auto-sampler injection needle to reduce the carry-over effect. The injection volume was 2 μL.

Mass spectrometric detection was carried out on a Thermo Vantage TSQ mass spectrometer equipped with an electrospray ionization interface (ESI) operated in positive ion mode. The mass monitoring was conducted in multiple reactions monitoring (MRM) mode with precursor-to-product transitions at m/z 429.2 >135.1 for capivasertib and m/z 458.2 > 387.2 for ipatasertib, respectively. The collision energy was set at 30 V for capivasertib and 32 V for ipatasertib. The ESI sourceparameters were optimized as following: spray voltage, 3.0 kV; vaporizer temperature, 250 oC; sheath gas flow rate, 40 arb; aux gas flow rate 10 arb; capillary temperature 300 oC. Data acquisition and processing were achieved with Xcalibur software (Version 2.3.1, Thermo Fisher Scientific, USA).

2.3. Preparation of calibration standards and quality control samples

The standard of capivasertib was accurately weighted and dissolved in acetonitrile-methanol (1:1, v/v) to yield the stock solution at a concentration of 1 mg/mL. The stock solution was stepwise diluted with acetonitrile, resulting in the working solutions at the concentrations of 1, 5, 10, 50, 100, 200, 500 and 1000 ng/mL. The working standard solution (30 μL) was individually spiked into a 1.5-mL polypropylene tube and then evaporated to dryness by nitrogen blowing. The residue was mixed with 30 μL of blank dog plasma to prepare calibration standards at the concentrations of 1, 5, 10, 50, 100, 200, 500 and 1000 ng/mL. The quality control (QC) samples for method validation were prepared from a separated stock solution by the same procedures at the concentrations of 2, 80 and 800 ng/mL. The final ISTD working solution (1 μg/mL) was diluted with acetonitrile from the stock solution of 1 mg/mL. All the solutions were stored at -20 oC until use.

2.4. Sample pretreatment

Acetonitrile-mediated protein precipitation was used for sample preparation. To an aliquot of 30 μL of each dog plasma, 10 μL of ISTD working solution (1 μg/mL) was spiked. After vortexing for 2 min, the mixture was then spiked with 150 μL of acetonitrile and then vortexed for 5 min. Afterwards, the sample was centrifuged at 15000 rpm for 10 min to remove the denatured protein. The resulting supernatant (100 μL) was mixed with equal volume of water. After centrifugation at 15000 rpm for 5 min, a 2 μL aliquot of the supernatant was submitted to LC-MS/MS for analysis.

2.5. Validation procedures

The newly developed method was validated in accordance with the Guidance: Bioanalytical Method Validation (Food and Drug Administration, 2018). The method was validated for selectivity, carry-over, precision, accuracy, linearity, sensitivity, extraction recovery, matrix effect, storage stability, dilution integrity and incurred sample reanalysis (ISR) testing.

The selectivity of the assay was examined to confirm the influence of endogenous substances located at the retention times of capivasertib and ISTD. Blank dog plasma, blank dog plasma spiked with capivasertib at lower limit of quantification (LLOQ) and ISTD, and the plasma samples collected at 2 h after oral administration were used to demonstrate the selectivity of the assay. The blank dog plasma used for selectivity confirmation was obtained from six different individuals.

To investigate whether the capivasertib or ISTD remaining in the analytical instrument would impact the quantification as it appeared at the next sample injection, the carry-over test was performed by injecting two blank dog plasma samples following the upper limit of
quantification (1000 ng/mL). The peak in the blank sample < 20% of the LLOQ or < 5% of the ISTD was acceptable. Calibration curves were constructed by plotting the peak area ratios of capivasertib to ISTD versus the nominal concentrations of capivasertib spiked into plasma. A weight (1/x2) least square regression was applied. The calibration curves should be linear over the concentration range of 1-1000 ng/mL with the correlation coefficient > 0.99 (r > 0.99). The acceptance criteria of the accuracy of the calibration standards was with ± 15%. The sensitivity of the assay was indicated by the LLOQ, at which the ratio of signal-to-noise should be more than 10 (S/N > 10), and the precision and accuracy had to meet the required limits (±15%).

The intra-day precision and accuracy were determined by analyzing six replicates of QC samples at four concentration levels (1, 2, 80 and 800 ng/mL) in the same day. The inter-day precision and accuracy were investigated by analyzing six replicates of QC samples at four concentration levels on three successive days. The precision was calculated as relative standard deviation (RSD%) which should be < 15%. The accuracy expressed as relative error (RE%) was suggested to be within ±15%. The extraction recovery of capivasertib was evaluated at three QC concentration levels (2, 80 and 800 ng/mL) by comparing the peak area of the regularly prepared QC samples in six replicates with that of pre-extraction of blank samples spiked at the corresponding concentrations. The recovery was suggested to be > 80%. The matrix effect was investigated by comparing the peak area of the analyte resolved in extracted matrix of blank plasma with those in water-substituted samples. If the value was >115% or < 85%, matrix effect was implied. The extraction recovery and matrix effect of ISTD were determined in the same way. The stability of capivasertib in dog plasma was assessed by analyzing the QC samples at low (1 ng/mL) and high (800 ng/mL) concentration levels under the different storage conditions, including the long-term stability at -20 oC for 30 days, the short-term stability at room temperature for 12 h and after three cycles of freeze (-20 oC)-thaw (room temperature). The post-preparative stability was investigated by placing the processed QC samples at auto- sampler (10 oC) for 8 h. The analyte was considered stable when 85-115% of the concentration was remained. The dilution effect was evaluated by diluting the QC samples at the concentration of 5 μg/mL to 500 ng/mL (10-fold dilution). The RE% should be within ±15% with RSD less than 15%.To ensure the reproducibility of the newly developed method, ISR testing was conducted. A total of 20 samples were determined and compared with initial analyzed values. A minimum of 67% of the repeated values should be within 85-115% of the original values. 2.6. Animal experiments and pharmacokinetic study Male beagle dogs with body weight of 8-10 kg were provided by the Animal Experimental Center of Tongji Medical College (Wuhan, China). Dogs were housed in an environmentally controlled breeding room (temperature of 25 ± 2 oC and humidity of 55-65%) and fed with food and water ad libitum. Before drug administration, the dogs were fasted for 12 h but water was available. All the animal experiments were approved by the Ethics Committee of Tongji Medical College (Wuhan, China). One group of dogs (n = 5) were orally administered with capivasertib formulated in 0.5% CMC-Na-0.5% DMSO-99% saline at a single dose of 5 mg/kg. The other group of dogs (n = 5) were intravenously administered with capivasertib formulated in 0.5% CMC-Na-0.5% DMSO-99% saline at a single dose of 1 mg/kg. The blood samples (1 mL) were collected into heparinized tubes at 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h post- dose. The collected blood samples were immediately centrifuged at 5000 rpm for 5 min. The resulting plasma samples were separated and stored at -20 oC until analysis. 2.7. Data acquisition and processing The plasma samples were processed and analyzed as described above. In company with actual plasma samples, QC samples at low, medium and high concentrations were distributed in the analytical run and simultaneously analyzed in duplicate. More than 67% of the QC samples should be within ± 15% of the nominal concentration, and more than 50% at each QC concentration level should meet the criteria.The pharmacokinetic parameters were calculated by using DAS 2.0 software based on non-compartmental analysis (Version 2.0, Chinese Pharmacology Society), including area under the curve (AUC), maximum plasma concentration (Cmax), time to reach Cmax (Tmax), mean time residence (MRT), half-life (T1/2), volume of distribution (Vd) and clearance (CL). The oral bioavailability was calculated using the following equation: F(%) = (AUCoral × Dose intravenous)/(AUCintravenous × Dose oral) × 100%. 3. Results and discussions 3.1. Method development This is the first report regarding the method development and validation for the determination of capivasertib in biological matrices. In this study, a simple and reliable LC-MS/MS assay was developed for the determination of capivasertib in dog plasma. Initially, the mass conditions were investigated and optimized. Capivasertib and ISTD showed strong mass signal in positive ion mode, with protonated molecule [M+H]+ at m/z 429.2 and 458.2, respectively. The MS/MS spectra were displayed in Figure 1. Capivasertib showed two abundant product ions at m/z 215.1 and 135.1. The MRM transitions of m/z 429.2 > 135.1 and m/z 429.2 > 215.1 were compared and we found that the transition of m/z 429.2 > 135.1 had a higher sensitivity. Therefore, transition of m/z 429.2 > 135.1 was used for quantification, while transition of m/z
429.2 > 215.1 was used as a qualifier. For ISTD, the most abundant product ion was m/z 387.2.

For sample preparation, protein precipitation and liquid-liquid extraction were tested. For liquid-liquid extraction, ethyl acetate and methylene chloride were involved. Unfortunately, both organic solvents showed the relatively low extraction recovery (~70-75%). Further, protein precipitation with methanol and acetonitrile were tested. Acetonitrile showed higher sensitivity and lower matrix effect than did methanol. The extraction recovery produced by acetonitrile was much higher (>85%) than did liquid-liquid extraction. Finally, acetonitrile was used as the organic solvent for sample preparation.

3.2. Method validation

3.2.1 Selectivity

Figure 2 displayed the representative MRM chromatograms of blank dog plasma, blank plasma spiked with capivasertib and ISTD, and incurred samples collected at 2 h after oral administration. There were no significant interferences impacting the determination of capivasertib and ISTD. Under the current conditions, capivasertib and ISTD were eluted at the retention times of 0.89 and 1.22 min, respectively.

3.2.2. Carry-over

There were no clearly visible peaks of the analytes in the blank dog plasma after injecting ULOQ sample, suggesting that the carry-over was negligible.

3.2.3. Calibration curve and linearity

The developed assay shows an excellent linearity over the concentration range of 1-1000 ng/mL, with correlation coefficient > 0.999 (r > 0.999). The linear regression equation was y = (0.0062 ± 0.00021) x + (0.0029 ± 0.00012), where y means the peak area ratio of capivasertib to ISTD and x is the concentration of capivasertib spiked in dog plasma. The back-calculated concentrations of the calibrators were in the range of 85-115% of the nominal concentrations.

3.2.4. Sensitivity

The developed assay provides LLOQ of 1 ng/mL, at which the signal-to-noise was >10 and the accuracy and precision met the required limits (Table 1). This result demonstrated that the developed assay was sensitive enough to quantify the concentration of capivasertib in dog plasma.

3.2.5. Precision and accuracy

Table 1 presents the intra- and inter-day precision and accuracy for capivasertib. The inter-day RE ranged from -9.50 to 8.45% with RSD of < 9.58%. The intra-day RE ranged from -10.60 to 12.50% with RSD of < 7.78%. These data indicated that this assay was reproducible and accurate for the determination of capivasertib in dog plasma. 3.2.6. Extraction Recovery and Matrix Effect Table 2 summarizes the extraction recovery and matrix effect of capivasertib. The extraction recovery ranged from 85.81 to 93.45%, and the extraction recovery of ISTD was 89.54%, suggesting that the developed assay had excellent extraction efficiency. Matrix effect for capivasertib at three concentration levels ranged from 97.41% to 112.19%, and the matrix effect of ISTD was 94.56%, suggesting that the co-eluted substances did not influence the ionization of the analytes. 3.2.7. Stability Capivasertib was demonstrated to be stable under the tested storage conditions. As shown in Table 3, the RE% values ranged from -5.50% to 11.35%, with RSD <15%, suggesting that the method can be applied to a pharmacokinetic study. 3.2.8. Dilution integrity The results of accuracy and precision of the dilution QC samples were within acceptable range (±15%), indicating that the plasma sample could be quantified reliably by 10-fold dilution with blank plasma if the concentration of the analyte was above the calibration range. 3.2.9. ISR testing A total of 20 samples were determined to measure ISR. All the repeated values were in the range of 85-115% of the original values, indicating that the assay was reproducible for analysis of capivasertib. 3.3. Pharmacokinetic study With the validated LC-MS/MS method, the plasma samples collected from dogs were analyzed. Figure 3 shows the plasma concentration-time profiles of capivasertib after oral administration of capivasertib at 5 mg/kg and tail vein injection at 1 mg/kg. The relevant pharmacokinetic parameters are summarized in Table 4. After oral administration, capivasertib was detected at the first sampling time point (5 min), suggesting that capivasertib was rapidly absorbed. Capivasertib reached the maximum plasma concentration (Cmax) around 1 h post dose with Cmax of 1059.80 ± 231.19 ng/mL. The half-life (T1/2) was calculated to be 4.24 ± 0.60 h. After intravenous injection, capivasertib showed low clearance (CL) from plasma with CL of 7.53 ± 1.31 mL/min/kg, which is far below the hepatic blood flow (31 mL/min/kg). The half-life time (3.19 ± 0.61 h) was comparable to that after oral administration. The oral bioavailability was calculated to be 47.04%, suggesting that capivasertib had good absorption. 4. Conclusion In summary, a simple and reliable LC-MS/MS method was developed and validated for the first time to determine capivasertib in dog plasma. The method required 30 μL of plasma sample, yet retained adequate sensitivity (LLOQ 1 ng/mL). The method was also demonstrated to be sufficiently accurate and reliable. The method was validated according to FDA guidance. Moreover, the developed method was successfully applied to pharmacokinetic study of capivasertib in dog plasma. Pharmacokinetic results demonstrated that capivasertib had good absorption and low clearance in dogs. To the best of knowledge,AZD5363 this is the first report on determination of capivasertib in biological matrices and the application to a pharmacokinetic study.