Abstract
Previously compound I showed great anti-glioblastoma activity without toxicity in a mouse xenograft study. In this study, a sensitive and rapid high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed and validated to investigate the pharmacokinetics and brain distribution of compound I in mice. The protein precipitation method was applied to extract the compound from mouse plasma and brain homogenates, and it was then separated using a Kinetex C18 column with a mobile phase consisting of acetonitrile–0.1% formic acid water (50:50, v/v). The analytes were detected with multiple reaction monitoring for the quantitative response of the compounds. The inter- and intra-day precisions were <8.29 and 3.85%, respectively, and the accuracy range was within ±7.33%. The method was successfully applied to evaluate the pharmacokinetics of compound I in mouse plasma and brain tissue. The peak concentration in plasma was achieved within 1 h. The apparent elimination half-life was 4.06 h. The peak concentration of compound I in brain tissue was 0.88 μg/g. The results indicated that compound I was rapidly distributed and could cross the blood–brain barrier. The pharmacokinetic profile summarized provides valuable information for the further investigation of compound I as a potential anti-glioblastoma agent.
Keywords: anti-glioblastoma, blood–brain barrier, HPLC–MS/MS, pharmacokinetics
1 ∣. INTRODUCTION
Glioblastoma (GBM) is a common brain cancer and is considered the most aggressive malignant brain cancer in adults. The prognosis of GBM remains poor even when the combination treatment of chemotherapy and radiotherapy are performed after surgical removal of the tumor (Simon et al., 2020). The median survival rate of the patients is about 15 months with the treatment because of the high recurrence rate and resistance to therapeutics (Davis, 2016). Temozolomide (TMZ), an oral alkylating chemotherapy prodrug, is often used after surgical resection in the current treatment (Stupp et al., 2009). However, recurrence usually occurs within months after TMZ treatment. Therefore, there is an urgent need to develop a new therapeutic approach to treatment of GBM.
Since the androgen receptor (AR) was first detected in astrocytoma in 1996 (Chung et al., 1996), many studies have indicated that AR could be a potential target for the treatment of GBM. Testosterone could stimulate the progression of GBM by promoting proliferation, migration and invasion via activating AR functions (Rodríguez-Lozano et al., 2019). Also, Zalcman et al. revealed that siRNA-induced AR gene silencing triggered GBM cell death, while enzalutamide (AR antagonist) reduced the GBM tumor growth in nude mice (Zalcman et al., 2018). However, the AR mutation (AR-V7), which commonly occurs in GBM, causes the AR antagonist agents to be less effective (Zalcman et al., 2018). HSP27 is a well-documented chaperone protein to stabilize AR (Kiliccioglu et al., 2019; J. Li et al., 2018; Liu et al., 2018), and inhibition of HSP27 results in AR degradation regardless of the mutation (Zoubeidi et al., 2007). Thus, HSP27 suppression could be a novel approach for AR overexpressed GBM treatment.
Compound I (Figure 1), an HSP27 inhibitor, has been identified to abolish AR in GBM (Y. Li et al., 2021). Compound I could significantly downregulate AR and AR-V7 at 50 nM, and the IC50 to inhibit GBM cell growth could reach 5 nM. Moreover, compound I could inhibit GBM tumor growth in the mouse xenograft model, and does not cause toxicity to animals even at much higher doses. Compound I could be a potential drug candidate treat GBM. However, whether compound I can cross the blood–brain barrier is a critical factor to consider for the treatment of GBM. Thus, a pre-clinical pharmacokinetic investigation and brain tissue detection of compound I are urgently needed to determine the pharmacokinetic profiles of compound I. In the present study, a sensitive and reliable HPLC–MS/MS method was established and validated for the determination of the concentration of compound I in mice plasma and brain tissue. The method was successfully applied to the pharmacokinetic study of compound I in mice after intraperitoneal injection.
FIGURE 1.
Full-scan product ion spectrum and proposed fragmentation pathways of (a) compound I and (b) compound 14 (internal standard, IS)
2 ∣. MATERIALS AND METHODS
2.1 ∣. Chemicals and reagents
Compound I and internal standard (IS) compound 14 (Figure 1) were prepared and characterized according to published methods (Zhong et al., 2013). MS-grade acetonitrile and ACS-grade methanol were purchased from Sigma-Aldrich (St Louis, MO, USA), and LC/MS-grade formic acid was purchased from Fisher Scientific (Waltham, MA, USA). Double-deionized water was prepared using a Barnstead Nanopure® water purification system from Thermo Scientific (Waltham, MA, USA).
2.2 ∣. Animals
C57BL/6 mouse (36 males, 20–25 g) bred at the animal facility of Cleveland State University were used for the study. All of the mice were given free access to water and a standard laboratory diet, and were maintained under standard conditions (25 ± 2°C, 12 h dark–light cycle, 50 ± 10% humidity) in accordance with the institutional guidelines for Animal Research Facilities. All mice fasted overnight but were allowed free access to water before the drug injection.
2.3 ∣. Instrumentations and HPLC–MS/MS conditions
The HPLC–MS/MS method was performed with a Shimadzu UPLC system (Columbia, MD, USA) which consisted of a Prominence DGU-20A3R inline degasser, two LC–30 AD pumps, a SIL-30 AC autosampler and a CBM-20A controller. The chromatographic separation was performed on a Kinetex C18 column (50 × 2.1 mm, 1.3 μm) with a mobile phase consisting of acetonitrile–0.1% formic acid and water (50:50, v/v) at a flow rate of 0.3 ml/min. The temperature of the column was maintained at 36°C. The injection volume was 5.0 μl.
Mass spectrometric detection was operated on an AB Sciex Qtrap 5500 mass spectrometer (Toronto, Canada) in negative electrospray ionization mode. The multiple reaction monitoring function was used for quantification with the transitions of compound I and IS compound 14, which were detected at m/z 485.1 → 256.1 and m/z 499.2 → 268.2, respectively. The proposed fragmentation pathways of compound I and compound 14 are shown in Figure 1. The optimized ion source parameters were set as follows: ion spray voltage, −4,200 V; temperature, 450°C; heating gas, nebulization gas and curtain gas, 40 psi. Compound parameters were as follows: declustering potential, −100 V; entrance potential, −10 V; collision energy, −35 V for compound I, −40 V for compound 14; collision exit potential, −15 V for compound I, −20 V for compound 14. Data acquisition and quantification were performed using Analyst software (version 1.6.2).
2.4 ∣. Preparation of standards and samples
2.4.1 ∣. Preparation of stock and working solution
The stock solutions were prepared by dissolving compound I and compound 14 in methanol at 1.0 mg/ml. Then, the stock solution of compound I was serially diluted with methanol into a concentration gradient: 1.0, 2.0, 5.0, 10, 20, 50, 100, 200, 500 and 1,000 ng/ml. Also, a 250 ng/ml working solution of compound 14 (IS) was prepared in methanol from the stock solution of compound 14. All of the solutions were stored at 4°C in the dark.
2.4.2 ∣. Preparation of calibration standards and quality control
The calibration standards were prepared as follows: after spiking with 100 μl of the corresponding standards solutions, 40 μl of compound 14 working solution, 100 μl of blank mouse plasma or brain homogenate (0.4 g blank brain tissue mixed with 2 ml phosphate-buffered saline, PBS) and 800 μl of methanol was transferred into a 1.5 ml tube. The mixture was then vortexed and centrifuged at 12,000g for 10 min. The supernatant was collected and transferred into a new 1.5 ml tube. After being dried with nitrogen, the residue was stored at −80°C and dissolved in 50% acetonitrile before analysis. Also, the quality control (QC) samples were independently prepared with the same blank mouse plasma or brain homogenates. The preparation method was the same as for the calibration standards. The QC samples were prepared at concentrations of 2 ng/ml (low), 50 ng/ml (medium) and 200 ng/ml (high), and were then divided into aliquots and stored in the freezer at −80°C before analysis.
2.4.3 ∣. Preparation of samples
A simple protein precipitation method was applied to extract compound I from mouse plasma or brain homogenate (0.4 g brain tissue mix with 2 ml PBS). Briefly, 100 μl of each sample, 40 μl of compound 14 (IS, 250 ng/ml) and 800 μl of methanol were combined in a 1.5 ml tube. Then, the mixture was vortexed and centrifuged at 12,000g for 5 min. The supernatant was collected and then transferred into a new 1.5 ml tube. The liquid was dried using a nitrogen-blowing instrument. The residue was stored at −80°C and dissolved with 100 μl 50% acetonitrile before analysis.
2.5 ∣. Method validation
In accordance with the US Food and Drug Administration bioanalytical method validation guidance, the HPLC–MS/MS method was validated in terms of selectivity, linearity and sensitivity, precision and accuracy, matrix effect, and recovery and stability (US Department of Health and Human Services, 2018).
2.5.1 ∣. Selectivity
The selectivity was determined by testing the blank plasma from six mice or blank brain tissue homogenate, blank matrices spiked with IS only, and plasma and brain samples obtained from mice dosed with compound I.
2.5.2 ∣. Linearity and sensitivity
The linearity of this method was examined via analysis of the standard curve containing 10 different concentrations (1–1,000 ng/ml). The standard curve was obtained by plotting the peak area ratios (compound I/compound 14, y-axis) vs. the concentrations (x-axis) of compound I and assessed by weighted least-squares linear regression using 1/x2 as the weighting factor. The lowest concentration (1.0 ng/ml) was considered as the lower limit of quantification (LLOQ).
2.5.3 ∣. Precision and accuracy
The precision and accuracy were investigated with the QC samples in five replicates, which were prepared and analyzed on three continuous days. The intra- and inter-day precision was represented as the RSD and the accuracy was expressed as the RE.
2.5.4 ∣. Extraction recovery and matrix effect
Extraction recovery was measured by calculating the ratio of the responses of QC samples spiked with analytes prior to extraction to the responses of those spiked with protein-precipitated blank plasma or brain homogenates. The matrix effect was evaluated by measuring the ratio of the responses in blank plasma or brain homogenates to those dissolved with 50% acetonitrile (v/v) at the same concentration. Generally, the extraction recovery should be >85% and the matrix effects should be <15% according to US Food and Drug Administration guidance for methodology development.
2.5.5 ∣. Stability
The stability was evaluated by testing five replicates of the samples at three QC levels under different conditions (24 h storage at room temperature, three freeze–thaw cycles and storage at −80° C for 30 days). The samples are considered stable if the average percentage concentration deviation is within 15% of the actual value.
2.6 ∣. Pharmacokinetic study
A dosing solution (5 mg/ml) of compound I was prepared in preclinical formulation vehicle [5% Cremophor EL (CrEL) with PBS]. The mouse received a 100 mg/kg dose of compound I by intraperitoneal (i.p.) administration. A 400 μl mouse blood sample and the whole brain tissue were collected at each time point by euthanizing the treatment group of mice at 0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 12 and 24 h, respectively. The blood was directly collected from heart, then followed by perfusion with PBS and brain tissue was collected consequently. The blood samples were centrifuged immediately at 3,000g for 10 min at 4°C, and the supernatant plasma was collected, and immediately stored at −80°C until analysis. The brain tissue was weighed out and mixed with PBS (1:5, m/v) in a Dounce Tissue Grinder (DWK Life Sciences), homogenized on ice. Then, aliquots of the homogenates were immediately frozen and stored at −80°C until analysis.
2.7 ∣. Data analysis
The plasma and brain homogenates samples were subjected to HPLC–MS/MS analysis as above and the concentrations of compound I were calculated according to the calibration curve. Microsoft Excel 2020 (Microsoft Co., USA) was used to calculate the pharmacokinetic parameters. GraphPad Prism 5.0 (GraphPad Software, USA) was used to plot the concentration–time curve. All of the results are expressed as means ± SD.
3 ∣. RESULTS AND DISCUSSION
3.1 ∣. Optimization of chromatography conditions and MS conditions
The chromatographic condition was optimized to improve peak shape, increase the signal response of analytes and shorten the retention time. In the current chromatographic separation, a Kinetex C18 column was used to separate the analytes. The composition of the mobile phase, especially the organic phase, was optimized in order to acquire good chromatographic separation. Compared with methanol–water, it was found that acetonitrile–water obtained a better peak shape. The signal response was significantly improved with 0.1% formic acid added to the water phase. Eventually, acetonitrile–0.1% formic acid and water (50:50, v/v) was adopted as the mobile phase at a flow rate of 0.3 ml/min. Under the optimized condition, sharp peaks of analyte and internal standard were acquired with consistent separation. The retention times were 1.51 min for compound I and 1.88 min for compound 14, respectively.
In order to optimize the mass spectrometer parameters, 200 ng/ml of compound I and 100 ng/ml of compound 14 in acetonitrile–0.1% formic acid and water (50:50, v/v) were injected into the mass spectrometer through direct infusion at 10 μl/min. Negative modes of electrospray ionization were assessed for compound I and compound 14 (Figure 1) and were used for the study. The results showed that the predominant deprotonated molecular ions [M − H]− were m/z 485.1 (compound I) and m/z 499.2 (compound 14). The multiple reaction monitoring function was used for quantification of the transitions of compound I and internal standard compound 14, which were detected at m/z 485.1 → 256.1 and m/z 499.2 → 268.2, respectively.
3.2 ∣. Method validation
3.2.1 ∣. Selectivity
Selectivity was used to evaluate the potential interference of endogenous substances with the detection of analytes. In this present study, the retention time was 1.51 min for compound I and 1.88 min for compound 14. Both the analyte and internal standard showed well-separated peaks with no significant interference from endogenous substances observed at their corresponding retention times under current detection condition. Typical chromatograms of blank mouse plasma or brain, blank plasma or brain homogenates spiked with IS (compound 14, 100 ng/ml) and plasma and brain samples from mice with i.p. administration of compound I in 5% CrEL at 0.5 h are shown in Figure 2.
FIGURE 2.
Typical chromatograms of different samples: (a) blank mouse plasma; (b) mouse plasma spiked with IS (compound 14, 100 ng/ml) only; (c) plasma samples from mice with i.p. administration of compound I in 5% Cremophor EL (CrEL) at 1.0 h; (d) blank brain homogenates; (e) brain homogenates spiked with IS (compound 14, 100 ng/ml) only; and (f) samples from mice administered i.p. with compound I in 5% CrEL at 1.0 h
3.2.2 ∣. Linearity and sensitivity
The calibration curve of the ratio of analyte/IS peak area (y) to the concentration (x) was plotted via a 1/x2 weighted linear least-square regression model. The calibration curve of compound I in mouse plasma was y = 0.1234x + 0.7208, which exhibited good linearity (r = 0.990). The calibration curve of compound I in brain tissue was y = 0.1460x + 0.4691, which exhibited good linearity (r = 0.996). The LLOQ for compound I was determined to be 1.0 ng/ml in different mouse plasma and brain tissue samples.
3.2.3 ∣. Precision and accuracy
The precision was demonstrated as the RSD of the QC samples at three concentration levels (2, 50 and 500 ng/ml) while the accuracy was expressed as the RE. The precisions of the intra- and inter-day accuracy are shown in Table 1. The RSDs for intra- and inter-day precisions are <3.85 and 8.29%, respectively. The RE for accuracy is within ± 7.33%. The results show that the method is acceptable for precision and accuracy.
TABLE 1.
Intra- and inter-assay precision, accuracy for the determining compound I in mouse plasma and brain tissue (n = 3 days, 5 replicates per day)
| Samples | Analyte concentration (ng/ml) | Intra-day (RSD %) | Inter-day (RSD %) | Accuracy (RE%) |
|---|---|---|---|---|
| 2 | 1.80 | 8.29 | −5.88 | |
| Plasma | 50 | 2.05 | 4.41 | −6.16 |
| 200 | 2.77 | 4.50 | 3.12 | |
| 2 | 1.81 | 4.85 | 6.21 | |
| Brain | 50 | 1.24 | 4.55 | −5.54 |
| 200 | 3.85 | 7.77 | −7.33 |
3.2.4 ∣. Extraction recovery and matrix effects
Extraction recovery and matrix effects of QC samples from mouse plasma and brain tissue at three different concentrations (2, 50 and 200 ng/ml) were evaluated. The results are listed in Table 2. The results show that the extraction recoveries were >93.16% for three different levels of QC samples. The results indicate that this extraction method was qualified by the requirement for determination of compound I drug concentration in plasma and brain samples. The matrix effects at three concentrations were within 92.24–111.24%, indicating minor matrix effects.
TABLE 2.
Extraction recovery and matrix effect of compound I in mouse plasma and brain tissue (n = 5)
| Samples | Analyte concentrations (ng/ml) | Extraction recovery (%) | RSD (%) | Matrix effect (%) | RSD (%) |
|---|---|---|---|---|---|
| 2 | 93.16 ± 12.86 | 13.80 | 96.23 ± 6.30 | 6.55 | |
| Plasma | 50 | 105.30 ± 2.57 | 2.44 | 104.22 ± 5.42 | 5.20 |
| 200 | 95.02 ± 4.46 | 9.74 | 92.37 ± 10.43 | 11.29 | |
| 2 | 110.54 ± 12.95 | 11.71 | 92.24 ± 7.23 | 7.84 | |
| Brain | 50 | 110.15 ± 8.88 | 8.06 | 99.38 ±9.11 | 9.17 |
| 200 | 117.45 ± 13.54 | 11.53 | 111.24 ± 10.42 | 9.37 |
3.2.5 ∣. Stability
The stability of compound I in mouse plasma and brain homogenates was investigated by measuring the concentration of compound I in QC samples with three different storage conditions, including three freeze–thaw cycles and maintenance at room temperature for 24 h (short term) and at −80°C for 30 days (long term). The results are shown in Table 3. The RE values for compound I in mouse plasma and brain homogenate at three different levels of QC samples were <8.7%. Compound I was found to be stable in mouse plasma and brain homogenate after three freeze–thaw cycles and after storing at room temperature for 24 h and at −80°C for 30 days.
TABLE 3.
Stability of compound I in mouse plasma and brain tissue (n = 5)
| Three freeze–thaw cycles |
Room temperature for 24 h |
Long-term (30 days, −80°C) |
|||||
|---|---|---|---|---|---|---|---|
| Samples | Analyte concentration (ng/ml) | RSD % | RE % | RSD % | RE % | RSD % | RE % |
| 2 | 11.59 | −8.7 | 9.56 | 6.1 | 12.44 | 7.5 | |
| Plasma | 50 | 9.97 | 5.8 | 10.21 | −2.7 | 8.63 | −6.4 |
| 200 | 8.66 | −3.5 | 6.64 | 6.5 | 6.76 | 5.6 | |
| 2 | 8.45 | 5.7 | 8.14 | −3.8 | 4.87 | 5.9 | |
| Brain | 50 | 8.58 | 7.2 | 10.51 | −4.4 | 12.42 | 7.8 |
| 200 | 8.51 | 4.5 | 5.71 | −5.5 | 9.52 | 7.1 | |
3.3 ∣. Pharmacokinetic study and brain tissue determination
The validated HPLC–MS/MS method was successfully used to evaluate the concentration of compound I in mouse plasma and brain tissue after the i.p. dose (100 mg/kg). The mouse plasma and brain tissue concentrations of compound I at different dosing times are exhibited in Figure 3. The pharmacokinetic parameters are listed as means ± SD in Table 4. The results showed that compound I was rapidly absorbed and distributed and the peak concentration (Cmax) was achieved within 1.0 h. The apparent elimination half-life (t1/2) was 4.06 h, indicating that compound I could be cleared rapidly from the mouse plasma. Also, the Cmax of compound I in mouse plasma reached 68.97 μg/ml. Moreover, compound I was detected in brain tissue after i.p. administration. The Cmax of compound I in brain tissue was 0.88 μg/g (1–2% of the concentration in blood), indicating that part of compound I in the blood circulation was able to pass the blood–brain barrier and accumulate in the brain tissue (Figure 3c and d).
FIGURE 3.
Plasma (a and b) and brain (c and d) tissue concentration–time profiles of compound I with i.p. administration in mice (mean ± SD, n = 4)
TABLE 4.
Noncompartmental pharmacokinetic parameters of compound I in mouse after i.p. administration (n = 4, mean ± SD)
| Pharmacokinetic parameters |
Value | |
|---|---|---|
| Plasma | Brain | |
| t 1/2 | 4.06 ± 1.65 (h) | 3.24 ± 0.62 (h) |
| C max | 68.97 ± 6.66 (μg/ml) | 0.88 ± 0.85 (μg/g) |
| AUC0–24 h | 65.44 ± 37.35 (μg·h/ml) | 1.54 ± 0.81 (μg·h/g) |
| AUC0–∞ h | 77.96 ± 20.42 (μg·h/ml) | 1.66 ± 0.78 (μg·h/g) |
t1/2, Elimination half-life; Cmax, peak concentration; AUC, area under concentration–time curve.
4 ∣. CONCLUSIONS
A reliable and sensitive HPLC–MS/MS method for the quantification of compound I in mouse plasma and brain tissue was developed. The method was accurate, efficient, reliable and successfully applied for evaluation of the pharmacokinetics of compound I in the in vivo study. The results indicated that compound I was rapidly distributed. Moreover, compound I could cross the blood–brain barrier, which indicates the compound could be delivered to the central nervous system. The pharmacokinetic profile summarized in the study provides valuable information for the further investigation of compound I as a potential anti-glioblastoma agent.
ACKNOWLEDGEMENT
This research was supported by grant from National Institute of Neurological Disorders and Stroke 1R15NS116766-01A1 (B. Su), National Science Foundation Major Research Instrumentation grants (CHE-0923398 and CHE-1126384) and the Faculty Research Development and the Center for Gene Regulation in Health and Disease of Cleveland State University.
Footnotes
DECLARATION OF COMPETING INTERESTS
The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
DATA AVAILABILITY STATEMENT
Original mass spectrum data are available upon request.
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Associated Data
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Data Availability Statement
Original mass spectrum data are available upon request.