MS method for determination of HZ08 in rat plasma and tissues: Application to a pharmacokinetic study of liposome injections

Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 246–252

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A rapid and sensitive UPLC–MS/MS method for determination of HZ08 in rat plasma and tissues: Application to a pharmacokinetic study of liposome injections Fang Yan a , Miaomiao Sun a , Taijun Hang a,∗ , Jing Sun a , Xia Zhou a , Xin Deng b , Liang Ge a , Hai Qian b , Ding Ya a , Wenlong Huang b a b

Department of Pharmacy, China Pharmaceutical University, Nanjing, PR China Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China

a r t i c l e

i n f o

Article history: Received 1 June 2014 Received in revised form 28 August 2014 Accepted 10 September 2014 Available online 19 September 2014 Keywords: Rat plasma Rat tissues HZ08 UPLC–MS/MS Pharmacokinetics

a b s t r a c t Overexpression of P-glycoprotein leads to tumor multidrug resistance (MDR). HZ08, a novel tetrahydroisoquinoline derivate, was discovered to inhibit the MDR in the cancer cell lines of MCF-7/ADM, K562/ADM and KBV in our previous studies. A rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometric method (UPLC–MS/MS) was developed and validated for determination of HZ08 in rat plasma and tissues after intravenous administration of HZ08 liposome injection at different doses. The analytes were extracted from plasma and tissues using protein precipitation by acetonitrile with clotrimazole as internal standard. The chromatographic separation was performed on a Thermo BDS HYPERSIL C18 column (100 mm × 4.6 mm, 2.4 ␮m) at a flow rate of 0.7 ml/min using 0.2% ammonium acetate solution (containing 0.1% formic acid) and methanol as mobile phase. The total run time was 4 min. The tandem mass detection was applied with electrospray ionization in positive ion selected reaction monitoring mode. The ion transitions monitored were m/z 523.5 to 342.3 for HZ08 and 277.1 to 165.1 for the internal standard, respectively. The calibration curves obtained were linear in different matrices, and the lower limit of quantification (LLOQ) achieved was 1 ng/ml for rat plasma and 0.25 ng/ml for rat tissues, respectively. The RSDs for intra- and inter-day precision were less than 15%. Extraction recovery, matrix effect and stability were satisfactory in rat plasma and tissues. The developed method was successfully applied to a pharmacokinetic study of HZ08 liposome injection following intravenous administration of 1, 3, 10 mg/kg to Sprague-Dawley rats. The data profiles revealed that HZ08 had linear pharmacokinetic properties at the tested doses, and was rapidly distributed into the systemic circulation with wide distribution throughout the body followed by a rapid elimination phase. The major distribution tissues of HZ08 in rats were lung, spleen and liver. These results provided constructive contribution to support the clinical evaluation. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Multidrug resistance (MDR) in cancer cells could lead to chemotherapeutic failure. The molecular mechanisms leading to MDR include the activation of transport and detoxification systems, enhancement of target repair activities, alterations of drug targets, and deregulation of cells death pathways [1–4]. With the aim of inhibiting MDR, a mount of chemicals were synthesized and discovered, with the ability to modulate relational targets [5–9]. Clinically, however, definitive benefit of cancer multidrug resistant inhibitor

∗ Corresponding author. Tel.: +86 02583271090; fax: +86 02583271269. E-mail address: [email protected] (T. Hang). http://dx.doi.org/10.1016/j.jpba.2014.09.017 0731-7085/© 2014 Elsevier B.V. All rights reserved.

still remains not to be established owing to the complex mechanism of MDR in anticancer drug treatment [10–12]. It is complicated by the fact that many chemomodulators were also involved in serious side effects or disappointing clinic effects [10–12], including the third-generation P-glycoprotein inhibitors (such as Zosuquidar (LY335979) [13,14] and Tariquidar (XR9576) [15]). HZ08, N-cyano-1-[(3,4-dimethoxyphenyl)methyl]-3,4-dihydro-6,7-dimethoxy-N -octyl-2(1H)-isoquinolinecarboximidamide, Fig. 1(A), was designed and synthesized as a novel MDR modulator on the basis of tetrahydroisoquinoline, which had lower activity of Ca2+ channel than verapamil. Our previous studies showed that HZ08 had strong effects on reversing MDR in vitro and in vivo, by competition of P-glycoprotein transport sites as substrate, enhancement of apoptosis induced by anticancer drugs,

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247

Fig. 1. Structure formula of HZ08 (A) and clotrimazole (B, internal standard).

generation of reactive oxygen species, depletion of GSH, potential depolarization of mitochondrial membrane, release of cytochrome c and activation of caspases [16–20]. Furthermore, HZ08 had little impact on cytochrome P450 activities in rat liver microsomes in vitro [21] and adriamycin pharmacokinetics of rats in vivo [22]. Therefore, it is possible that HZ08 would be a new strategy for the reversal of tumor MDR in clinic. In order to provide the systematic and scientific basis on clinical application of HZ08, its ADME characteristics should be determination in vivo in experimental animals. In our previous report, a high-performance liquid chromatographic method coupled with ion spray tandem mass spectrometry detection (HPLC–MS/MS) was developed for the determination of HZ08 in rat plasma with liquid–liquid extraction and gradient elution [23]. However, the complex sample preparation and long column equilibrium time of gradient elution decreased the work efficiency in the analysis of large amount of biological samples. Meanwhile, its sensitivity with the LLOQ of 5 ng/ml is not enough to meet the very low concentration of HZ08 in rat plasma and tissues, when experimental animals were intravenously (i.v.) administrated at low dose of 1 mg/kg HZ08 liposome injection (low dose of 2 mg/kg in our previous study). Recently, an improvement in chromatographic performance has been achieved by the introduction of UPLC with superior resolution and simpler analysis method [24]. In this research, an UPLC–MS/MS assay was established with good sensitivity (the LLOQs of 1 ng/ml in rat plasma and 0.25 ng/ml in rat tissues) and simple sample preparation to study the plasma pharmacokinetics and tissue distribution of HZ08 after intravenous administration of 1, 3, 10 mg/kg.

from 1 to 10,000 ng/ml. The clotrimazole solution was prepared in methanol at a concentration of 50 ng/ml. The stock and standard solutions were stored at 4 ◦ C before use and stable for at least 30 days (RE < 0.5%). 2.2. UPLC–MS/MS conditions An Acquity ultra-performance liquid chromatography (UPLC, Waters Corp., Milford, MA, USA) was coupled to an Acquity triplequadrupole system with an electrospray ion source and MassLynx 4.0 software (Waters Corp, Milford, MA, USA). The UPLC system consisted of a binary pump, a column heater and a cooling autosampler set at 4 ◦ C. UPLC separation was performed on a Thermo BDS HYPERSIL C18 analytical column (100 mm × 4.6 mm, 2.4 ␮m) at temperature of 35 ◦ C. The mobile phase consisted methanol and 0.1% formic acid solution (containing 0.2% ammonium acetate) (82:18, v/v) at the flow rate of 0.7 ml/min. The sample injection volume was 10 ␮l. Split injection of 50% of the eluent was introduced into the inlet of the mass spectrometer. The tandem MS detections were operated with positive electrospray ionization and multiple reaction monitoring (MRM) of [M+H]+ ions for both HZ08 and clotrimazole. The MRM transitions selected for determination were m/z 523.5 to 342.3 for HZ08 and m/z 277.1 to 165.1 for clotrimazole. The detection parameters were optimized as following: source temperature 150 ◦ C, nitrogen desolvation gas 350 ◦ C with a flow rate of 550 l/h, the argon collision energy 32 eV for both HZ08 and clotrimazole.

2. Materials and methods

2.3. Method validation

2.1. Reagents and chemicals

Full validation of the analytical method was conducted according to the US Food and Drug Administration guidelines for bioanalytical method validation [25]. Specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect, and stability were evaluated. The accuracy of all validation tests was expressed as the relative error (RE%) obtained by calculating the percentage difference between the found concentration over that of the spiked value. The precision was denoted by the relative standard deviation (RSD%). The accuracy should not exceed 15% and the precision was required to be within ±15% for all levels (±20% for LLOQ). The specificity was assessed by confirming non-interference at retention times of HZ08 and internal standard from the endogenous components in six blank plasma and tissue samples using the proposed extraction procedure and UPLC–MS/MS conditions. Sensitivity of analysis was defined as LLOQ in the calibration ranges for both plasma and tissue samples with acceptable precision and accuracy set by the guideline for bioassay (1 ng/ml in plasma and 0.25 ng/ml in tissues; n = 5), while the upper limits of quantification (ULOQs) set as the highest levels of concentration in the

The HZ08 liposome injection (60 mg HZ08 per vial) in freezedried form for intravenous administration and the chemical reference substance of HZ08 (Batch No. 201004061) with purity >99.0% were supplied by Professor Wenlong Huang in the Drug Discovery Center of China Pharmaceutical University (Nanjing, China). The HZ08 liposome injection was reconstituted in 5% (w/v) glucose injection before intravenous administration. The chemical reference substance of clotrimazole (internal standard, Fig. 1B, Batch No. 100037) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC grade methanol was obtained from Tedia Company Inc. (Fairfield, OH, USA). All other chemicals were of analytical reagent grade from Nanjing Chemical Reagent Factory (Nanjing, PR China). Triple-deionized water was used to prepare all aqueous solutions (Millipore, Bedford, MA, USA). Stock solution of HZ08 (400 ␮g/ml) was prepared in methanol, which was further diluted stepwise with methanol to obtain a series of standard solutions in the range

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calibration curves (10,000 ng/ml in rat plasma and 2500 ng/ml in rat tissues with five replicate injections). The linearity was evaluated by preparing and assaying a series of calibration standards with HZ08 concentration range of 1–10000 ng/ml in rat plasma and 0.25–3750 ng/ml in rat tissues. The calibration standard curves were obtained by plotting the peak area ratios of HZ08 to internal standard versus the concentrations of HZ08 with 1/C2 weighted least-squares linear regression analysis. The acceptance criterion of a calibration curve was a correlation coefficient (r) of 0.99 or better and the REs and RSDs for each level should be within ±15% for all levels (20% for the LLOQ). Intra-day and inter-day precision and accuracy were performed at five quality control (QC) levels of 2, 4, 100, 1000 and 10,000 ng/ml in rat plasma and 0.5, 1, 25, 250, 2500 ng/ml in rat tissue, respectively. The intra-day and inter-day precision and accuracy were calculated according to all above mentioned. The recovery was obtained at five concentration levels (2, 4, 100, 1000 and 10,000 ng/ml in rat plasma and 0.5, 1, 25, 250, 2500 ng/ml in rat tissue) by comparing the peak responses of the analytes in the five samples with the responses of the equivalent amount of the analytes in standard solutions spiked in post-extracted blank samples. The matrix effect was measured via comparison of the peak responses of the analytes in the extracted blank matrix to those obtained from neat standard solutions at equivalent concentrations. The stability of HZ08 at post-preparation (room temperature), long-term storage (−80 ◦ C), short-term storage (−4 ◦ C) and freeze-thaw cycles were investigated with the samples of 50 and 5000 ng/ml in rat plasma and tissues. 2.4. Sample preparation Plasma samples were thawed and vortex-mixed before use. After thawed, rat tissue samples were minced and homogenized with deionized water (heart, muscle, and testis, 1:5, w/v; brain, stomach, duodenum, fat, skin and uterus, 1:10, w/v; lung, liver, spleen and kidney, 1:30, w/v; ovaries, 1:30, w/v) thoroughly. An aliquot of 50 ␮l rat plasma sample or 200 ␮l rat tissue samples was spiked with 50 ␮l of 50% acetonitrile solution, 50 ␮l of internal standard solutions (50 ng/ml clotrimazole) and different volume of acetonitrile (200 ␮l for rat plasma samples or 450 ␮l for rat tissue samples) in 1.5 ml Eppendorf tube. After vortex for 2 min and centrifugation at 16,000 rpm for 10 min, 10 ␮l of the supernatant solution was injected into the UPLC–MS/MS system. The same procedures of sample preparation were applied for linearity, recovery, precision, accuracy and stability tests. 2.5. Animals Sixty-two Sprague-Dawley rats with the body weights of 193 ± 14 g, were purchased from the Animal Central of the Chinese Academy of Science in Shanghai (Shanghai, China). All the animals were housed in an air-conditioned animal quarter at a temperature of 22 ± 2 ◦ C and a relative humidity of 50 ± 10%, and kept on 12 h light/dark cycle with free access to food. The animals were fasted overnight for 12 h prior to dosing. All animal experiments were carried out according to the Guidelines of the Committee on the Care and Use of Laboratory Animals in China, and approved by the Animal Ethics Committee of China Pharmaceutical University. 2.6. Rat pharmacokinetics and tissue distribution For the pharmacokinetic study, the rats were randomly divided into three groups by intravenous administration of 1 (5 male, 5 female), 3 (5 male, 5 female) and 10 (5 male, 5 female) mg/kg HZ08 liposome injection. Rat blood samples (0.2 ml) were collected in heparinized Eppendorf tubes via the oculi chorioideae vein at 0

(pre-dose), 0.033, 0.083, 0.167, 0.25, 0.33, 0.50, 0.75, 1, 2, 4 and 6 h post-dose, respectively. The plasma samples were separated by centrifugation at 12,000 rpm for 4 min and stored at −80 ◦ C until analysis. To study the tissue distribution of HZ08, all the rats were randomly divided into four groups, with 8 (4 male, 4 female) in each. After intravenous administration of HZ08 liposome injection at a dose of 3 mg/kg to each group, brain, heart, lung, liver, spleen, kidney, stomach, duodenum, fat, muscle, skin, uterus, ovaries and testis samples were collected at 0.033, 0.167, 1, 4 h post-dose, respectively. Tissue samples were rinsed with normal saline solution to remove the blood, dried with Whatman filter paper (Grade No. 1), weighed rapidly and stored at −80 ◦ C until analysis. 2.7. Data analysis For the pharmacokinetic study, non-compartmental pharmacokinetic analysis of all data was performed using the DAS software (Version 2.0, Medical College of Wannan, China). The maximum concentration (Cmax ) of HZ08 in plasma and the time to reach the maximum concentration (Tmax ) were directly obtained from the observed values. Other non-compartmental parameters, such as AUC0−t , mean residence time (MRT), terminal elimination half-life (t1/2 ), mean residence time (MRT) and clearance (CLZ ), were calculated by standard formula in previous studies [24]. For tissue distribution studies, the concentrations of HZ08 in rat tissue samples were expressed in terms of ␮g/g tissue and calculated by equation: Ct = CS VS /P, where Ct represented the tissue concentration (␮g/g), CS , VS and P were the concentration (␮g/ml), the volume (ml) and the weight (g) of the sample, respectively. All the data were expressed as mean ± standard deviation (S.D.). Results were analyzed using one-way ANOVA followed by Turkey post hoc test or Student’s t-test. Differences with P values less than 0.05 were considered to be significant. 3. Results and discussion 3.1. Method development The internal standard was selected according to its chemical structure and chromatographic behavior. In the previous study, drotaverine was reported as IS since it and HZ08 were both the derivatives of tetrahydroisoquinoline [23]. However, the polarity of HZ08 was weaker than drotaverine due to the octylamine branch of HZ08, which caused the significant difference of the chromatographic retention between drotaverine and HZ08. Though gradient elution was applied to shorten the analytical time, the continuous change of the organic phase proportion might influence the ionization procedure of HZ08 and drotaverine, which led to the quantitative deviation of HZ08 in biological samples. Considering that it was a kind of alkaloid with weak polarity, clotrimazole was suitable for its chromatographic behavior and mass spectrometry response of IS. Mobile phase was optimized to achieve satisfactory chromatographic behavior, such as symmetrical peak shape, short run time and good separation from interferences. In this study, methanol was chosen as the organic phase because it provided higher responses than acetonitrile. Due to the similar structure of HZ08 with tetrahydroisoquinoline, sensitivity and peak symmetry of HZ08 was remarkably improved, when the aqueous phase contained 0.1% formic and 0.2% ammonium acetate. The concentrations of formic acid and ammonium acetate in mobile phase were investigated from 0.01% to 0.3%. The addition of 0.1% formic acid and 0.2% ammonium acetate led to enough strong response for HZ08 and IS. Moreover, the superior resolution and shorter analysis time were

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Table 1 Precision, accuracy, recovery and matrix effect for the determination of HZ08 in rat plasma (n = 5) (mean ± S.D.). Nominal concentration (ng/ml)

Intra-day RSD (%)

Inter-day RSD (%)

Accuracy RE (%)

Recovery (%)

2 4 100 1000 10,000

10.3 12.8 13.7 6.6 2.3

5.9 6 2.4 2.3 2.8

10.6 8.5 −8.9 2.4 7.5

97.5 101.9 96.6 96.3 95.8

± ± ± ± ±

5.6 3.7 5.6 4.0 2.8

Matrix effect (%) 112 104.7 101.5 102.9 104.9

± ± ± ± ±

4.1 8.9 5.5 2.4 0.6

The sample size of n represents the number of spiked plasma and tissues samples for each concentration level.

achieved by the same mobile phase with isocratic elution instead of gradient elution. Finally, methanol and 0.1% formic acid solution (containing 0.2% ammonium acetate) (82:18, v/v) was selected as the mobile phase with an isocratic flow rate of 0.7 ml/min for 4 min. To optimize ESI conditions, HZ08 and IS were dissolved in mobile phase and directly injected into the mass spectrometer. Both HZ08 and IS represented higher responses in positive mode than negative mode. Full scan Q1 MS spectra for HZ08 and IS predominantly contained protonated precursor [M+H]+ ions at m/z 523.5 and 277.1, respectively. The most abundant and stable product ions in Q3 MS spectra for HZ08 and IS were observed at m/z 342.3 and 165.1, respectively. Protein precipitation method was attempted as sample preparation with methanol and acetonitrile for simplicity and time saving. Compared with the liquid–liquid extraction method reported previously [23], protein precipitation possessed higher recovery and better precision. Though protein precipitation might decrease the sensitivity due to increasing the sample volume, the UPLC–MS/MS method with the lower LLOQs of 1 ng/ml for rat plasma and 0.25 ng/ml for rat tissues was satisfactory to determine HZ08 in different matrices. Consequently, the addition of acetonitrile was applied because acetonitrile could absolutely precipitate protein with the less volume than methanol.

3.2. Method validation 3.2.1. Specificity The specificity of the method was evaluated by the analysis of blank rat plasma and tissues from six different sources. The detection of HZ08 and IS by MRM mode of MS detector was highly

selective with no interference from the endogenous substances. Typical chromatograms of blank plasma, blank plasma spiked with HZ08 and IS, and rat plasma collected at 0.5 h after i.v. injection of 3 mg/kg HZ08 liposome, are shown in Fig. 2 (typical chromatograms of rat tissues are shown in Supplementary Material). The retention times of HZ08 and IS were 3.03 min and 3.25 min, respectively.

3.2.2. Calibration curve and LLOQ The calibration curves demonstrated good linearity over the concentration ranges of 1–10000 ng/ml and 0.25–3750 ng/ml for HZ08 in rat plasma and tissues. Mean calibration equations were Y = 0.004944X + 0.002064 (r = 0.9990) in rat plasma, Y = 0.01236X + 0.0006317 (r = 0.9983) in rat liver tissue, Y = 0.01174X + 0.001009 (r = 0.9985) in rat brain tissue, Y = 0.01288X + 0.0002751 (r = 0.9984) in rat muscle tissue and Y = 0.01243X + 0.0006154 (r = 0.9986) in rat testis tissue, where Y represented the peak area ratio of HZ08 to clotrimazole (internal standard) and X represented the sample concentrations of HZ08. The RE and RSD for each level were within ±15% for all levels (20% for the LLOQ) in different matrix. The present method offered a LLOQ of 1 ng/ml with RSD of 8.1% and the accuracy of 99.37% in only 50 ␮l rat plasma. The estimated LLOQ for a 200 ␮l aliquot of liver, brain, muscle and testis samples were each 0.25 ng/ml, with RSD of 7.2%, 9.1%, 8.1% and 4.9%, and accuracy of 99.0%, 103.4%, 103.5% and 110.9%, respectively. The ULOQs were 10,000 ng/ml in rat plasma with RSD and precision of 1.2% and 100.7%, and 3750 ng/ml in liver, brain, muscle and testis samples with RSD of 3.7%, 2.5%, 7.3% and 1.5%, and precision of 102.1%, 99.7%, 102.4% and 101.8%, respectively. The LLOQs were sufficient for the pharmacokinetic and tissue distribution studies of HZ08 in rats.

Table 2 Precision, accuracy, recovery and matrix effect for the determination of HZ08 in rat tissues (n = 5) (mean ± S.D.). Tissues

Nominal concentration (ng/ml)

Intra-day RSD (%)

Inter-day RSD (%)

Accuracy RE (%)

Recovery (%)

Liver

0.5 1 25 250 2500

14.3 5.7 4.2 6.9 5.4

7.0 6.0 2.7 2.5 3.6

0.2 −1.3 4.8 1.2 −7.1

98.5 102.5 93.3 94.8 95.3

± ± ± ± ±

7.6 8.6 1.6 1.0 5.9

95.8 97.6 101.5 100.3 99.5

± ± ± ± ±

11.0 8.8 3.6 2.1 0.6

Brain

0.5 1 25 250 2500

11.5 14.4 4.7 8.3 8.0

4.5 5.6 2.2 1.9 1.8

2.7 3.5 7.5 3.1 −3.8

94.6 93.4 97.1 100.8 96.4

± ± ± ± ±

4.7 4.9 2.7 1.9 4.7

102 100 101.5 99.3 92.5

± ± ± ± ±

2.0 6.6 0.3 3.5 0.9

Muscle

0.5 1 25 250 2500

15.6 17.6 8.2 4.7 0.3

4.9 5.2 3.0 3.9 3.1

1.2 1.0 1.9 −5.7 −7.5

104.9 95.3 105.9 98.1 106.7

± ± ± ± ±

7.4 3.8 4.9 4.9 2.5

97.3 103.8 101.5 91.8 94.9

± ± ± ± ±

7.3 15.3 0.4 1.2 0.8

Testis

0.5 1 25 250 2500

20.2 2.8 5.9 4.1 4.6

3.8 5.0 3.4 3.8 3.2

0.2 −1.4 0.1 −3.2 −6.6

103.7 92.7 101.1 98.6 105.3

± ± ± ± ±

4.6 6.8 7.7 5.1 4.2

103.8 100.4 101.5 100.2 98.5

± ± ± ± ±

3.4 2.1 1.5 2.5 3.1

The sample size of n represents the number of spiked plasma and tissues samples for each concentration level.

Matrix effect (%)

250

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Fig. 2. Typical MRM chromatograms of HZ08 in rat plasma samples. (A) Blank rat plasma. (B) Plasma spiked with 0.25 ng/ml HZ08 (tR = 3.03 min) and 50 ng/ml clotrimazole (tR = 3.25 min). (C) Plasma sample after administration of 3 mg/kg HZ08 liposome injection at 0.5 h.

3.2.3. Precision and accuracy The precision and accuracy of the method were assessed at five levels of QC samples in different matrices (2, 4, 100, 1000 and 10,000 ng/ml in rat plasma; 0.5, 1, 25, 250 and 2500 ng/ml in rat tissues). The intra- and inter-day precision (RSD) for the analysis of HZ08 in different matrices were less than 15% (Tables 1 and 2). Mean inter- and intra-day accuracy (RE) in rat plasma and different rat tissues, also met the requirement of the analysis of the biological sample (<15%, data shown in Tables 1 and 2), which indicated that the assay was reproducible and accurate for the determination HZ08 in rat plasma and tissues. 3.2.4. Recovery and matrix effect The recovery of HZ08 at five different concentration levels were within 90–110% in spiked rat plasma and tissues (Tables 1 and 2) with the intra- and inter-level RSD less than 15%, which met the requirement of a pharmacokinetic study of HZ08 liposome injection in rat. As shown in Tables 1 and 2, the matrix effect values at the five QC concentration levels of HZ08 ranged from 95% to 110% in rat plasma and tissues with the RSDs lower than 10%, which indicated that no significant matrix effects were observed at each concentration level. 3.2.5. Stability The stability of HZ08 in different matrices were summarized in Table 3, which demonstrated that HZ08 was stable for over 3 months kept at −80 ◦ C, 8 h at −4 ◦ C, up to three freeze-thaw cycles and at least 8 h at room temperature without obvious changes in the concentrations tested within the time period under the indicated storage conditions. 3.3. Pharmacokinetic study The validated analytical method was successfully applied to investigate the pharmacokinetics of HZ08 after a single i.v. (1, 3, 10 mg/kg) administration. The corresponding pharmacokinetic parameters calculated using non-compartmental model are listed

Fig. 3. Mean plasma concentration–time profiles of HZ08 in rat plasma after intravenous administration at three doses.

in Table 4. The mean plasma concentration–time profiles were depicted in Fig. 3 demonstrating that HZ08 was eliminated rapidly from rat plasma. With regard to the dose response in plasma kinetics of HZ08 in rats, AUC0−t and Cmax increased proportionally within the dose range from 1 to 10 mg/kg implicating linear pharmacokinetics. There were no significant differences for other parameters, including t1/2 , CLz and MRT among the three dosages analyzed by one-way ANOVA (P > 0.05). In different sexes groups, the main pharmacokinetic parameters including Cmax , t1/2 , MRT, CLz and AUC0−t were not statistically different at the three dosages (P > 0.05). 3.4. Tissue distribution in rats The tissue concentrations of HZ08 determined at 0.033, 0.167, 1 and 4 h after i.v. administration at a dose of 3 mg/kg, are shown in Fig. 4, which indicated that HZ08 was distributed rapidly in tissues. At 0.033 h, after i.v. administration, the highest tissue concentration of HZ08 was observed in heart (3107 ng/g), lung (35,787 ng/g), brain (528.0 ng/g), fat (268.8 ng/g), muscle (245.2 ng/g), skin (219.5 ng/g), uterus (281.6 ng/g), ovaries (1701 ng/g) and testis (153.5 ng/g), except liver, spleen, kidney, stomach and duodenum. At 0.167 h, the tissue concentrations of HZ08 in liver (38,170 ng/g), spleen (12,159 ng/g), kidney (4050 ng/g), stomach (995.4 ng/g) and duodenum (785.3 ng/g), reached the highest levels. At 4 h, the amount

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Table 3 The stability of HZ08 in rat plasma and tissues (n = 5). Conditions

Tissues

Nominal concentration (ng/ml)

Accuracy RSD (%) RE (%)

Long-term stability (3 months, −80 ◦ C)

Plasma

50 5000 50 5000 50 5000 50 5000 50 5000

1.2 2.9 0.3 1.5 4.4 1.6 8.9 0.2 8.3 −2.4

2.1 1.3 4.9 1.9 3.9 1.7 4.2 2.6 4.4 2.0

50 5000 50 5000 50 5000 50 5000 50 5000

−0.3 2.4 −4.4 14.4 4.0 0.1 9.1 2.3 8.1 0.6

0.8 1.6 4.7 5.6 4.5 0.8 4.8 1.8 4.8 1.9

50 5000 50 5000 50 5000 50 5000 50 5000

−2.8 −0.6 2.9 1.4 5.6 −4.7 3.4 0.5 −0.6 −9.4

1.9 2.3 4.0 3.6 4.7 2.3 3.4 2.3 3.6 3.9

50 5000 50 5000 50 5000 50 5000 50 5000

0.7 5.5 5.9 0.7 5.6 8.3 5.9 −2.1 3.6 −7.3

0.7 5.3 3.6 1.1 4.7 1.9 3.4 2.2 3.3 4.0

Liver Brain Muscle Testis Plasma

Short-term stability (8 h, −4 ◦ C)

Liver Brain Muscle Testis Plasma

Freeze-thaw stability (3 cycles)

Liver Brain Muscle Testis Post-preparative stability (8 h, room temperature)

Plasma Liver Brain Muscle Testis

The sample size of n represents the number of spiked plasma and tissues samples for each concentration level.

of HZ08 was few in all the collected tissues. Compared with the corresponding plasma concentrations, the tissues of liver, spleen and lung exhibited the higher exposure. The distributions in brain, heart, kidney, stomach, duodenum, fat, muscle, skin, uterus, ovaries and testis, were approximate to or lower than that in plasma. Meanwhile, the exposure of different tissues was similar between male and female. Notably, the amount of HZ08 in ovaries was half of the corresponding plasma concentrations at the same time point, leading to possible reproductive toxicity in female rats. Tissue distribution study showed a specific uptake and

Table 4 Mean pharmacokinetic parameters for HZ08 in rats (n = 10) (mean ± S.D.). Parameters

Dose (mg/kg) 1

Tmax (h) Cmax (ng/ml) AUC0−t (h ng/ml) MRT (h) CLz (L/h/kg) t1/2 (h)

0.033 780 136 0.77 7.4 1.8

3 ± ± ± ± ± ±

0 176 32 0.2 1.7 0.4

0.033 3655 603 0.52 7.0 1.4

10 ± ± ± ± ± ±

0 747 104 0.1 1.0 0.4

0.033 6160 1343 0.66 7.9 1.3

± ± ± ± ± ±

0 1125 398 0.1 2.3 0.3

Data expressed as mean ± S.D., with n as the number of rats included in the study.

Fig. 4. The distributed amount of HZ08 in rat main tissues at 0.033, 0.167, 1 and 4 h post-dose following intravenous administration of HZ08 liposome injection at 3 mg/kg.

accumulative character of HZ08 liposome injection. Most of the knowledge concerning the behavior of liposome injection in vivo, indicated that liposome given intravenously, was absorbed onto the surface of vesicles mediated their endocytosis by fixed macrophages of the reticuloendothelial system and circulating monocytes, and the liver, spleen and lung took up nearly all liposome given by the i.v. route [26–28]. Therefore, HZ08 liposome after i.v. administration was mainly distributed in lung, liver and spleen. However, it is worth noting that the accumulation of HZ08 in ovaries possibly led to reproductive toxicity in female rats. 4. Conclusion In the present study, a simple and sensitive UPLC–MS/MS method was developed and validated, which was successfully applied to the pharmacokinetics of HZ08 liposome injection in plasma and tissues of rats, including plasma kinetics and tissue distribution. The data profiles showed that HZ08 had linear pharmacokinetic properties with rapid absorption and elimination, and was widely distributed throughout the rat body after intravenous administration at three doses. The major distribution tissues of HZ08 in rats were lung, spleen and liver. These pharmacokinetic results provide constructive contribution to the future clinical investigations. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.09.017. References [1] H. Ishii, M. Iwatsuki, K. Ieta, D. Ohta, N. Haraguchi, K. Mimori, M. Mori, Cancer stem cells and chemoradiation resistance, Cancer Sci. 99 (2008) 1871–1877. [2] B.C. Baguley, Multiple drug resistance mechanisms in cancer, Mol. Biotechnol. 46 (2010) 308–316. [3] K.G. Chen, B.I. Sikic, Molecular pathways: regulation and therapeutic implications of multidrug resistance, Clin. Cancer Res. 18 (2012) 1863–1869. [4] J.P. Gillet, M.M. Gottesman, Mechanisms of multidrug resistance in cancer, Methods Mol. Biol. 596 (2010) 47–76. [5] M. Ito, K. Kajino, M. Abe, T. Fujimura, R. Mineki, T. Ikegami, T. Ishikawa, O. Hino, NP-1250, an ABCG2 inhibitor, induces apoptotic cell death in mitoxantrone-resistant breast carcinoma MCF7 cells via a caspase-independent pathway, Oncol. Rep. 29 (2013) 1492–1500. [6] D. He, X.Q. Zhao, X.G. Chen, Y. Fang, S. Singh, T.T. Talele, H.J. Qiu, Y.J. Liang, X.K. Wang, G.Q. Zhang, Z.S. Chen, L.W. Fu, BIRB796, the inhibitor of p38 mitogen-activated protein kinase, enhances the efficacy of chemotherapeutic agents in ABCB1 overexpression cells, PLOS ONE 8 (2013) e54181. [7] Y. Lei, J. Tan, M. Wink, Y. Ma, N. Li, G. Su, An isoquinoline alkaloid from the Chinese herbal plant Corydalis yanhusuo W.T. Wang inhibits P-glycoprotein and multidrug resistance-associate protein 1, Food Chem. 136 (2013) 1117–1121.

252

F. Yan et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 246–252

[8] Y.Z. Zhao, D.D. Dai, C.T. Lu, L.J. Chen, M. Lin, X.T. Shen, X.K. Li, M. Zhang, X. Jiang, R.R. Jin, X. Li, H.F. Lv, L. Cai, P.T. Huang, Epirubicin loaded with propylene glycol liposomes significantly overcomes multidrug resistance in breast cancer, Cancer Lett. 330 (2013) 74–83. [9] S.G. Das, D.L. Hermanson, N. Bleeker, X. Lowman, Y. Li, A. Kelekar, C. Xing, Ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3 -carboxylate (CXL017): a novel scaffold that resensitizes multidrug resistant leukemia cells to chemotherapy, ACS Chem. Biol. 8 (2013) 327–335. [10] B. Goldman, Multidrug resistance: can new drugs help chemotherapy score against cancer? J. Natl. Cancer Inst. 95 (2003) 255–257. [11] C.F. Higgins, Multiple molecular mechanisms for multidrug resistance transporters, Nature 446 (2007) 749–757. [12] Z. Binkhathlan, A. Lavasanifar, P-glycoprotein inhibition as a therapeutic approach for overcoming multidrug resistance in cancer: current status and future perspectives, Curr. Cancer Drug Targets 13 (2013) 326–346. [13] P. Ruff, D.A. Vorobiof, J.P. Jordaan, G.S. Demetriou, S.D. Moodley, A.L. Nosworthy, I.D. Werner, J. Raats, L.J. Burgess, A randomized, placebo-controlled, double-blind phase 2 study of docetaxel compared to docetaxel plus zosuquidar (LY335979) in women with metastatic or locally recurrent breast cancer who have received one prior chemotherapy regimen, Cancer Chemother. Pharmacol. 64 (2009) 763–768. [14] L. Pusztai, P. Wagner, N. Ibrahim, E. Rivera, R. Theriault, D. Booser, F.W. Symmans, F. Wong, G. Blumenschein, D.R. Fleming, R. Rouzier, G. Boniface, G.N. Hortobagyi, Phase II study of tariquidar, a selective P-glycoprotein inhibitor, in patients with chemotherapy-resistant, advanced breast carcinoma, Cancer 104 (2005) 682–691. [15] L.D. Cripe, H. Uno, E.M. Paietta, M.R. Litzow, R.P. Ketterling, J.M. Bennett, J.M. Rowe, H.M. Lazarus, S. Luger, M.S. Tallman, Zosuquidar, a novel modulator of P-glycoprotein, does not improve the outcome of older patients with newly diagnosed acute myeloid leukemia: a randomized, placebo-controlled trial of the Eastern Cooperative Oncology Group 3999, Blood 116 (2010) 4077–4085. [16] F. Yan, Y. Jiang, Y.M. Li, X. Zhen, J. Cen, W.R. Fang, Reversal of P-glycoprotein and multidrug resistance-associated protein 1 mediated multidrug resistance in cancer cells by HZ08 isomers, tetrataisohydroquinolin derivatives, Biol. Pharm. Bull. 31 (2008) 1258–1264. [17] J. Cen, Y. Qi, Y.F. Tao, Y. Deng, W.R. Fang, Y.M. Li, L.Y. Zhang, W.L. Huang, HZ08, a great regulator to reverse multidrug resistance via cycle arrest and apoptosis sensitization in MCF-7/ADM, Eur. J. Pharmacol. 647 (2010) 21–30.

[18] J. Cen, Y.L. Zhu, Y. Yang, J.R. Zhu, W.R. Fang, W.L. Huang, Y.M. Li, Y.F. Tao, Effects of the multidrug resistance modulator HZ08 on the apoptosis pathway in human chronic leukaemia cell line K562/A02, Arzneimittelforschung 61 (2011) 622–630. [19] Y.L. Zhu, J. Cen, Y.Y. Zhang, Y.D. Feng, Y. Yang, Y.M. Li, W.L. Huang, The multidrug resistant modulator HZ08 reverses multidrug resistance via P-glycoprotein inhibition and apoptosis sensitization in human epidermoid carcinoma cell line KBV200, Arzneimittelforschung 62 (2012) 236–242. [20] Y. Feng, Y. Hu, J. Cen, K.N. Darshika, W. Fang, Y. Li, W. Huang, HZ08 inhibits the multi-drug resistance on multiple sites as the substrate of P-glycoprotein, Eur. J. Pharmacol. 712 (2013) 53–59. [21] F. Yan, Y.M. Li, Q.J. Wang, W.R. Fang, K. Kang, Effects of HZ08, a novel P-glycoprotein inhibitor, on the reversal of P-glycoprotein mediated multidrug resistance in nude mice and cytochrome P-450 activities in rat liver microsomes, J. Chin. Pharm. Univ. 39 (2008) 447–452. [22] F. Yan, W.R. Fang, J. Cen, T.J. Hang, Y.M. Li, Effect of HZ08, a novel modulator of tumor multidrug-resistance, on adriamycin pharmacokinetics in rats, Chin. J. News Drugs 19 (2010) 2170–2196. [23] J.Y. Weng, M. Song, T.J. Hang, W.L. Huang, Y. Du, Pharmacokinetics of HZ08 in rats by liquid chromatography–tandem mass spectrometry, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 856 (2007) 29–34. [24] M.I. Churchwell, N.C. Twaddle, L.R. Meeker, D.R. Doerge, Improving LC–MS sensitivity through increases in chromatographic performance: comparisons of UPLC–ES/MS/MS to HPLC–ES/MS/MS, J Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 825 (2005) 134–143. [25] US Department of Health and Human Services, Guidance for Industry Analytical Procedures and Methods Validation for Drugs and Biologics, 2014, Available at: www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/ guidances/ucm386366.pdf [26] R.T. Gomes, R.P. Camargos, A.P. Viotti, A.P. Tavares, M.P. Revelo, T.V. Freitas, Comparison of the biodistribution of free or liposome-entrapped Crotalus durissus terrificus (South American rattlesnake) venom in mice, Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 131 (2002) 295–301. [27] H. Shmeeda, Y. Amitay, D. Tzemach, J. Gorin, A. Gabizon, Liposome encapsulation of zoledronic acid results in major changes in tissue distribution and increase in toxicity, J. Control. Release 167 (2013) 265–275. [28] N. Mirahmadi, M.H. Babaei, A.M. Vali, S. Dadashzadeh, Effect of liposome size on peritoneal retention and organ distribution after intraperitoneal injection in mice, Int. J. Pharm. 383 (2010) 7–13.