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In vivo microdialysis with LC–MS for analysis of spinosin and its interaction with cyclosporin A in rat brain, blood and bile
Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 22–29
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In vivo microdialysis with LC–MS for analysis of spinosin and its interaction with cyclosporin A in rat brain, blood and bile Rong-Hua Ma a,1 , Jie Yang a,1 , Lian-Wen Qi a , Gui-Zhong Xin a , Chong-Zhi Wang b , Chun-Su Yuan b , Xiao-Dong Wen a,∗ , Ping Li a,∗∗ a
State Key Laboratory of Natural Medicines (China Pharmaceutical University), Nanjing 210009, China Tang Center for Herbal Medicine Research and Department of Anesthesia & Critical Care, The Pritzker School of Medicine, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA b
a r t i c l e
i n f o
Article history: Received 13 September 2011 Received in revised form 11 November 2011 Accepted 12 November 2011 Available online 22 November 2011 Keywords: Spinosin In vivo microdialysis LC–/MS Cyclosporin A Drug–drug interactions
a b s t r a c t Spinosin, a major bioactive herbal ingredient isolated from Semen Ziziphi Spinosae, plays an important role in sedation and hypnosis. However, the pharmacokinetic behavior of spinosin in special sites has not been reported. Microdialysis (MD) technique, as a continuous, realtime monitoring sampling technique, is very suitable for the evaluation of the disposition of diverse drugs. To obtain more useful information on spinosin, an in vivo microdialysis sampling technique with High Performance Liquid Chromatography–mass spectrograph (HPLC–MS) method was developed to investigate the pharmacokinetics of spinosin and its interaction with cyclosporin A (CsA) in the brain, blood and bile of rats. The method was validated in terms of selectivity, linearity and sensitivity, and showed advantages in monitoring the pharmacokinetic behavior of drugs. The results revealed that CsA has obvious effects on the pharmacokinetic process of spinosin. When co-administered, the area under the curve (AUC) of spinosin in blood, bile and brain increased from 205.70 to 673.51 mg min/L, 7.77 × 104 to 1.25 × 105 mg min/L, and 2.09 to 5.58 mg min/L, respectively. The t1/2 values of spinosin in blood, bile and brain also changed from 48.07 to 95.04 min, from 97.20 to 152.21 and from 42.18 to 73.83 min, respectively. These results demonstrated that the CsA decreased the efflux of spinosin through the inhibition of P-glycoprotein (P-gp) efflux transporter and it might be used as a group of P-gp substrate. Other transporters or pathways may also be involved in the metabolism of spinosin. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Pharmacokinetic (PK) behavior of drugs plays a crucial role in the determination of their pharmacological actions. Any alterations in pharmacokinetic parameters will affect their therapeutic efficacy. Spinosin (2 -ˇ-O-glucopyranosyl swertisin, C28 H38 O15 , Fig. 1), a bioactive herbal ingredient isolated from Semen Ziziphi Spinosae, plays an important role in sedation and hypnosis [1,2]. It significantly potentiated the hypnotic effect of pentobarbital by decreasing sleep latency, increasing sleeping time, and enhancing the rate of sleep onset induced by subhypnotic doses of pentobarbital [3]. The mechanism of this action may be related to postsynaptic 5-HT1A receptors [4]. Although spinosin possesses significant activities, previous pharmacokinetic studies [5–9] are not sufficient for illustrating its mechanism of action and supplying research
∗ Corresponding author. Tel.: +86 25 8327 1379; fax: +86 25 8327 1379. ∗∗ Corresponding author. Tel.: +86 25 8327 1382; fax: +86 25 8327 1382. E-mail addresses: [email protected] (X.-D. Wen), [email protected] (P. Li). 1 These authors contributed equally to this work. 0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.11.014
information for clinical use. The pharmacokinetic behavior of spinosin in special sites has not been reported elsewhere. In recent decades, the crucial role of efflux transporters in drug absorption and disposition has gained considerable attention [10]. The inhibition or induction of transporters can lead to significant drug–drug interaction by affecting various pharmacokinetic parameters of the drug [11]. P-glycoprotein (P-gp), the most extensively studied ATP-binding cassette transporter, functions as a biological barrier by extruding toxic substances and xenobiotics out of cells [12]. The hypothesis that inhibition of P-gp improves the bioavailability of drugs that are substrate for this efflux transporter is gaining widespread recognition [13]. However, the role of P-gp efflux transporter in spinosin metabolism and disposition has not been investigated. In many clinical cases, determining the total drug concentration does not always produce the designed data because only the free drug concentration is responsible for the distribution, elimination, and therapeutic effect of drugs [14]. MD is a sampling technique that not only enables direct measurement of free drug levels at the biophase and continuous sampling without net fluid loss, but also permits monitoring of local concentrations of drugs
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Fig. 1. The chemical structure of spinosin and puerarin (internal standard).
and metabolites at specific sites [15]. Recently, there has been growing interest in the use of microdialysis to evaluate the disposition of diverse drugs [16–18]. PK involving MD commonly requires an efficient and sensitive analytical method to measure considerably low concentration of the analyte in the microdialysate [15]. HPLC–MS with its high stability and sensitivity is particularly suitable for determination of compounds at low levels in biological fluids. Thus, microdialysis sampling techniques coupled with the HPLC–MS method provides an attractive tool for pharmacokinetic research. To obtain more useful information on the metabolic fate of spinosin, this study aimed to investigate the pharmacokinetics of unbound spinosin in the brain, blood and bile of rats. For this purpose, an in vivo microdialysis sampling technique with the HPLC–MS method was developed and validated. The role of P-gp efflux transporter in spinosin metabolism and disposition was also investigated by comparing the PK of spinosin with and without CsA, a well-established P-gp inhibitor [19,20]. 2. Experimental 2.1. Chemicals and materials Spinosin was isolated from Semen Ziziphi Spinosae in the authors’ laboratories and its structure in Fig. 1 was elucidated by Infrared Spectroscopy (IR), mass spectrography (MS), Carbon Nuclear Magnetic Resonance Spectroscopy (1 H NMR) and Nuclear Magnetic Resonance Spectroscopy (13 C NMR) methods and by comparison with the data in references [21]. The purity of this compound was determined to be higher than 98% by normalization of the peak area detected by HPLC. The internal standard (IS) Puerarin (Fig. 1) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). CsA was purchased from Novartis Pharma Stein AG (Basel, Switzerland). Acetonitrile was of HPLC grade from Merck (Darmstadt, Germany). Purified water prepared by the Millipore system (Millipore, Bedford, MA, USA) was used for all the preparations. Other chemicals were of analytical grade.
Institutional Animal Experimentation Committee of China Pharmaceutical University (Nanjing, China). The rats were anesthetized with urethane 1 g/kg (i.p.) and remained anesthetized throughout the experimental period. The femoral vein was exposed for further drug administration. The rat’s body temperature was maintained at 37 ◦ C during the experimental procedure. 2.3. Microdialysis experiments Blood, brain and bile microdialysis systems consisted of a 3syringe bracket microdialysis pump with a Bee Syringe Pump Controller (Bioanalytical System Inc., West Lafayette, IN, USA). Blood and bile duct microdialysis probes were constructed inhouse based on a previous design [16]. A dialyzing membrane of 10 and 65 mm were used for blood and bile sampling, respectively. Under urethane anesthesia, the flexible blood microdialysis probe was implanted into the jugular vein toward the rat’s right atrium, and then perfused with anticoagulant citrate dextrose (ACD) solution (3.5 mM citric acid, 7.5 mM sodium citrate, and 13.6 mM dextrose) at a flow rate of 2 L/min. The bile microdialysis probe was perfused with Ringer’s solution (147 mM NaCl, 2.4 mM CaCl2 , and 4.0 mM KCl with a pH of 7.3) at a flow rate of 2.0 L/min. For brain sampling, the rat was mounted on a stereotaxic frame (RWD Life Science Co. Ltd, Shenzhen China). An incision was made in the scalp, and a small hole was drilled for striatum implantation of a rigid brain microdialysis probe. Stereotaxic coordinates from bregma for ventral hippocampus (HC): −5.6 mm rostral, 5.0 mm medial, −3.4 mm ventral from the top of the skull according to the Paxinos and Watson. A microdialysis probe (MD-2204, 4 mm membrane, Bioanalytical system, Inc., USA) was lowered into the hippocampus of the anesthetized rat. Perfusion of the microdialysis probe with filtered Ringer’s solution (147 mM NaCl, 2.4 mM CaCl2 , and 4.0 mM KCl with a pH of 7.3) was begun shortly before probe insertion and continued for the duration of the experiment delivered by a microinjection pump at a constant flow-rate of 2 L/min. The blood, bile and brain samples were drawn at every 20 min after administration of spinosin until 6 h (interval of 10 min periods for the first 120 min). The midpoints of the sampling times were used for blood, bile and brain spinosin concentration-time profiles.
2.2. Experimental animals 2.4. Drug administration Sprague–Dawley rats (280 ± 20 g) were obtained from SinoBritish Sippr/BK Lab. Animal Co. Ltd. (Shanghai, China) and housed with unlimited access to food and water except for fasting 12 h before the experiment. The animals were maintained on a 12-h light/12-h dark cycle (light on at 8:00) at ambient temperature (22–25 ◦ C) and 60% relative humidity. All experimental animal surgery procedures were reviewed and approved by the
Spinosin (2 mg/mL) was dissolved in saline (containing 0.5% dimethyl sulfoxide (DMSO)). CsA was diluted with 0.9% (w/v) sodium chloride to 10 mg/mL. For the control group, 5 mg/kg of spinosin was administrated through the femoral vein to each rat 2 h after the blood, brain and bile probe were implanted for the equilibrium of the dialysis membrane. For CsA co-administration
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groups, 10 mg/kg CsA was given through the femoral vein 10 min prior to spinosin injection. The volume of injection was 1 mL/kg. 2.5. Sample preparation A stock solution of an internal standard (IS) of puerarin for blood was prepared in a concentration of 0.5 g/mL in ACD solution. The blood analysis samples were prepared by spiking 3 L (0.5 g/mL) of the corresponding IS stock solutions into 12 L dialysate followed by vortexing. And stock IS solutions of puerarin for bile and brain were prepared in Ringer’s solution at 0.11 and 0.15 g/mL, respectively. The bile analysis samples were prepared by spiking 18 L (0.11 g/mL) of the corresponding IS stock solutions into 2 L dialysate followed by vortexing. And the brain analysis samples were prepared by spiking 3 L (0.15 g/mL) of the corresponding IS stock solutions into 12 L dialysate followed by vortexing. 2.6. HPLC–MS system
ACD with Ringer’s solution and forming different serial concentrations (2.00 × 10−2 , 5.00 × 10−2 , 1.00, 5.00, 10.0, 20.0, 50.0 g/mL and 5 × 10−3 , 8 × 10−3 , 1.00 × 10−2 , 5.00 × 10−2 , 1.00 × 10−1 , 2.00 × 10−1 , 5.00 × 10−1 g/mL in bile and brain, respectively), bile and brain calibration samples were prepared just as the same as blood samples. Standard working solutions were prepared every day, immediately before use. The quality control (QC) samples with low, middle and high concentrations for blood (3.12 × 10−2 , 3.12 × 10−1 and 3.12 g/mL), bile (5.00 × 10−2 , 1.00, 20.0 g/mL) and brain (1.00 × 10−2 , 1.00 × 10−1 , 5.00 × 10−1 g/mL) were also prepared one by one and daily in the same manner. All solutions were stored at −20 ◦ C before use. For a standard curve, the ratio of the chromatographic peaks area (analytes/IS) as ordinate variables were plotted versus the concentration of spinosin as abscissa. The limit of detection (LOD) and limit of quantification (LOQ) determinations were considered as the final concentration producing a signal-to-noise ratio of 3 and 10, respectively. Both were measured with at least five replications.
Chromatographic analysis was performed on an Agilent 1100 series LC system (Agilent, Germany) equipped with a binary pump and a thermostatically controlled column apartment. Chromatographic separation was carried out at 25 ◦ C on an Angilent extend-C18 column (50 mm × 4.6 mm I.D., 1.8 m) with a C18 guard column (Hanbang, Jiangsu, China). The mobile phase consisted of 0.1% formic acid water (Solvent A) and acetonitrile (Solvent B). The injection volume of blood, brain, bile samples are 10 L, 10 L, 5 L, respectively. The chromatograph was developed with a 10 min linear gradient from 15% Solvent A to 50% Solvent B followed by a 5 min re-equilibration. The flow rate was set at 0.5 mL/min. To avoid ion suppression, a post-column Diverter valve was applied to remove the ions in microdialysate. Detections were performed by a single quadrupole mass spectrometer (Product No. G2710BA, Agilent Corp, Santa Clara, CA, USA) equipped with an ESI source. The mass range was set at m/z 100–1000. The ESI-MS data were acquired in positive mode and conditions of MS analysis were as follows: drying gas (N2 ) flow-rate, 10 L/min; drying gas temperature, 300 ◦ C; nebulizing gas (N2 ) pressure, 16 psi; capillary voltage, 3000 V; quad temperature, 100 ◦ C; fragmentor, 100 V. The samples from the fast HPLC column were analyzed in selective ion monitoring (SIM) mode by monitoring the molecular ions m/z 417 for puerarin and m/z 609 for spinosin. SIM for each compound were restricted to specific retention time windows: 0–10 min, m/z 417, m/z 609 (retention time: 6.3 min, 7.4 min). All the operations, the acquiring and analysis of data were controlled by Chemstation software (Agilent Technologies, USA).
2.7.3. Accuracy and precision The intra-day precision and accuracy of the method were determined by analysis of the QC samples five times in a single day while its inter-day precision and accuracy were estimated by analysis of the QC samples three times in three consecutive days. The accuracy was calculated as mean percent deviation (RE) of the observed concentration (Cobs ) from the nominal concentration (Cnom ), accuracy (%RE) = [(Cobs − Cnom )/Cnom ] × 100. The precision was expressed as percent of coefficient of variation (CV), precision (%CV) = [Standard deviation (SD)/mean Cobs ) × 100. The acceptable intraday and interday precision is required to be less than 15% and the acceptable accuracy is required to be within 15% for all QC samples.
2.7. Method development
2.7.6. Recoveries of microdialysis probes A retrodialysis calibration technique was applied to recovery determination [22]. The experiment was carried as described in Section 2.3 except the perfusate was replaced by the spinosin ACD solution (for blood microdialysis) or Ringer’s solution (for bile and brain microdialysis) in certain concentrations. The perfusate (Cperf ) and dialysate (Cdial ) concentrations of spinosin were determined by the HPLC–MS system. The in vivo relative recovery (Rdial ) of spinosin across the microdialysis probe was calculated by the following equation: Rdial = (Cperf − Cdial )/Cperf . Spinosin microdialysate concentrations (Cm ) were converted to unbound concentration (Cu ) by the estimated in vivo recoveries (Rdial ) as follows: Cu = Cm × n/Rdial (n: microdialysis samples dilute multiples, the dilution multiples of samples are 1.25, 10 and 1.25 in blood, bile and brain, respectively).
2.7.1. Selectivity The selectivity of the method was tested by analyzing drugfree blood, bile and brain dialysate from five different rats. Each dialysate sample was tested using the HPLC–MS conditions proposed to insure no interference with spinosin and IS. 2.7.2. Calibration curves Because of the differences in concentration ranges and matrix between blood, brain and bile dialysates, the calibration curves were prepared separately. The stock solution of spinosin for blood at 1.04 mg/mL was prepared in ACD solution (containing 10% DMSO), and then diluted with ACD solution to make the working standard solutions of different serial concentrations. Then, 10 L of IS for blood and 10 L of the corresponding working standard solutions were mixed with 80 L of blank blood dialysate to form serial concentrations (1.04 × 10−2 , 3.12 × 10−2 , 1.04 × 10−1 , 3.12 × 10−1 , 1.04 and 3.12 g/mL). Except for replacing
2.7.4. Matrix effect Since the ionization of the analyte may influenced by the coeluting, undetected endogenous matrix compounds, the matrix effect of analytes must be investigated during the analysis. It was evaluated by comparing the peak area of IS and spinosin in the blank dialysate with that at the same concentrations of IS and spinosin in methanol. 2.7.5. Stability The stability of analytes was evaluated by analyzing QC samples at three concentrations exposed to different time and temperature conditions: modeling three freeze/thaw cycles (−20 ◦ C to ambient temperature), 24 h storage at room temperature and frozen at −70 ◦ C for 1 month.
2.8. Data analysis Pharmacokinetic parameters in rats were estimated by a noncompartmental method using Drug and Statistics 2.0 (DAS
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Fig. 2. Selected ion monitoring chromatograms from (A) blank blood dialysate; (B) blank blood dialysate spiked with spinosin and puerarin, the concentrations of spinosin and puerarin 0.624 g/mL and 0.1 g/mL, respectively; (C) blood dialysate collected at 20 min after administration, showing a spinosin concentration of 0.559 g/mL; and (D) blood dialysate at 20 min after spinosin and CsA co-administration, showing a spinosin concentration of 1.247 g/mL.
2.0) software package (Mathematical Pharmacology Professional Committee of China, Shanghai, China). The area under the concentration-time curve (AUC) was calculated according to the log linear trapezoidal method. The half-life (t1/2 ) was calculated as follows: t1/2 = 0.693/k (k is the elimination rate constant). All data were presented as mean ± S.D. Microsoft Excel was used for analysis with the t-test and p < 0.05 or p < 0.01 was used to determine significant differences between grouped measures. 3. Results and discussion 3.1. Method validation 3.1.1. Selectivity Typical chromatograms of an extract from a dosed rat’s dialysate (blood, bile and brain) and the blank samples are shown in Figs. 2–4. The retention times of spinosin and IS were 7.4 and 6.3 min, respectively. The results demonstrated that there is no interference in the determination of the analytes, suggesting good selectivity of the developed method. 3.1.2. Linearity and sensitivity Three linear regressions of spinosin in blood, bile and brain dialysate exhibited good linear relationships over the range of 0.0104–3.12, 0.02–50 and 0.005–0.5 g/mL, respectively. Each standard point was back calculated with the calibration, and all the non-zero samples showed less than 10.2% deviation except the LOQ of bile was less than 19.5%. The mean values of regression equation of the analyte in rat blood, bile and brain were: y = 2.0755x + 0.0083 (r2 = 0.9999), y = 2.1603x + 0.04 (r2 = 0.9954) and y = 7.0339x − 0.0145 (r2 = 0.9984), respectively. The LOD and LOQ for spinosin were found to be 3 and 10 ng/mL in blood dialysate, 10 and 20 ng/mL in bile dialysate, 1 and 5 ng/mL in
brain dialysate, all of which were acceptable in the current assay. Intra-day accuracy and precision were determined by analyzing five replicates at three different concentration levels, five times at each concentration (Table 1). For all the samples evaluated, the variability was less than 15% (R.S.D.). 3.1.3. Matrix effect The peak area ratio of the standard solutions mixed with methanol compared to that spiking into dialysates expresses the matrix effect in the sample matrix. The ratio value of 1 represented the response in the mobile phase and in the dialysate was the same and no significant matrix effect was observed. The average matrix effects of spinosin at three levels in blood (0.03, 0.3 and 3 g/mL), bile (0.05, 1, 20 g/mL) and brain (0.01, 0.1, 0.5 g/mL) were 0.69 ± 0.02, 0.74 ± 0.04 and 0.71 ± 0.03, respectively. These results showed that endogenous substances slightly affected the ionization of the analytes under the present chromatographic and extraction conditions when the ESI interface was utilized. It reminds us that matrices ion suppression is an interesting point that we may develop new methods to reduce the effects. 3.1.4. Stability Stability data are summarized in Table 2. The measured concentration in bile dialysate at 0.05 g/mL is obviously lower than 0.05 g/mL, suggesting spinosin is not stable at this concentration. Meanwhile, the solutions of spinosin at 1 g/mL or 20 g/mL were stable for three freeze/thaw cycles, 24 h at room temperature and at least 1 month when kept frozen at −70 ◦ C. Thus, the concentration of spinosin in all the samples we used is higher than 1 g/mL. 3.1.5. In vivo recovery of spinosin from microdialysis probe Table 3 shows the average in vivo recovery levels of spinosin were 21.0% in blood dialysate, 59.3% in bile dialysate, and 22.35% in
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Fig. 3. Selected ion monitoring chromatograms from (A) blank bile dialysate; (B) blank bile dialysate spiked with spinosin and puerarin, the concentrations of spinosin and puerarin 0.5 g/mL and 0.1 g/mL, respectively; (C) bile dialysate collected at 10 min after administration, showing a spinosin concentration of 4.807 g/mL; and (D) bile dialysate at 10 min after spinosin and CsA co-administration, showing a spinosin concentration of 0.141 g/mL.
Fig. 4. Selected ion monitoring chromatograms from (A) blank brain dialysate; (B) blank brain dialysate spiked with spinosin and puerain, the concentrations of spinosin and puerarin 0.2 g/mL and 0.03 g/mL, respectively; (C) brain dialysate collected at 20 min after administration, showing a spinosin concentration of 6.677 ng/mL; and (D) brain dialysate at 20 min after spinosin and CsA co-administration, showing a spinosin concentration of 26.4 ng/mL.
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Table 1 Intra- and inter-assay precision (% R.S.D.) and accuracy (% bias) of the method for the determination of spinosin (n = 5). Biological specimen Blood Intra-day
Inter-day
Bile Intra-day
Inter-day
Brain Intra-day
Inter-day
Nominal concentration (g/mL)
Observed concentration (g/mL)
Precision (% RSD)
Accuracy (% bias)
0.03 0.3 3 0.03 0.3 3
2.80 × 10−2 3.30 × 10−1 3.12 3.00 × 10−2 3.40 × 10−1 3.22
± ± ± ± ± ±
1.20 × 10−3 2.80 × 10−3 5.50 × 10−2 6.70 × 10−4 2.50 × 10−3 5.20 × 10−2
4.41 0.85 1.78 2.22 0.73 1.35
−6.67 9.77 4.04 0.27 13.01 7.20
0.05 1 20 0.05 1 20
4.60 × 10−2 1.05 22.80 4.80 × 10−2 1.03 19.95
± ± ± ± ± ±
1.50 × 10−3 1.10 × 10−2 1.40 × 10−1 5.00 × 10−3 1.10 × 10−2 4.30 × 10−1
3.27 0.73 0.61 13.02 0.85 2.16
−7.61 4.57 13.4 −3.99 3.39 0.23
0.01 0.1 0.5 0.01 0.1 0.5
1.10 × 10−2 9.60 × 10−2 4.90 × 10−1 1.50 × 10−2 9.70 × 10−2 4.76 × 10−1
± ± ± ± ± ±
7.30 × 10−4 4.20 × 10−4 1.30 × 10−2 3.80 × 10−4 2.70 × 10−3 1.00 × 10−2
6.52 0.43 2.71 0.03 3.45 2.37
10.26 −3.69 −2.6 5.32 2.63 4.88
Table 2 Stability of spinosin in dialysate (n = 5). Microdialysis sampling
Blood
Bile
Brain
Nominal concentration (g/mL)
0.03 0.3 3 0.05 1 20 0.01 0.1 0.5
Measured concentration (g/mL) (RSD %) (RE %)
After three freeze/thaw cycles in dialysate
At room temperature for 24 h in dialysate
0.32 1.27 1.68 12.82 0.85 2.16 6.37 0.51 4.22
4.29 0.85 1.76 2.6 1.13 0.84 6.73 0.44 2.65
brain dialysate. Sample concentrations were corrected by the probe recovery before pharmacokinetic data analysis.
3.2. Pharmacokinetics of spinosin in blood, bile and brain The concentration versus time curves for spinosin in rat blood, bile, and brain dialysate after spinosin administration are shown in Fig. 5A–C. The peak concentration of spinosin in bile dialysate, which were significantly higher than that in blood and brain dialysate, was observed at about 15 min and could be detected until 6 h. The pharmacokinetic data of spinosin in blood, bile and brain dialysate are shown in Table 4. The AUC of spinosin in blood, bile and brain were 205.70 ± 80.79 mg min/L, 7.77 × 104 ± 2.13 × 104 mg min/L and 2.09 ± 0.03 mg min/L, respectively. The amount of spinosin, as estimated from the AUC, T1/2 , in bile set against the concentration gradient was significantly greater than that in blood and brain,
3.33 23.33 21.00 −22.00 30.00 −0.25 10.00 −16.00 −10.00
−6.67 10 4 −30 33 0.7 10 −4 −2
At −70 ◦ C for 1 month in dialysate −30 16.7 3.67 −22 30 −0.25 30 −21 −14
6.19 0.77 1.79 0.13 0.01 0.02 0.25 0.03 0.02
suggesting that spinosin might be actively excreted into the bile. The CL of spinosin in brain is significantly greater than that in blood and bile. The results indicated that spinosin entered into the brain rapidly (within 10 min) and it could be detected until 50 min after drug administration, suggesting that spinosin can effectively and rapidly penetrate the blood–brain barrier (BBB). This observation is consistent with a previous study [8]. It also supports the claim that the drug exerts it pharmacodynamic effect on the central nervous system [4].
3.3. Interaction of CsA with spinosin After 10 mg/kg CsA was co-administered, the spinosin concentration in blood, bile and brain dialysate was significantly altered (Table 4 and Fig. 5). The AUC of spinosin in blood, bile and brain dialysate, at the dose of 5 mg/kg, increased from 205.70 to 673.51 mg min/L, 7.77 × 104 to 1.25 × 105 mg min/L and 2.09 to
Table 3 In vivo microdialysate recoveries (%) of spinosin from rat blood, bile and brain (n = 5). Microdialysis sampling
Perfused concentration (g/mL)
Recovery (%)
Blood
0.02 1 15 150 0.01 0.3
25.10 16.90 59.06 59.54 30.04 14.65
Bile Brain
± ± ± ± ± ±
8.17 2.97 9.96 6.71 16.90 1.62
Average 21.0 ± 5.57 59.30 ± 8.34 22.35 ± 9.28
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spinosin spinosin+CsA (10mg/kg)
1
0.1
0.01 0
40
80 120 160 200 240 280 320 360
B
C 1000
unbound spinosin (10-6 g/ml)
unbound spinosin (10-6 g/ml)
A
unbound spinosin (10-6 g/ml)
28
spinosin spinosin+CsA (10mg/kg)
800 600 400 200 0 0
0.16
spinosin spinosin+CsA (10mg/kg)
0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0
40 80 120 160 200 240 280 320 360
Time (min)
Time (min)
20
40
60
80
100
Time (min)
Fig. 5. Mean concentration–time profiles from (A) spinosin in rat blood dialysates after single administration of spinosin (5 mg/kg) and co-administration of spinosin and CsA (10 mg/kg); (B) spinosin in rat bile dialysates after single administration of spinosin (5 mg/kg) and co-administration of spinosin and CsA (10 mg/kg); and (C) spinosin in rat brain dialysates after single administration of spinosin (5 mg/kg) and co-administration of spinosin and CsA (10 mg/kg).
Table 4 Mean pharmacokinetic parameters of spinosin in dialysate after administration of spinosin (5 mg/kg). Treatment Blood Spinosin, 5 mg/kg +Cyclosprine, 10 mg/kg Bile Spinosin, 5 mg/kg +Cyclosprine, 10 mg/kg Brain Spinosin, 5 mg/kg +Cyclosprine, 10 mg/kg
Cmax (mg/L)
AUC (mg min/L)
5.59 ± 2.65 7.76 ± 4.51
205.70 ± 80.79 673.51 ± 327.19*
695.40 ± 162.90 689.90 ± 191.10
7.77 × 104 ± 2.13 × 104 1.25 × 105 ± 2.14 × 104 **
5.10 × 10−2 ± 7.5 × 10−3 9.00 × 10−2 ± 3.1 × 10−2
2.09 ± 0.03 5.58 ± 1.58**
T1/2 (min) 48.07 ± 4.71 95.04 ± 30.39** 97.20 ± 37.63 152.21 ± 70.26
CL (L/min/kg)
V (L/kg)
3.00 × 10−2 ± 5.10 × 10−3 9.00 × 10−3 ± 1.60 × 10−3 **
2.08 ± 0.35 1.25 ± 0.19
1.00 × 10−2 ± 2.00 × 10−3 2.00 × 10−3 ± 3.00 × 10−4 **
9.40 × 10−2 ± 1.80 × 10−2 7.00 × 10−2 ± 1.15 × 10−2
42.18 ± 13.71 73.83 ± 20.60*
1.72 ± 0.28 0.31 ± 4.9 × 10−2 **
101.67 ± 16.45 134.34 ± 17.77
Data expressed as means ± S.D. (n = 5). Cmax : maximum concentration; AUC: area under the concentration vs. time curve; t1/2 : elimination half-life. * p < 0.05 compared with single 5 mg/kg spinosin administration group. ** p < 0.01 compared with single 5 mg/kg spinosin administration group.
5.58 mg min/L respectively. The t1/2 values of spinosin in blood, bile and brain dialysate also changed, respectively, from 48.07 to 95.04 min, from 97.20 to 152.21 and from 42.18 to 73.83 min when the drug was co-administered with CsA. According to the results above, the AUC and t1/2 values of spinosin in blood, bile and brain dialysate were significantly increased in CsA-added group, which suggested that CsA has obvious effects on the pharmacokinetic process of spinosin. The increase of AUC and t1/2 values of spinosin in the blood and brain could be due to the P-gp inhibition by CsA. CsA inhibited the efflux of spinosin by P-gp located on the membrane of the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (BCSF) barrier, resulting in an increase of the spinosin concentration in the brain. Meanwhile, the inhibition of CsA on the excretion of spinosin may lead to the increase of its AUC in blood. It has been extensively verified that CsA inhibits the hepatobiliary excretion of P-gp substrates from the liver into the bile [23]. Compared with the control group (given 5 mg/kg spinosin alone), the concentration of spinosin in bile dialysate was decreased in the period of 0–40 min when CsA was co-administered, suggesting CsA inhibits the P-gp transporter and thus decrease the hepatobiliary excretion of spinosin. Interestingly, after 40 min, the concentration of spinosin in bile was higher than that of control group, indicating that some other transporters, such as multispecific organic anion transporters [24], non-bile acid organic anion transporters [25], or conjugate export pumps [26] might be involved in the hepatobiliary excretion of spinosin. In addition, the results of the present study indicate that the combining CsA with spinosin could provide an opportunity to achieve enhanced therapeutic effects of spinosin because of the increase of its concentration in blood and brain. The multi-drug treatment might be a useful way to reduce the dose of drugs and achieve better therapeutic effects in clinical practice.
4. Conclusions In this study, a microdialysis technique with HPLC–MS method was developed to investigate the pharmacokinetics of unbound spinosin in the brain, blood and bile dialysate of rats. The work demonstrated this method can be conveniently and sensitively applied to monitor the pharmacokinetic behavior of drugs. The results revealed that CsA has obvious effects on pharmacokinetic process of spinosin since the AUC and t1/2 values of spinosin in blood, bile and brain dialysate were significantly increased after drug co-administration. Since CsA is a well-established Pgp inhibitor, spinosin is also a substrate for P-gp transporter. Because hepatobiliary excretion of spinosin is also increased after co-administration, other transporters or pathways may also be involved in the metabolism of spinosin. Acknowledgements The authors greatly appreciate the financial support from the research project (No. 30973884) of the National Science Foundation of China, the program for Changjiang Scholars and Innovative Research Team in University, No. IRT0868, and the Postgraduate Innovative Projects of China Pharmaceutical University to Dr. Gui-Zhong Xin (No. ZJ10059). References [1] Y.J. Li, K.S. Bi, Study on the therapeutic material basis of traditional Chinese medicinal preparation Suanzaoren Decoction, Chem. Pharm. Bull. 56 (2006) 847–851. [2] K. Kawashima, K. Saito, A. Yamada, S. Obara, T. Ozaki, Y. Kano, Pharmacological properties of traditional medicines. XXIII. Searching for active compounds in the blood and bile of rats after oral administrations of extracts of sansohnin, Biol. Pharm. Bull. 20 (1997) 1171–1174.
R.-H. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 22–29 [3] L.E. Wang, Y.J. Bai, X.R. Shi, X.R. Shi, X.Y. Cui, S.Y. Cui, F. Zhang, Q.Y. Zhang, Y.H. Zhang, Y.Y. Zhao, Spinosin, a C-glycoside flavonoid from semen Ziziphi Spinozae, potentiated pentobarbital-induced sleep via the serotonergic system, Pharmacol. Biochem. Behav. 90 (2008) 399–403. [4] L.E. Wang, X.Y. Cui, S.Y. Cui, J.X. Cao, J. Zhang, Y.H. Zhang, Q.Y. Zhang, Y.J. Bai, Y.Y. Zhao, Potentiating effect of spinosin, a C-glycoside flavonoid of Semen Ziziphi spinosae, on pentobarbital-induced sleep may be related to postsynaptic 5HT(1A) receptors, Phytomedicine 17 (2010) 404–409. [5] Y.J. Li, X.M. Liang, H.B. Xiao, K.S. Bi, Determination of spinosin in rat plasma by reversed-phase high-performance chromatography after oral administration of Suanzaoren decoction, J. Chromatogr. B 787 (2003) 421–425. [6] Y.J. Li, M.C. Yao, S. Cheng, Quantitative determination of spinosin in rat plasma by liquid chromatography–tandem mass spectrometry method, J. Pharm. Biomed. Anal. 48 (2008) 1169–1173. [7] Y.J. Li, X.M. Liang, H.B. Xiao, K.S. Bi, Pharmacokinetic study on spinosin in rat plasma after oral administration of suanzaoren extract at a single dose, Acta Pharm. Sin. 38 (2003) 448–450. [8] Y.J. Li, Y.H. Dai, Y.L. Yu, Y. Li, Y.L. Deng, Pharmacokinetics and tissue distribution of spinosin after intravenous administration in rats, Yakugaku Zasshi 127 (2007) 1231–1235. [9] K.D. Bao, P. Li, L.W. Qi, H.J. Li, L. Yi, W. Wang, Y.Q. Wang, Characterization of flavonoid metabolites in rat plasma, urine, and feces after oral administration of Semen Ziziphi Spinosae extract by HPLC–diode-array detection (DAD) and ion-trap mass spectrometry (MS(n)), Chem. Pharm. Bull. 57 (2009) 144–148. [10] V.R. Tandon, B. Kapoor, G. Bano, S. Gupta, Z. Gillani, S. Gupta, D. Kour, P-glycoprotein: pharmacological relevance, Indian J. Pharmacol. 38 (2006) 13–24. [11] M. Dorababu, A. Nishimura, T. Prabha, K. Naruhashi, N. Sugioka, K. Takada, N. Shibata, Effect of cyclosporine on drug transport and pharmacokinetics of nifedipine, Biomed. Pharmacother. 63 (2009) 697–702. [12] J.H. Lin, Drug–drug interaction mediated by inhibition and induction of Pglycoprotein, Adv. Drug Deliv. Rev. 55 (2003) 53–81. [13] J.H. Hamman, P.H. Demana, E.I. Olivier, Targeting receptors, transporters and site of absorption to improve oral drug delivery, Drug Target Insights 2 (2007) 71–81. [14] J.D. Wright, F.D. Boudinot, M.R. Ujhelyi, Measurement and analysis of unbound drug concentrations, Clin. Pharmacokinet. 30 (1996) 445–462.
29
[15] M.L.T. Vieira, R.P. Singh, H. Derendorf, Simultaneous, HPLC analysis of triamcinolone acetonide and budesonide in microdialysate and rat plasma: application to a pharmacokinetic study, J. Chromatogr. B 878 (2010) 2967–2973. [16] Q. Wu, X.D. Wen, L.W. Qi, W. Wang, L. Yi, Z.M. Bi, P. Li, An in vivo microdialysis measurement of harpagoside in rat blood and bile for predicting hepatobiliary excretion and its interaction with cyclosporin A and verapamil, J. Chromatogr. B 877 (2009) 751–756. [17] C.F. Chien, Y.T. Wu, W.C. Lee, L.C. Lin, T.H. Tsai, Herb–drug interaction of Andrographis paniculata extract and andrographolide on the pharmacokinetics of theophylline in rats, Chem. Biol. Interact. 184 (2010) 458–465. [18] W.H. Sheu, H.C. Chuang, S.M. Cheng, M.R. Lee, C.C. Chou, F.C. Cheng, Microdialysis combined blood sampling technique for the determination of rosiglitazone and glucose in brain and blood of gerbils subjected to cerebral ischemia, J. Pharm. Biomed. Anal. 54 (2011) 759–764. [19] L. Kagan, T. Dreifinger, D. Mager, A. Hoffman, Role of p-glycoprotein in regionspecific gastrointestinal absorption of talinolol in rats, Drug. Metab. Dispos. 38 (2010) 1560–1566. [20] X.Y. Chu, K. Bleasby, J. Yabut, X.X. Cai, G.H. Chan, M.J. Hafey, S.Y. Xu, A.J. Bergman, M.P. Braun, D.C. Dean, R. Evers, Transport of the dipeptidyl peptidase-4 inhibitor sitagliptin by human organic anion transporter 3, organic anion transporting polypeptide 4C1, and multidrug resistance P-glycoprotein, J. Pharmacol. Exp. Ther. 321 (2007) 673–683. [21] W.S. Woo, S.S. Kang, S.H. Shim, H. Wagner, V.M. Chari, O. Seligmann, G. Obermeier, The structure of spinosin (2 -O--glucosylswertisin) from Zizyphus vulgaris var.Spinosus, Phytochemistry 18 (1979) 353–355. [22] T.H. Tsai, Assaying protein unbound drugs using microdialysis techniques, J. Chromatogr. B 797 (2003) 161–173. [23] X.Y. Chu, Y. Kato, Y. Sugiyama, Possible involvement of P-glycoprotein in biliary excretion of CPT-11 in rats, Drug Metab. Dispos. 27 (1999) 440–441. [24] Y. Kobayashi, R. Sakai, N. Ohshiro, M. Ohbayashi, N. Kohyama, T. Yamamoto, Possible involvement of organic anion transporter 2 on the interaction of theophylline with erythromycin in the human liver, Drug Metab. Dispos. 33 (2005) 619–622. [25] C.D. Klaassen, L.M. Aleksunes, Xenobiotic, bile acid, and cholesterol transporters: function and regulation, Pharmacol. Rev. 62 (2010) 1–96. [26] M.J. Zamek-Gliszczynski, K.A. Hoffmaster, K. Nezasa, M.N. Tallman, K.L.R. Brouwer, Integration of hepatic drug transporters and phase II metabolizing enzymes: mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites, Eur. J. Pharm. Sci. 27 (2006) 447–486.
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
In vivo microdialysis with LC–MS for analysis of spinosin and its interaction with cyclosporin A in rat brain, blood and bile Rong-Hua Ma a,1 , Jie Yang a,1 , Lian-Wen Qi a , Gui-Zhong Xin a , Chong-Zhi Wang b , Chun-Su Yuan b , Xiao-Dong Wen a,∗ , Ping Li a,∗∗ a
State Key Laboratory of Natural Medicines (China Pharmaceutical University), Nanjing 210009, China Tang Center for Herbal Medicine Research and Department of Anesthesia & Critical Care, The Pritzker School of Medicine, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA b
a r t i c l e
i n f o
Article history: Received 13 September 2011 Received in revised form 11 November 2011 Accepted 12 November 2011 Available online 22 November 2011 Keywords: Spinosin In vivo microdialysis LC–/MS Cyclosporin A Drug–drug interactions
a b s t r a c t Spinosin, a major bioactive herbal ingredient isolated from Semen Ziziphi Spinosae, plays an important role in sedation and hypnosis. However, the pharmacokinetic behavior of spinosin in special sites has not been reported. Microdialysis (MD) technique, as a continuous, realtime monitoring sampling technique, is very suitable for the evaluation of the disposition of diverse drugs. To obtain more useful information on spinosin, an in vivo microdialysis sampling technique with High Performance Liquid Chromatography–mass spectrograph (HPLC–MS) method was developed to investigate the pharmacokinetics of spinosin and its interaction with cyclosporin A (CsA) in the brain, blood and bile of rats. The method was validated in terms of selectivity, linearity and sensitivity, and showed advantages in monitoring the pharmacokinetic behavior of drugs. The results revealed that CsA has obvious effects on the pharmacokinetic process of spinosin. When co-administered, the area under the curve (AUC) of spinosin in blood, bile and brain increased from 205.70 to 673.51 mg min/L, 7.77 × 104 to 1.25 × 105 mg min/L, and 2.09 to 5.58 mg min/L, respectively. The t1/2 values of spinosin in blood, bile and brain also changed from 48.07 to 95.04 min, from 97.20 to 152.21 and from 42.18 to 73.83 min, respectively. These results demonstrated that the CsA decreased the efflux of spinosin through the inhibition of P-glycoprotein (P-gp) efflux transporter and it might be used as a group of P-gp substrate. Other transporters or pathways may also be involved in the metabolism of spinosin. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Pharmacokinetic (PK) behavior of drugs plays a crucial role in the determination of their pharmacological actions. Any alterations in pharmacokinetic parameters will affect their therapeutic efficacy. Spinosin (2 -ˇ-O-glucopyranosyl swertisin, C28 H38 O15 , Fig. 1), a bioactive herbal ingredient isolated from Semen Ziziphi Spinosae, plays an important role in sedation and hypnosis [1,2]. It significantly potentiated the hypnotic effect of pentobarbital by decreasing sleep latency, increasing sleeping time, and enhancing the rate of sleep onset induced by subhypnotic doses of pentobarbital [3]. The mechanism of this action may be related to postsynaptic 5-HT1A receptors [4]. Although spinosin possesses significant activities, previous pharmacokinetic studies [5–9] are not sufficient for illustrating its mechanism of action and supplying research
∗ Corresponding author. Tel.: +86 25 8327 1379; fax: +86 25 8327 1379. ∗∗ Corresponding author. Tel.: +86 25 8327 1382; fax: +86 25 8327 1382. E-mail addresses: [email protected] (X.-D. Wen), [email protected] (P. Li). 1 These authors contributed equally to this work. 0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.11.014
information for clinical use. The pharmacokinetic behavior of spinosin in special sites has not been reported elsewhere. In recent decades, the crucial role of efflux transporters in drug absorption and disposition has gained considerable attention [10]. The inhibition or induction of transporters can lead to significant drug–drug interaction by affecting various pharmacokinetic parameters of the drug [11]. P-glycoprotein (P-gp), the most extensively studied ATP-binding cassette transporter, functions as a biological barrier by extruding toxic substances and xenobiotics out of cells [12]. The hypothesis that inhibition of P-gp improves the bioavailability of drugs that are substrate for this efflux transporter is gaining widespread recognition [13]. However, the role of P-gp efflux transporter in spinosin metabolism and disposition has not been investigated. In many clinical cases, determining the total drug concentration does not always produce the designed data because only the free drug concentration is responsible for the distribution, elimination, and therapeutic effect of drugs [14]. MD is a sampling technique that not only enables direct measurement of free drug levels at the biophase and continuous sampling without net fluid loss, but also permits monitoring of local concentrations of drugs
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Fig. 1. The chemical structure of spinosin and puerarin (internal standard).
and metabolites at specific sites [15]. Recently, there has been growing interest in the use of microdialysis to evaluate the disposition of diverse drugs [16–18]. PK involving MD commonly requires an efficient and sensitive analytical method to measure considerably low concentration of the analyte in the microdialysate [15]. HPLC–MS with its high stability and sensitivity is particularly suitable for determination of compounds at low levels in biological fluids. Thus, microdialysis sampling techniques coupled with the HPLC–MS method provides an attractive tool for pharmacokinetic research. To obtain more useful information on the metabolic fate of spinosin, this study aimed to investigate the pharmacokinetics of unbound spinosin in the brain, blood and bile of rats. For this purpose, an in vivo microdialysis sampling technique with the HPLC–MS method was developed and validated. The role of P-gp efflux transporter in spinosin metabolism and disposition was also investigated by comparing the PK of spinosin with and without CsA, a well-established P-gp inhibitor [19,20]. 2. Experimental 2.1. Chemicals and materials Spinosin was isolated from Semen Ziziphi Spinosae in the authors’ laboratories and its structure in Fig. 1 was elucidated by Infrared Spectroscopy (IR), mass spectrography (MS), Carbon Nuclear Magnetic Resonance Spectroscopy (1 H NMR) and Nuclear Magnetic Resonance Spectroscopy (13 C NMR) methods and by comparison with the data in references [21]. The purity of this compound was determined to be higher than 98% by normalization of the peak area detected by HPLC. The internal standard (IS) Puerarin (Fig. 1) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). CsA was purchased from Novartis Pharma Stein AG (Basel, Switzerland). Acetonitrile was of HPLC grade from Merck (Darmstadt, Germany). Purified water prepared by the Millipore system (Millipore, Bedford, MA, USA) was used for all the preparations. Other chemicals were of analytical grade.
Institutional Animal Experimentation Committee of China Pharmaceutical University (Nanjing, China). The rats were anesthetized with urethane 1 g/kg (i.p.) and remained anesthetized throughout the experimental period. The femoral vein was exposed for further drug administration. The rat’s body temperature was maintained at 37 ◦ C during the experimental procedure. 2.3. Microdialysis experiments Blood, brain and bile microdialysis systems consisted of a 3syringe bracket microdialysis pump with a Bee Syringe Pump Controller (Bioanalytical System Inc., West Lafayette, IN, USA). Blood and bile duct microdialysis probes were constructed inhouse based on a previous design [16]. A dialyzing membrane of 10 and 65 mm were used for blood and bile sampling, respectively. Under urethane anesthesia, the flexible blood microdialysis probe was implanted into the jugular vein toward the rat’s right atrium, and then perfused with anticoagulant citrate dextrose (ACD) solution (3.5 mM citric acid, 7.5 mM sodium citrate, and 13.6 mM dextrose) at a flow rate of 2 L/min. The bile microdialysis probe was perfused with Ringer’s solution (147 mM NaCl, 2.4 mM CaCl2 , and 4.0 mM KCl with a pH of 7.3) at a flow rate of 2.0 L/min. For brain sampling, the rat was mounted on a stereotaxic frame (RWD Life Science Co. Ltd, Shenzhen China). An incision was made in the scalp, and a small hole was drilled for striatum implantation of a rigid brain microdialysis probe. Stereotaxic coordinates from bregma for ventral hippocampus (HC): −5.6 mm rostral, 5.0 mm medial, −3.4 mm ventral from the top of the skull according to the Paxinos and Watson. A microdialysis probe (MD-2204, 4 mm membrane, Bioanalytical system, Inc., USA) was lowered into the hippocampus of the anesthetized rat. Perfusion of the microdialysis probe with filtered Ringer’s solution (147 mM NaCl, 2.4 mM CaCl2 , and 4.0 mM KCl with a pH of 7.3) was begun shortly before probe insertion and continued for the duration of the experiment delivered by a microinjection pump at a constant flow-rate of 2 L/min. The blood, bile and brain samples were drawn at every 20 min after administration of spinosin until 6 h (interval of 10 min periods for the first 120 min). The midpoints of the sampling times were used for blood, bile and brain spinosin concentration-time profiles.
2.2. Experimental animals 2.4. Drug administration Sprague–Dawley rats (280 ± 20 g) were obtained from SinoBritish Sippr/BK Lab. Animal Co. Ltd. (Shanghai, China) and housed with unlimited access to food and water except for fasting 12 h before the experiment. The animals were maintained on a 12-h light/12-h dark cycle (light on at 8:00) at ambient temperature (22–25 ◦ C) and 60% relative humidity. All experimental animal surgery procedures were reviewed and approved by the
Spinosin (2 mg/mL) was dissolved in saline (containing 0.5% dimethyl sulfoxide (DMSO)). CsA was diluted with 0.9% (w/v) sodium chloride to 10 mg/mL. For the control group, 5 mg/kg of spinosin was administrated through the femoral vein to each rat 2 h after the blood, brain and bile probe were implanted for the equilibrium of the dialysis membrane. For CsA co-administration
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groups, 10 mg/kg CsA was given through the femoral vein 10 min prior to spinosin injection. The volume of injection was 1 mL/kg. 2.5. Sample preparation A stock solution of an internal standard (IS) of puerarin for blood was prepared in a concentration of 0.5 g/mL in ACD solution. The blood analysis samples were prepared by spiking 3 L (0.5 g/mL) of the corresponding IS stock solutions into 12 L dialysate followed by vortexing. And stock IS solutions of puerarin for bile and brain were prepared in Ringer’s solution at 0.11 and 0.15 g/mL, respectively. The bile analysis samples were prepared by spiking 18 L (0.11 g/mL) of the corresponding IS stock solutions into 2 L dialysate followed by vortexing. And the brain analysis samples were prepared by spiking 3 L (0.15 g/mL) of the corresponding IS stock solutions into 12 L dialysate followed by vortexing. 2.6. HPLC–MS system
ACD with Ringer’s solution and forming different serial concentrations (2.00 × 10−2 , 5.00 × 10−2 , 1.00, 5.00, 10.0, 20.0, 50.0 g/mL and 5 × 10−3 , 8 × 10−3 , 1.00 × 10−2 , 5.00 × 10−2 , 1.00 × 10−1 , 2.00 × 10−1 , 5.00 × 10−1 g/mL in bile and brain, respectively), bile and brain calibration samples were prepared just as the same as blood samples. Standard working solutions were prepared every day, immediately before use. The quality control (QC) samples with low, middle and high concentrations for blood (3.12 × 10−2 , 3.12 × 10−1 and 3.12 g/mL), bile (5.00 × 10−2 , 1.00, 20.0 g/mL) and brain (1.00 × 10−2 , 1.00 × 10−1 , 5.00 × 10−1 g/mL) were also prepared one by one and daily in the same manner. All solutions were stored at −20 ◦ C before use. For a standard curve, the ratio of the chromatographic peaks area (analytes/IS) as ordinate variables were plotted versus the concentration of spinosin as abscissa. The limit of detection (LOD) and limit of quantification (LOQ) determinations were considered as the final concentration producing a signal-to-noise ratio of 3 and 10, respectively. Both were measured with at least five replications.
Chromatographic analysis was performed on an Agilent 1100 series LC system (Agilent, Germany) equipped with a binary pump and a thermostatically controlled column apartment. Chromatographic separation was carried out at 25 ◦ C on an Angilent extend-C18 column (50 mm × 4.6 mm I.D., 1.8 m) with a C18 guard column (Hanbang, Jiangsu, China). The mobile phase consisted of 0.1% formic acid water (Solvent A) and acetonitrile (Solvent B). The injection volume of blood, brain, bile samples are 10 L, 10 L, 5 L, respectively. The chromatograph was developed with a 10 min linear gradient from 15% Solvent A to 50% Solvent B followed by a 5 min re-equilibration. The flow rate was set at 0.5 mL/min. To avoid ion suppression, a post-column Diverter valve was applied to remove the ions in microdialysate. Detections were performed by a single quadrupole mass spectrometer (Product No. G2710BA, Agilent Corp, Santa Clara, CA, USA) equipped with an ESI source. The mass range was set at m/z 100–1000. The ESI-MS data were acquired in positive mode and conditions of MS analysis were as follows: drying gas (N2 ) flow-rate, 10 L/min; drying gas temperature, 300 ◦ C; nebulizing gas (N2 ) pressure, 16 psi; capillary voltage, 3000 V; quad temperature, 100 ◦ C; fragmentor, 100 V. The samples from the fast HPLC column were analyzed in selective ion monitoring (SIM) mode by monitoring the molecular ions m/z 417 for puerarin and m/z 609 for spinosin. SIM for each compound were restricted to specific retention time windows: 0–10 min, m/z 417, m/z 609 (retention time: 6.3 min, 7.4 min). All the operations, the acquiring and analysis of data were controlled by Chemstation software (Agilent Technologies, USA).
2.7.3. Accuracy and precision The intra-day precision and accuracy of the method were determined by analysis of the QC samples five times in a single day while its inter-day precision and accuracy were estimated by analysis of the QC samples three times in three consecutive days. The accuracy was calculated as mean percent deviation (RE) of the observed concentration (Cobs ) from the nominal concentration (Cnom ), accuracy (%RE) = [(Cobs − Cnom )/Cnom ] × 100. The precision was expressed as percent of coefficient of variation (CV), precision (%CV) = [Standard deviation (SD)/mean Cobs ) × 100. The acceptable intraday and interday precision is required to be less than 15% and the acceptable accuracy is required to be within 15% for all QC samples.
2.7. Method development
2.7.6. Recoveries of microdialysis probes A retrodialysis calibration technique was applied to recovery determination [22]. The experiment was carried as described in Section 2.3 except the perfusate was replaced by the spinosin ACD solution (for blood microdialysis) or Ringer’s solution (for bile and brain microdialysis) in certain concentrations. The perfusate (Cperf ) and dialysate (Cdial ) concentrations of spinosin were determined by the HPLC–MS system. The in vivo relative recovery (Rdial ) of spinosin across the microdialysis probe was calculated by the following equation: Rdial = (Cperf − Cdial )/Cperf . Spinosin microdialysate concentrations (Cm ) were converted to unbound concentration (Cu ) by the estimated in vivo recoveries (Rdial ) as follows: Cu = Cm × n/Rdial (n: microdialysis samples dilute multiples, the dilution multiples of samples are 1.25, 10 and 1.25 in blood, bile and brain, respectively).
2.7.1. Selectivity The selectivity of the method was tested by analyzing drugfree blood, bile and brain dialysate from five different rats. Each dialysate sample was tested using the HPLC–MS conditions proposed to insure no interference with spinosin and IS. 2.7.2. Calibration curves Because of the differences in concentration ranges and matrix between blood, brain and bile dialysates, the calibration curves were prepared separately. The stock solution of spinosin for blood at 1.04 mg/mL was prepared in ACD solution (containing 10% DMSO), and then diluted with ACD solution to make the working standard solutions of different serial concentrations. Then, 10 L of IS for blood and 10 L of the corresponding working standard solutions were mixed with 80 L of blank blood dialysate to form serial concentrations (1.04 × 10−2 , 3.12 × 10−2 , 1.04 × 10−1 , 3.12 × 10−1 , 1.04 and 3.12 g/mL). Except for replacing
2.7.4. Matrix effect Since the ionization of the analyte may influenced by the coeluting, undetected endogenous matrix compounds, the matrix effect of analytes must be investigated during the analysis. It was evaluated by comparing the peak area of IS and spinosin in the blank dialysate with that at the same concentrations of IS and spinosin in methanol. 2.7.5. Stability The stability of analytes was evaluated by analyzing QC samples at three concentrations exposed to different time and temperature conditions: modeling three freeze/thaw cycles (−20 ◦ C to ambient temperature), 24 h storage at room temperature and frozen at −70 ◦ C for 1 month.
2.8. Data analysis Pharmacokinetic parameters in rats were estimated by a noncompartmental method using Drug and Statistics 2.0 (DAS
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Fig. 2. Selected ion monitoring chromatograms from (A) blank blood dialysate; (B) blank blood dialysate spiked with spinosin and puerarin, the concentrations of spinosin and puerarin 0.624 g/mL and 0.1 g/mL, respectively; (C) blood dialysate collected at 20 min after administration, showing a spinosin concentration of 0.559 g/mL; and (D) blood dialysate at 20 min after spinosin and CsA co-administration, showing a spinosin concentration of 1.247 g/mL.
2.0) software package (Mathematical Pharmacology Professional Committee of China, Shanghai, China). The area under the concentration-time curve (AUC) was calculated according to the log linear trapezoidal method. The half-life (t1/2 ) was calculated as follows: t1/2 = 0.693/k (k is the elimination rate constant). All data were presented as mean ± S.D. Microsoft Excel was used for analysis with the t-test and p < 0.05 or p < 0.01 was used to determine significant differences between grouped measures. 3. Results and discussion 3.1. Method validation 3.1.1. Selectivity Typical chromatograms of an extract from a dosed rat’s dialysate (blood, bile and brain) and the blank samples are shown in Figs. 2–4. The retention times of spinosin and IS were 7.4 and 6.3 min, respectively. The results demonstrated that there is no interference in the determination of the analytes, suggesting good selectivity of the developed method. 3.1.2. Linearity and sensitivity Three linear regressions of spinosin in blood, bile and brain dialysate exhibited good linear relationships over the range of 0.0104–3.12, 0.02–50 and 0.005–0.5 g/mL, respectively. Each standard point was back calculated with the calibration, and all the non-zero samples showed less than 10.2% deviation except the LOQ of bile was less than 19.5%. The mean values of regression equation of the analyte in rat blood, bile and brain were: y = 2.0755x + 0.0083 (r2 = 0.9999), y = 2.1603x + 0.04 (r2 = 0.9954) and y = 7.0339x − 0.0145 (r2 = 0.9984), respectively. The LOD and LOQ for spinosin were found to be 3 and 10 ng/mL in blood dialysate, 10 and 20 ng/mL in bile dialysate, 1 and 5 ng/mL in
brain dialysate, all of which were acceptable in the current assay. Intra-day accuracy and precision were determined by analyzing five replicates at three different concentration levels, five times at each concentration (Table 1). For all the samples evaluated, the variability was less than 15% (R.S.D.). 3.1.3. Matrix effect The peak area ratio of the standard solutions mixed with methanol compared to that spiking into dialysates expresses the matrix effect in the sample matrix. The ratio value of 1 represented the response in the mobile phase and in the dialysate was the same and no significant matrix effect was observed. The average matrix effects of spinosin at three levels in blood (0.03, 0.3 and 3 g/mL), bile (0.05, 1, 20 g/mL) and brain (0.01, 0.1, 0.5 g/mL) were 0.69 ± 0.02, 0.74 ± 0.04 and 0.71 ± 0.03, respectively. These results showed that endogenous substances slightly affected the ionization of the analytes under the present chromatographic and extraction conditions when the ESI interface was utilized. It reminds us that matrices ion suppression is an interesting point that we may develop new methods to reduce the effects. 3.1.4. Stability Stability data are summarized in Table 2. The measured concentration in bile dialysate at 0.05 g/mL is obviously lower than 0.05 g/mL, suggesting spinosin is not stable at this concentration. Meanwhile, the solutions of spinosin at 1 g/mL or 20 g/mL were stable for three freeze/thaw cycles, 24 h at room temperature and at least 1 month when kept frozen at −70 ◦ C. Thus, the concentration of spinosin in all the samples we used is higher than 1 g/mL. 3.1.5. In vivo recovery of spinosin from microdialysis probe Table 3 shows the average in vivo recovery levels of spinosin were 21.0% in blood dialysate, 59.3% in bile dialysate, and 22.35% in
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Fig. 3. Selected ion monitoring chromatograms from (A) blank bile dialysate; (B) blank bile dialysate spiked with spinosin and puerarin, the concentrations of spinosin and puerarin 0.5 g/mL and 0.1 g/mL, respectively; (C) bile dialysate collected at 10 min after administration, showing a spinosin concentration of 4.807 g/mL; and (D) bile dialysate at 10 min after spinosin and CsA co-administration, showing a spinosin concentration of 0.141 g/mL.
Fig. 4. Selected ion monitoring chromatograms from (A) blank brain dialysate; (B) blank brain dialysate spiked with spinosin and puerain, the concentrations of spinosin and puerarin 0.2 g/mL and 0.03 g/mL, respectively; (C) brain dialysate collected at 20 min after administration, showing a spinosin concentration of 6.677 ng/mL; and (D) brain dialysate at 20 min after spinosin and CsA co-administration, showing a spinosin concentration of 26.4 ng/mL.
R.-H. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 22–29
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Table 1 Intra- and inter-assay precision (% R.S.D.) and accuracy (% bias) of the method for the determination of spinosin (n = 5). Biological specimen Blood Intra-day
Inter-day
Bile Intra-day
Inter-day
Brain Intra-day
Inter-day
Nominal concentration (g/mL)
Observed concentration (g/mL)
Precision (% RSD)
Accuracy (% bias)
0.03 0.3 3 0.03 0.3 3
2.80 × 10−2 3.30 × 10−1 3.12 3.00 × 10−2 3.40 × 10−1 3.22
± ± ± ± ± ±
1.20 × 10−3 2.80 × 10−3 5.50 × 10−2 6.70 × 10−4 2.50 × 10−3 5.20 × 10−2
4.41 0.85 1.78 2.22 0.73 1.35
−6.67 9.77 4.04 0.27 13.01 7.20
0.05 1 20 0.05 1 20
4.60 × 10−2 1.05 22.80 4.80 × 10−2 1.03 19.95
± ± ± ± ± ±
1.50 × 10−3 1.10 × 10−2 1.40 × 10−1 5.00 × 10−3 1.10 × 10−2 4.30 × 10−1
3.27 0.73 0.61 13.02 0.85 2.16
−7.61 4.57 13.4 −3.99 3.39 0.23
0.01 0.1 0.5 0.01 0.1 0.5
1.10 × 10−2 9.60 × 10−2 4.90 × 10−1 1.50 × 10−2 9.70 × 10−2 4.76 × 10−1
± ± ± ± ± ±
7.30 × 10−4 4.20 × 10−4 1.30 × 10−2 3.80 × 10−4 2.70 × 10−3 1.00 × 10−2
6.52 0.43 2.71 0.03 3.45 2.37
10.26 −3.69 −2.6 5.32 2.63 4.88
Table 2 Stability of spinosin in dialysate (n = 5). Microdialysis sampling
Blood
Bile
Brain
Nominal concentration (g/mL)
0.03 0.3 3 0.05 1 20 0.01 0.1 0.5
Measured concentration (g/mL) (RSD %) (RE %)
After three freeze/thaw cycles in dialysate
At room temperature for 24 h in dialysate
0.32 1.27 1.68 12.82 0.85 2.16 6.37 0.51 4.22
4.29 0.85 1.76 2.6 1.13 0.84 6.73 0.44 2.65
brain dialysate. Sample concentrations were corrected by the probe recovery before pharmacokinetic data analysis.
3.2. Pharmacokinetics of spinosin in blood, bile and brain The concentration versus time curves for spinosin in rat blood, bile, and brain dialysate after spinosin administration are shown in Fig. 5A–C. The peak concentration of spinosin in bile dialysate, which were significantly higher than that in blood and brain dialysate, was observed at about 15 min and could be detected until 6 h. The pharmacokinetic data of spinosin in blood, bile and brain dialysate are shown in Table 4. The AUC of spinosin in blood, bile and brain were 205.70 ± 80.79 mg min/L, 7.77 × 104 ± 2.13 × 104 mg min/L and 2.09 ± 0.03 mg min/L, respectively. The amount of spinosin, as estimated from the AUC, T1/2 , in bile set against the concentration gradient was significantly greater than that in blood and brain,
3.33 23.33 21.00 −22.00 30.00 −0.25 10.00 −16.00 −10.00
−6.67 10 4 −30 33 0.7 10 −4 −2
At −70 ◦ C for 1 month in dialysate −30 16.7 3.67 −22 30 −0.25 30 −21 −14
6.19 0.77 1.79 0.13 0.01 0.02 0.25 0.03 0.02
suggesting that spinosin might be actively excreted into the bile. The CL of spinosin in brain is significantly greater than that in blood and bile. The results indicated that spinosin entered into the brain rapidly (within 10 min) and it could be detected until 50 min after drug administration, suggesting that spinosin can effectively and rapidly penetrate the blood–brain barrier (BBB). This observation is consistent with a previous study [8]. It also supports the claim that the drug exerts it pharmacodynamic effect on the central nervous system [4].
3.3. Interaction of CsA with spinosin After 10 mg/kg CsA was co-administered, the spinosin concentration in blood, bile and brain dialysate was significantly altered (Table 4 and Fig. 5). The AUC of spinosin in blood, bile and brain dialysate, at the dose of 5 mg/kg, increased from 205.70 to 673.51 mg min/L, 7.77 × 104 to 1.25 × 105 mg min/L and 2.09 to
Table 3 In vivo microdialysate recoveries (%) of spinosin from rat blood, bile and brain (n = 5). Microdialysis sampling
Perfused concentration (g/mL)
Recovery (%)
Blood
0.02 1 15 150 0.01 0.3
25.10 16.90 59.06 59.54 30.04 14.65
Bile Brain
± ± ± ± ± ±
8.17 2.97 9.96 6.71 16.90 1.62
Average 21.0 ± 5.57 59.30 ± 8.34 22.35 ± 9.28
R.-H. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 22–29
10
spinosin spinosin+CsA (10mg/kg)
1
0.1
0.01 0
40
80 120 160 200 240 280 320 360
B
C 1000
unbound spinosin (10-6 g/ml)
unbound spinosin (10-6 g/ml)
A
unbound spinosin (10-6 g/ml)
28
spinosin spinosin+CsA (10mg/kg)
800 600 400 200 0 0
0.16
spinosin spinosin+CsA (10mg/kg)
0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0
40 80 120 160 200 240 280 320 360
Time (min)
Time (min)
20
40
60
80
100
Time (min)
Fig. 5. Mean concentration–time profiles from (A) spinosin in rat blood dialysates after single administration of spinosin (5 mg/kg) and co-administration of spinosin and CsA (10 mg/kg); (B) spinosin in rat bile dialysates after single administration of spinosin (5 mg/kg) and co-administration of spinosin and CsA (10 mg/kg); and (C) spinosin in rat brain dialysates after single administration of spinosin (5 mg/kg) and co-administration of spinosin and CsA (10 mg/kg).
Table 4 Mean pharmacokinetic parameters of spinosin in dialysate after administration of spinosin (5 mg/kg). Treatment Blood Spinosin, 5 mg/kg +Cyclosprine, 10 mg/kg Bile Spinosin, 5 mg/kg +Cyclosprine, 10 mg/kg Brain Spinosin, 5 mg/kg +Cyclosprine, 10 mg/kg
Cmax (mg/L)
AUC (mg min/L)
5.59 ± 2.65 7.76 ± 4.51
205.70 ± 80.79 673.51 ± 327.19*
695.40 ± 162.90 689.90 ± 191.10
7.77 × 104 ± 2.13 × 104 1.25 × 105 ± 2.14 × 104 **
5.10 × 10−2 ± 7.5 × 10−3 9.00 × 10−2 ± 3.1 × 10−2
2.09 ± 0.03 5.58 ± 1.58**
T1/2 (min) 48.07 ± 4.71 95.04 ± 30.39** 97.20 ± 37.63 152.21 ± 70.26
CL (L/min/kg)
V (L/kg)
3.00 × 10−2 ± 5.10 × 10−3 9.00 × 10−3 ± 1.60 × 10−3 **
2.08 ± 0.35 1.25 ± 0.19
1.00 × 10−2 ± 2.00 × 10−3 2.00 × 10−3 ± 3.00 × 10−4 **
9.40 × 10−2 ± 1.80 × 10−2 7.00 × 10−2 ± 1.15 × 10−2
42.18 ± 13.71 73.83 ± 20.60*
1.72 ± 0.28 0.31 ± 4.9 × 10−2 **
101.67 ± 16.45 134.34 ± 17.77
Data expressed as means ± S.D. (n = 5). Cmax : maximum concentration; AUC: area under the concentration vs. time curve; t1/2 : elimination half-life. * p < 0.05 compared with single 5 mg/kg spinosin administration group. ** p < 0.01 compared with single 5 mg/kg spinosin administration group.
5.58 mg min/L respectively. The t1/2 values of spinosin in blood, bile and brain dialysate also changed, respectively, from 48.07 to 95.04 min, from 97.20 to 152.21 and from 42.18 to 73.83 min when the drug was co-administered with CsA. According to the results above, the AUC and t1/2 values of spinosin in blood, bile and brain dialysate were significantly increased in CsA-added group, which suggested that CsA has obvious effects on the pharmacokinetic process of spinosin. The increase of AUC and t1/2 values of spinosin in the blood and brain could be due to the P-gp inhibition by CsA. CsA inhibited the efflux of spinosin by P-gp located on the membrane of the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (BCSF) barrier, resulting in an increase of the spinosin concentration in the brain. Meanwhile, the inhibition of CsA on the excretion of spinosin may lead to the increase of its AUC in blood. It has been extensively verified that CsA inhibits the hepatobiliary excretion of P-gp substrates from the liver into the bile [23]. Compared with the control group (given 5 mg/kg spinosin alone), the concentration of spinosin in bile dialysate was decreased in the period of 0–40 min when CsA was co-administered, suggesting CsA inhibits the P-gp transporter and thus decrease the hepatobiliary excretion of spinosin. Interestingly, after 40 min, the concentration of spinosin in bile was higher than that of control group, indicating that some other transporters, such as multispecific organic anion transporters [24], non-bile acid organic anion transporters [25], or conjugate export pumps [26] might be involved in the hepatobiliary excretion of spinosin. In addition, the results of the present study indicate that the combining CsA with spinosin could provide an opportunity to achieve enhanced therapeutic effects of spinosin because of the increase of its concentration in blood and brain. The multi-drug treatment might be a useful way to reduce the dose of drugs and achieve better therapeutic effects in clinical practice.
4. Conclusions In this study, a microdialysis technique with HPLC–MS method was developed to investigate the pharmacokinetics of unbound spinosin in the brain, blood and bile dialysate of rats. The work demonstrated this method can be conveniently and sensitively applied to monitor the pharmacokinetic behavior of drugs. The results revealed that CsA has obvious effects on pharmacokinetic process of spinosin since the AUC and t1/2 values of spinosin in blood, bile and brain dialysate were significantly increased after drug co-administration. Since CsA is a well-established Pgp inhibitor, spinosin is also a substrate for P-gp transporter. Because hepatobiliary excretion of spinosin is also increased after co-administration, other transporters or pathways may also be involved in the metabolism of spinosin. Acknowledgements The authors greatly appreciate the financial support from the research project (No. 30973884) of the National Science Foundation of China, the program for Changjiang Scholars and Innovative Research Team in University, No. IRT0868, and the Postgraduate Innovative Projects of China Pharmaceutical University to Dr. Gui-Zhong Xin (No. ZJ10059). References [1] Y.J. Li, K.S. Bi, Study on the therapeutic material basis of traditional Chinese medicinal preparation Suanzaoren Decoction, Chem. Pharm. Bull. 56 (2006) 847–851. [2] K. Kawashima, K. Saito, A. Yamada, S. Obara, T. Ozaki, Y. Kano, Pharmacological properties of traditional medicines. XXIII. Searching for active compounds in the blood and bile of rats after oral administrations of extracts of sansohnin, Biol. Pharm. Bull. 20 (1997) 1171–1174.
R.-H. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 22–29 [3] L.E. Wang, Y.J. Bai, X.R. Shi, X.R. Shi, X.Y. Cui, S.Y. Cui, F. Zhang, Q.Y. Zhang, Y.H. Zhang, Y.Y. Zhao, Spinosin, a C-glycoside flavonoid from semen Ziziphi Spinozae, potentiated pentobarbital-induced sleep via the serotonergic system, Pharmacol. Biochem. Behav. 90 (2008) 399–403. [4] L.E. Wang, X.Y. Cui, S.Y. Cui, J.X. Cao, J. Zhang, Y.H. Zhang, Q.Y. Zhang, Y.J. Bai, Y.Y. Zhao, Potentiating effect of spinosin, a C-glycoside flavonoid of Semen Ziziphi spinosae, on pentobarbital-induced sleep may be related to postsynaptic 5HT(1A) receptors, Phytomedicine 17 (2010) 404–409. [5] Y.J. Li, X.M. Liang, H.B. Xiao, K.S. Bi, Determination of spinosin in rat plasma by reversed-phase high-performance chromatography after oral administration of Suanzaoren decoction, J. Chromatogr. B 787 (2003) 421–425. [6] Y.J. Li, M.C. Yao, S. Cheng, Quantitative determination of spinosin in rat plasma by liquid chromatography–tandem mass spectrometry method, J. Pharm. Biomed. Anal. 48 (2008) 1169–1173. [7] Y.J. Li, X.M. Liang, H.B. Xiao, K.S. Bi, Pharmacokinetic study on spinosin in rat plasma after oral administration of suanzaoren extract at a single dose, Acta Pharm. Sin. 38 (2003) 448–450. [8] Y.J. Li, Y.H. Dai, Y.L. Yu, Y. Li, Y.L. Deng, Pharmacokinetics and tissue distribution of spinosin after intravenous administration in rats, Yakugaku Zasshi 127 (2007) 1231–1235. [9] K.D. Bao, P. Li, L.W. Qi, H.J. Li, L. Yi, W. Wang, Y.Q. Wang, Characterization of flavonoid metabolites in rat plasma, urine, and feces after oral administration of Semen Ziziphi Spinosae extract by HPLC–diode-array detection (DAD) and ion-trap mass spectrometry (MS(n)), Chem. Pharm. Bull. 57 (2009) 144–148. [10] V.R. Tandon, B. Kapoor, G. Bano, S. Gupta, Z. Gillani, S. Gupta, D. Kour, P-glycoprotein: pharmacological relevance, Indian J. Pharmacol. 38 (2006) 13–24. [11] M. Dorababu, A. Nishimura, T. Prabha, K. Naruhashi, N. Sugioka, K. Takada, N. Shibata, Effect of cyclosporine on drug transport and pharmacokinetics of nifedipine, Biomed. Pharmacother. 63 (2009) 697–702. [12] J.H. Lin, Drug–drug interaction mediated by inhibition and induction of Pglycoprotein, Adv. Drug Deliv. Rev. 55 (2003) 53–81. [13] J.H. Hamman, P.H. Demana, E.I. Olivier, Targeting receptors, transporters and site of absorption to improve oral drug delivery, Drug Target Insights 2 (2007) 71–81. [14] J.D. Wright, F.D. Boudinot, M.R. Ujhelyi, Measurement and analysis of unbound drug concentrations, Clin. Pharmacokinet. 30 (1996) 445–462.
29
[15] M.L.T. Vieira, R.P. Singh, H. Derendorf, Simultaneous, HPLC analysis of triamcinolone acetonide and budesonide in microdialysate and rat plasma: application to a pharmacokinetic study, J. Chromatogr. B 878 (2010) 2967–2973. [16] Q. Wu, X.D. Wen, L.W. Qi, W. Wang, L. Yi, Z.M. Bi, P. Li, An in vivo microdialysis measurement of harpagoside in rat blood and bile for predicting hepatobiliary excretion and its interaction with cyclosporin A and verapamil, J. Chromatogr. B 877 (2009) 751–756. [17] C.F. Chien, Y.T. Wu, W.C. Lee, L.C. Lin, T.H. Tsai, Herb–drug interaction of Andrographis paniculata extract and andrographolide on the pharmacokinetics of theophylline in rats, Chem. Biol. Interact. 184 (2010) 458–465. [18] W.H. Sheu, H.C. Chuang, S.M. Cheng, M.R. Lee, C.C. Chou, F.C. Cheng, Microdialysis combined blood sampling technique for the determination of rosiglitazone and glucose in brain and blood of gerbils subjected to cerebral ischemia, J. Pharm. Biomed. Anal. 54 (2011) 759–764. [19] L. Kagan, T. Dreifinger, D. Mager, A. Hoffman, Role of p-glycoprotein in regionspecific gastrointestinal absorption of talinolol in rats, Drug. Metab. Dispos. 38 (2010) 1560–1566. [20] X.Y. Chu, K. Bleasby, J. Yabut, X.X. Cai, G.H. Chan, M.J. Hafey, S.Y. Xu, A.J. Bergman, M.P. Braun, D.C. Dean, R. Evers, Transport of the dipeptidyl peptidase-4 inhibitor sitagliptin by human organic anion transporter 3, organic anion transporting polypeptide 4C1, and multidrug resistance P-glycoprotein, J. Pharmacol. Exp. Ther. 321 (2007) 673–683. [21] W.S. Woo, S.S. Kang, S.H. Shim, H. Wagner, V.M. Chari, O. Seligmann, G. Obermeier, The structure of spinosin (2 -O--glucosylswertisin) from Zizyphus vulgaris var.Spinosus, Phytochemistry 18 (1979) 353–355. [22] T.H. Tsai, Assaying protein unbound drugs using microdialysis techniques, J. Chromatogr. B 797 (2003) 161–173. [23] X.Y. Chu, Y. Kato, Y. Sugiyama, Possible involvement of P-glycoprotein in biliary excretion of CPT-11 in rats, Drug Metab. Dispos. 27 (1999) 440–441. [24] Y. Kobayashi, R. Sakai, N. Ohshiro, M. Ohbayashi, N. Kohyama, T. Yamamoto, Possible involvement of organic anion transporter 2 on the interaction of theophylline with erythromycin in the human liver, Drug Metab. Dispos. 33 (2005) 619–622. [25] C.D. Klaassen, L.M. Aleksunes, Xenobiotic, bile acid, and cholesterol transporters: function and regulation, Pharmacol. Rev. 62 (2010) 1–96. [26] M.J. Zamek-Gliszczynski, K.A. Hoffmaster, K. Nezasa, M.N. Tallman, K.L.R. Brouwer, Integration of hepatic drug transporters and phase II metabolizing enzymes: mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites, Eur. J. Pharm. Sci. 27 (2006) 447–486.