- Email: [email protected]
MS
Journal of Chromatography B 1113 (2019) 77–83
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
A novel xanthine oxidase inhibitor WSJ-557 study on pharmacokinetics and tissue distribution in rats by UPLC–MS/MS Donghu Zhang, Tian Yang, Jianyang Lin
T
⁎
Department of Pharmacy, the First Hospital of China Medical University, 155 Nanjing North Street, Shenyang 110001, Liaoning, China School of Pharmaceutical Science, China Medical University, 77 Puhe Road, Shenyang 110122, Liaoning, China
ARTICLE INFO
ABSTRACT
Keywords: WSJ-557 Pharmacokinetics Tissue distribution Bioavailability UPLC-MS/MS Rats
As a novel non-purine xanthine oxidase inhibitor, WSJ-557 is a potential drug for gout. To determine the WSJ557 concentration in plasma and various tissues of rats, a fast and sensitive method was first established by the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in this paper. The liquid–liquid extraction of ethyl acetate was adopted for the sample preparation, and carbamazepine was taken as the internal standard. In the process of chromatographic separation, MRM transitions for WSJ-557 and carbamazepine (internal standard, IS) were m/z 316.1 → 260.0 and m/z 237.0 → 194.0, correspondingly. The great linearity of WSJ-557 in all bio-samples was found in the corresponding concentration range (r > 0.99). The intra- and inter-day precision (RSD%) were below 9.5% in various tissues and plasma, whose accuracy (RE%) was within ± 9.2%. This method was resoundingly employed to the WSJ-557 study on rat pharmacokinetics and tissue distribution after the intravenous administration and oral administration. The average absolute bioavailability (F) of WSJ-557 was 6.48%. The highest distribution level of gastric and intestinal tissues indicated that WSJ-557 was first absorbed in the stomach and intestine. Moreover, this analytical method provides a significant approach for the further development and investigation of WSJ-557.
1. Introduction Gout is an abnormal metabolism resulted from the accumulation of urate crystals in joints or soft tissues, leading to an acute or no inflammatory response [1]. The long-term treatment of gout is concentrated on urate-lowering therapy (ULT), with which the sUA (serum uric acid) can be controlled in a sub-saturation range (usually < 6.0 mg·dL−1) [2,3]. To this end, two pharmacological approaches to ULT are adopted: the improvement of urinary excretion of uric acid and the inhibition of urate production with XO inhibitor [4,5]. XO inhibitors, such as febuxostat and allopurinol, have been currently approved for gout treatment. However, the drug-induced adverse reactions limit their long-term application [6]. Accordingly, new agents that possess potent XO inhibiting effects, lowered toxicity, and little adverse drug reactions are expected to prevent hyperuricemia or gout. WSJ-557, 2(4-(sec-butoxy)-3-cyanophenyl)-1-hydroxy-4-methyl-1H-imidazole-5carboxylic acid, is a novel non-purine XO inhibitor, which was synthesized by Shenyang Pharmaceutical University. It has a different structure from WSJ-537 [7] with a dominant XO inhibitory activity
⁎
(IC50 = 0.003 μM), which is much higher than febuxostat (IC50 = 0.01 μM) and WSJ-537 (IC50 = 0.005 μM) [8] in vitro pharmacological studies. Considering this remarkable activity, it is imperative to conduct a comprehensive study on vivo pharmacokinetics and distribution of WSJ-557 in the appropriate animal model. It is universally acknowledged that the study of pharmacokinetics and tissue distribution are considerable for the drug development [9], because it is helpful for prognosticating various issues related to drug efficacy and toxicity [10]. These issues were closely related to systemic concentration and exposure time. Previous LC-MS/MS methods have been achieved for WSJ-537, while WSJ-557 has a different structure and chromatographic performance from WSJ-537. Therefore, a new sensitive and reliable bio-analytical assays is essential for quantifying WSJ-557 concentrations biological matrices for further research. To our best acquaintance, there is no bioassay for the WSJ-557 determination in rat plasma and tissues. Thus, an fast, specific and sensitive UPLC-MS/MS method was first verified to investigate intravenous (i.v.), oral pharmacokinetics of WSJ-557 and tissue distribution after the oral administration in rats.
Corresponding author at: Department of Pharmacy, the First Hospital of China Medical University, 155 Nanjing North Street, Shenyang 110001, Liaoning, China. E-mail address: [email protected] (J. Lin).
https://doi.org/10.1016/j.jchromb.2019.03.013 Received 4 December 2018; Received in revised form 3 February 2019; Accepted 13 March 2019 Available online 15 March 2019 1570-0232/ © 2019 Elsevier B.V. All rights reserved.
D. Zhang, et al.
Journal of Chromatography B 1113 (2019) 77–83
2. Experimental process
2.5. Sample preparation
2.1. Animals
A biological sample of 100 μL aliquot (tissue sample or plasma sample) was transferred into a clean glass tube with 50 μL of IS solution (92 ng·mL−1) and 50 μL of methanol, respectively. The mixture was derived from 2.0 mL of ethyl acetate by 90 s vortex-mixing. After 10 min centrifugation at 3500 rpm, the supernatant was moved to another pure glass tube. Under the 40 °C nitrogen stream, the supernatant was dispersed. The remains were redissolved in 100 μL mobile phase by 30s vortex-mixing. Finally, 10 μL aliquot was transfused into UPLC-MS/ MS for the following analysis.
Before the experiment, Sprague-Dawley rats have been raised in SPF grade room of Shenyang Pharmaceutical University for a week, and then fasted for 12 h [11,12]. With the approval of Animal Ethics Committee of Shenyang Pharmaceutical University, this animal study was conducted in accordance with relevant regulations. 2.2. Reagents and chemicals WSJ-557 (purity 99.0%) was obtained from the pharmaceutical laboratory of Shenyang Pharmaceutical University. As an internal standard (IS), the purity 99.3% Carbamazepine was procured from National Institute for Food and Drug Control (Beijing, China). Ethyl acetate and methanol (HPLC grade), ammonium acetate (HPLC grade) were procured from Concord Technology Co., Ltd. (Tianjin, China) and Dikma (Richmond Hill, NY, USA), respectively. Ultrapure water was procured from Wahaha (Hangzhou, Jiangsu, China) in this experiment. Other chemicals and reagents were chromatographic grades and merchantable.
2.6. Verification procedures According to FDA guidelines [13] for verifying bio-analytical method, this method was validated in different properties. (1) Selectivity To study the selectivity of the proposed method, the chromatograms of blank rat plasma and liver tissue homogenate in 6 different batches were compared with those of QC (LLOQ) samples and biological samples subjected to the oral administration of 12 mg·kg−1 WSJ-557.
2.3. Equipments and operating conditions
(2) Linearity and sensitivity
2.3.1. Mass spectrometry In this experiment, triple-quadrupole tandem mass spectrometry was performed on the Micromass® Quattro micro™ API mass spectrometer (manufactured by Waters Corp., Milford, MA, USA) with an ESI source. Positive MRM modes were carried out in the quantification. MRM transitions for WSJ-557 and carbamazepine (IS) were m/z 316.1 → 260.0 and m/z 237.0 → 194.0, accordingly. Capillary voltage and cone voltage were 4.0 kV and 25 V, accordingly. Desolvation temperature and source temperature were maintained at 400 °C and 120 °C, correspondingly. Nitrogen was selected as the desolvation and cone gas, whose flowing rates were 450 and 30 L·h−1. The collision gas was highpurity argon. Optimized collision energy for WSJ-557 was set at 15 eV. All data were collected and analyzed by MassLynx™ NT 4.1 software.
To study the linearity of calibration curves, the method of weighted (1/x2) least-squares linear regression analysis, was used by plotting the peak area ratio of WSJ-557 to that of IS against the corresponding concentration of standard WSJ-557. Characterized as the lowest concentration on the standard curve, the LLOQ of the assay can be concluded with accuracy and precision. The accuracy of LLOQ should be within ± 20%, and the precision should not exceed 20%. (3) Accuracy and precision Six duplicates were assessed at the LLOQ and the 3 QC levels on the same day to obtain the intra-day precision and accuracy. The inter-day precision and accuracy were measured by assessing LLOQ and 3 QC levels within 3 consecutive days. In agreement with the guidance of FDA, the intra-day and inter-day precision were characterized as RSD within 15%, and the accuracy was characterized as the percentage variation between the calculated and the nominal concentration within 15%. LLOQ should be below 20% of precision and within ± 20% of accuracy.
2.3.2. Liquid chromatography The ACQUITY™ UPLC system was adopted with cooling auto-sampler and column oven in the chromatographic separation, guaranteeing the temperature control. The ACQUITY™ UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) was used as the analytical column, and the temperature was set at 40 °C. Methanol (A) and 5 mmol·L−1 ammonium acetate (B) constituted the mobile phase. This gradient elution program was operated as follows: 0–0.5 min, 50% A; 0.5–2.5 min, 80% A; 2.5–4.0 min, 50% A. Besides, flow rate was 0.20 mL·min−1, auto-sampler temperature was 4 °C, and injection volume was 10 μL.
(4) Extraction recovery and matrix effect Three QC concentrations (LQC, MQC and HQC) were all used to evaluate recoveries and matrix effects. Extraction recovery was assessed by contrasting the peak areas of extracted QC samples with unextracted QC samples. Matrix effects were estimated by contrasting peak areas of samples where blank samples were spiked with WSJ-557 after liquidliquid extraction to that where QC samples are spiked in the mobile phase.
2.4. Preparations of standard solutions and QC samples The stock solution of 50 μg·mL−1 WSJ-557 was provided by dissolving the certain amount of the chemical reference substance in methanol. A range of WSJ-557 working solutions were arranged by serially diluting stock solutions with methanol to the appropriate concentration. The 920 ng·mL−1 stock solution of carbamazepine was diluted with methanol for the preparation of IS working solution (92 ng·mL−1). The optimum temperature for storage of all solutions was 4 °C. To yield final concentrations of 5.0–20,000 ng·mL−1, the calibration standard was provided by spiking 100 μL of blank biological samples with appropriate amounts of working solutions. Quality control (QC) samples were served in the identical method with low, medium and high concentrations, correspondingly. The storage temperature for all standard solutions and QC samples was set at 4 °C.
(5) Stability The test on WSJ-557 stability in biological samples was carried out at two QC levels (LQC, HQC) under different conditions: after 4hours short-term storage at room temperature, after 6-hours posttreatment in auto-sampler at 4 °C, after 3 freeze-thaws cycles (from −20 °C to room temperature) and the long-term storage for 15 days at −20 °C. 78
D. Zhang, et al.
Journal of Chromatography B 1113 (2019) 77–83
2.7. Pharmacokinetic study
acetonitrile with different concentrations of ammonium acetate and formic acid. Because of shorter chromatographic running time and lower ground noise, methanol was selected as the organic portion of the mobile phase. 5 mmol·L−1 ammonium acetate was added to the mobile phase, so as to improve peak shapes without affecting the sensitivity of MS detection. Consequently, the mobile phase was composed of 5 mmol·L−1 ammonium acetate and methanol in water. Gradient elution was chosen for the better separation and symmetric peak shapes.
16 Male Sprague-Dawley (SD) rats (230 ± 20 g) were randomly branched into 2 groups (n = 8). The oral dose suspended in 0.5% CMCNa was employed for the first group by oral gavage, and the intravenous (i.v.) dose was employed for the second group through the caudal vein. The i.v. formulation was arranged by dissolving WSJ-557 in a mixture of castor oil: DMSO: ethanol in a ratio of 83:2:15. 0.3 mL of blood samples, which were gathered into heparinized tubes via the suborbital vein after oral administration of 12 mg·kg−1 WSJ-557 at 0, 0.333, 0.667, 1, 1.5, 2, 3, 4, 8, 12, 16 and 24 h, and after the intravenous administration of 12 mg·kg−1 WSJ-557 at 0, 0.017, 0.083, 0.17, 0.33, 0.5, 1, 2, 4, 6, 8 and 12 h. Blood samples were centrifuged at 13000 rpm for 10 min immediately. Before the analysis, all samples were reserved at −20 °C. All pharmacokinetic parameters were measured by Drug and Statistics, issued by Mathematical Pharmacology Professional Committee of China, Shanghai, China.
3.1.2. Improvement of sample preparations To extract the analyte and IS from tissue homogenate and plasma, protein precipitation and liquid–liquid extraction were investigated. In this study, liquid-liquid extraction was found to produce cleaner biological sample extracts with a high recovery and selectivity. Different extracting solvents were analyzed, including dichloromethane, ethyl ether, ethyl acetate, and MTBE (methyl tert-butyl ether). In the end, ethyl acetate was selected for its higher recovery and lower background absorption.
2.8. Tissue distribution studies To investigate the WSJ-557 tissue distribution, 32 SD rats (300 ± 30 g) were randomly classified into 4 groups, namely 8 rats in each group (female and male are equal in number). Subjected to 12 mg·kg−1 WSJ-557 oral administration, 8 rats were sacrificed at each time point (0.5, 4, 8 and 12 h). Their tissues were collected, including liver, heart, brain, spleen, kidney, lung, stomach, large intestinal, small intestinal, fat, muscle, ovary or testicle (the tissue of fat was get from the enterocoelia). Tissue samples from the rats were cleansed thoroughly, removing the blood or content by physiological saline. Then tissue samples were dried by filter papers, and deposited at −20 °C. 0.5 g tissue samples (if the total weight of the tissue is < 0.5 g, it is accepted totally and recorded the weight precisely) was accurately weighed, homogenized in 0.5 mL methanol by using tissue homogenizer, and stored at −20 °C for the following analysis.
3.2. Method verification (1) Selectivity The typical chromatograms of the blank rat plasma are shown in Fig. 2. WSJ-557 and IS were spiked with the blank rat plasma. After a single oral dose of 12 mg·kg−1 WSJ-557, real plasma sample was kept for 1 h and liver sample 0.5 h. No obvious interrupting endogenous peaks were observed within the retention time of WSJ-557 and IS. (2) Calibration curve and linearity A great linearity over the range of 10.0–20,000 ng·mL−1 in plasma and 5.0–10,000 ng·mL−1 for rat tissues was observed from the standard calibration curve. Table 1 presents typical calibration curves for different tissues and plasma. It should be noted that correlation coefficients (r) exceeded 0.99 completely.
2.9. Data analysis DAS 2.0 software, provided by the Pharmacological Society of China (Beijing, China), was used to calculate WSJ-557 pharmacokinetic parameters in rats. The oral absolute bioavailability (F) was calculated as [(AUCpo × Doseiv) / (AUCiv × Dosepo)] × 100%.
(3) Accuracy and precision Table 2 shows the intra- and inter-day precision and accuracy results of LLOQ and three levels of QC samples. In this study, the accuracy (RE) ranged from −5.8% to 9.2% in all plasma samples and liver homogenates, and the intra- and inter-day precision (RSD) were 3.0%–9.5% and 3.4%–9.5%.
3. Results and discussion 3.1. Method establishment 3.1.1. Improvement of mass conditions and chromatography Negative ion and positive ion modes of WSJ-557 ionization were analyzed on an ESI ion source, in order to determine the WSJ-557 accurately in the MS/MS method. The signal strength of the deprotonated molecule ion [M − H]− was weaker than that of the protonated molecule ion [M + H]+. Carbamazepine was chosen as the most appropriate IS owing to its identical mass spectrometric and chromatographic behaviors to WSJ-557 in the positive ion mode. As a result, the monitoring mode of the positive ion was selected for WSJ-557 and IS. Fig. 1 shows the spectrum of WSJ-557 and IS with chemical structures. After giving certain collision energy, the most plentiful and constant product ions for WSJ-557 and IS were monitored at m/z 260.0 and m/z 194.0, respectively. MRM transitions for WSJ-557 and IS were m/z 316.1 → 260.0 and m/z 237.0 → 194.0, which were employed for quantitative analysis. Parameters were improved by the highest intensity observed for product ions, including the source temperature, collision energy, cone, capillary voltage. To achieve the high sensitivity, symmetric peak and a short elution time in each WSJ-557 and IS analysis, the mobile phase optimization was obtained by investigating various proportion of methanol or
(4) Matrix effect and extraction recovery Table 2 also shows the extraction recovery and matrix effect of WSJ557 in plasma and liver homogenates. The RSD was within 12.9%, and the average WSJ-557 extraction recovery of QC samples at three levels was 77.1%–98.4% in plasma and 89.1%–97.2% in liver homogenates, indicating the reliability of this method. The RSD was within 6.3%, and values of matrix effect were 89.1%–112.4% in plasma and 93.7%–109.9% in liver homogenates, accordingly. The result shows that the liquid-liquid extraction efficiency was acceptable and no significant interference was found from the plasma or liver homogenates. (5) Stability The WSJ-557 stability in rat biological samples was performed from two QC levels (LQC, HQC) under different conditions. As shown in Table 3, WSJ-557 was proved to be steady in the rat plasma and liver homogenates during the storage and extraction. 79
Journal of Chromatography B 1113 (2019) 77–83
D. Zhang, et al.
Fig. 1. WSJ-557 and IS ion scanning mass spectrogram (A) WSJ-557 (B) IS.
Fig. 2. Representative MRM chromatograms for WSJ-557 and IS: (A) blank rat plasma; (B) blank rat plasma spiked with 10 ng·mL−1 of WSJ-557 and IS; (C) a plasma sample after 1 h administration of WSJ-557 to rat; (D) a liver homogenate sample after 0.5 h administration of WSJ-557 to rat.
80
Journal of Chromatography B 1113 (2019) 77–83
D. Zhang, et al.
Table 1 Calibration curve parameters of WSJ-557 in different biological samples. Tissues
Slope
Intercept
Plasma Liver Heart Brain Spleen Lung Kidney Stomach Small intestinal Large intestinal Fat Muscle Ovary Testicle
0.2359 0.2584 0.3819 0.3680 0.3614 0.4274 0.3966 0.3051 0.5278 0.2685 0.2518 0.3563 0.3346 0.3745
10.58 1.639 13.54 8.533 15.81 14.51 14.57 9.674 17.73 4.642 8.835 4.932 31.89 20.45
Table 4 Main pharmacokinetic parameters of WSJ-557 in rats after oral or i.v. administration (n = 8).
Linear ranges (ng·mL−1)
r 0.9990 0.9913 0.9912 0.9946 0.9918 0.9921 0.9906 0.9926 0.9940 0.9915 0.9902 0.9943 0.9947 0.9923
10.0–20,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000
Parameters
Unit
Cmax Tmax T1/2 AUC0−t AUC0−∞ MRT0−∞ CL Vd
ng·mL−1 h h ng·h·mL−1 ng·h·mL−1 h L·h−1·kg−1 L·kg−1
Oral administration
Intravenous administration
128.2 ± 34.7 3.38 ± 0.74 5.47 ± 1.08 1318.8 ± 296.5 1400.6 ± 287.9 9.65 ± 1.23 8.87 ± 1.68 71.09 ± 22.39
9519.4 ± 3600 0.017 3.76 ± 2.05 19,916 ± 12,331 21,632 ± 12,298 4.57 ± 2.70 0.70 ± 0.33 4.13 ± 3.21
plasma concentration-time curves (n = 8) of WSJ-557 in rats. Table 4 reveals the corresponding main pharmacokinetic parameters defied as mean ± SD. The average time of reaching the maximum concentration (Tmax) was 3.38 ± 0.74 h by oral administration, indicating the slow absorption of circulation in rats. The T1/2 values indicated that the elimination of WSJ-557 from plasma was a slow process. Compared with WSJ-537 [7], the longer Tmax and T1/2 contribute to a lasting effect of WSJ-557 on gout and may reduce the administration frequency so as to improve patients' compliance. The apparent distribution volume (Vd)
3.3. Pharmacokinetic research and bioavailability After the oral and i.v. administration at a single dose of 12 mg·kg−1, the UPLC-MS/MS method was resoundingly employed in the pharmacokinetic study of WSJ-557 in biological samples. Fig. 3 reveals average
Table 2 Precision, accuracy, recovery and matrix effect for the determination of WSJ-557 in rat biological samples (intra-day: n = 6; inter-day: n = 6 series per day, 3 days). Samples
Analyte
Plasma
WSJ-557
Liver
WSJ-557
Concentrations (ng·mL−1)
Precision (RSD, %)
Added
Found (mean ± SD)
Intra-day
Inter-day
10 25 800 16,000 5.0 12.5 333.3 8333
9.8 ± 0.9 25.4 ± 2.1 820.6 ± 63.4 15,065.6 ± 1036.5 5.1 ± 0.4 12.6 ± 1.1 331.6 ± 16.6 9097.4 ± 311.3
9.5 7.6 7.1 3.0 7.0 8.5 4.3 4.4
9.5 8.4 7.7 6.9 8.3 8.6 5.0 3.4
Accuracy (RE, %)
−2.4 1.6 2.6 −5.8 2.1 1.0 −0.5 9.2
Recovery
Matrix effect
Mean ± SD (%)
RSD (%)
Mean ± SD (%)
RSD (%)
– 77.1 ± 4.9 84.6 ± 10.9 98.4 ± 7.0 – 97.2 ± 5.3 89.5 ± 4.7 89.1 ± 4.8
– 6.4 12.9 7.1 – 5.4 5.2 5.4
– 112.4 ± 2.4 89.1 ± 5.3 94.9 ± 5.3 – 109.9 ± 5.3 96.2 ± 6.0 93.7 ± 3.6
– 2.2 6.0 5.6 – 4.8 6.3 3.8
Table 3 Stability of WSJ-557 in rat plasma and liver homogenate under various storage conditions (n = 3). Samples
Concentration (ng·mL−1)
4 h at room temperature
Three freeze-thaw cycles
6 h storage in the autosampler
15 days at −20 °C
Plasma
25 16,000 12.5 8333
99.3 ± 9.2 94.6 ± 7.9 107.8 ± 7.9 104.1 ± 3.6
97.0 ± 9.3 93.9 ± 8.4 101.2 ± 5.2 101.7 ± 4.2
96.0 ± 12.6 92.1 ± 7.9 109.1 ± 6.2 105.6 ± 0.7
103.6 ± 8.4 95.5 ± 11.0 104.4 ± 5.9 98.8 ± 2.6
Liver
Fig. 3. Concentration-time curve of WSJ-557 after oral (A) or i.v. (B) administration in rats (n = 8).
81
Journal of Chromatography B 1113 (2019) 77–83
D. Zhang, et al.
Fig. 4. The concentration–time profile of WSJ-557 in various tissue homogenates after the oral administration of WSJ-557 (12 mg·kg−1) to rats.
of WSJ-557 was 71.09 ± 22.39 L·kg−1, indicating that the drug distribution was extensive in tissues than that in plasma. The absolute bioavailability (F) of WSJ-557 was estimated as only 6.48% in rats. The low oral bioavailability of WSJ-557 was caused by the high intestinal bacterial biotransformation and first-pass metabolism. The new dosage forms will be used to improve the WSJ-557 bioavailability in future study.
highest. While WSJ-557 can hardly cross BBB in rat. The high distribution and low absolute oral bioavailability (6.48%) were observed in the intestine, suggesting the high first-pass metabolism and intestinal bacterial biotransformation probably. The parameters obtained from the study of WSJ-557 provide helpful information for further research and clinical references on rational use of drugs. Acknowledgments
3.4. Tissue distribution studies
This project is supported by National Natural Science Foundation of China under Grant 81302841 and University Outstanding Talent Support Plan Foundation of Liaoning Province under Grant LJQ2014086. The determination method of WSJ-557 in rat plasma by UPLC–MS/ MS in this report is the subject of a pending Chinese patent filed by The First Hospital of China Medical University in Shenyang City with application number CN201710052486.7.
Fig. 4 shows the WSJ-557 concentration in various tissues after the oral administration at different times. The drug was detected in most of the rat tissues at 0.5 h, indicating that it was extensively distributed in rat tissues after the oral administration. From the results, it was found that the concentration of WSJ-557 in stomach, small intestine and large intestine were much greater than other tissues, indicating that the drug was quickly absorbed from the gastrointestinal tract probably. The lower concentration of WSJ-557 was exhibited in the spleen, heart and kidney, indicating that blood flow and perfusion rate of the organ did not have an important function in the WSJ-557 distribution probably. In addition, the WSJ-557 was hardly detected in the rat brain, ovary or testicle, illustrating that WSJ-557 may not go through the blood-brain barrier (BBB).
References [1] G. Ragab, M. Elshahaly, T. Bardin, Gout: an old disease in new perspective – a review, J. Adv. Res. 8 (2017) 495–511. [2] H.R. Schumacher, M.A. Becker, E. Lloyd, P.A. MacDonald, C. Lademacher, Febuxostat in the treatment of gout: 5-yr findings of the FOCUS efficacy and safety study, Rheumatology 48 (2009) 188–194. [3] B.W. Coburn, K.A. Bendlin, H. Sayles, K.S. Hentzen, M.M. Hrdy, T.R. Mikuls, Target serum urate: do gout patients know their goal? Arthritis Care Res. 68 (2016) 1028–1035. [4] W. Zhang, M. Doherty, T. Bardin, E. Pascual, V. Barskova, P. Conaghan, J. Gerster, J. Jacobs, B. Leeb, F. Lioté, G. McCarthy, P. Netter, G. Nuki, F. Perez-Ruiz, A. Pignone, J. Pimentão, L. Punzi, E. Roddy, T. Uhlig, I. Zimmermann-Gòrska, EULAR evidence based recommendations for gout. Part II: management. Report of a task force of the EULAR Standing Committee for International Clinical Studies Including Therapeutics (ESCISIT), Ann. Rheum. Dis. 65 (2006) 1312–1324. [5] D. Khanna, P.P. Khanna, J.D. Fitzgerald, M.K. Singh, M. Kaldas, M. Gogia, F. Perez-ruiz, W. Taylor, F. Lioté, 2012 American college of rheumatology guidelines for management of gout part II: therapy and anti-inflammatory prophylaxis of acute gouty arthritis, Arthritis Care Res. 64 (2013) 1447–1461. [6] A.F. Rheumatology, P.K. Rheumatologist, The management of gout, Aust. Prescr. 39 (2016) 119–122.
4. Conclusions In this study, a fast, specific and sensitive UPLC-MS/MS method was first proposed to determine the WSJ-557 in plasma and tissue of rat. The pretreatment of the large number of plasma and tissue homogenates samples was obtained by the simple sample preparation procedure. The LLOQ was 5.0 ng·mL−1 in this method. Owing to the short running time of 4.0 min, the method will be useful for further preclinical studies of WSJ-557. After the oral administration of WSJ-557, it was widely spread in various tissues, where the gastrointestinal tract is
82
Journal of Chromatography B 1113 (2019) 77–83
D. Zhang, et al. [7] J. Lin, T. Yang, D. Zhang, Development of a selective and fast LC-MS/MS for determination of WSJ-537, an xanthine oxidase inhibitor, in rat plasma: application to a pharmacokinetic study, J. Chromatogr. B 1028 (2016) 51–55. [8] S. Chen, T. Zhang, J. Wang, F. Wang, H. Niu, C. Wu, S. Wang, Synthesis and evaluation of 1-hydroxy/methoxy-4-methyl-2-phenyl-1H-imidazole-5-carboxylic acid derivatives as non-purine xanthine oxidase inhibitors, Eur. J. Med. Chem. 103 (2015) 343–353. [9] E.A. Lakota, J.C. Bader, C.M. Rubino, Ensuring quality pharmacokinetic analyses in antimicrobial drug development programs[J], Curr. Opin. Pharmacol. 36 (2017) 139–145. [10] L. Zhu, J. Wu, M. Zhao, et al., Mdr1a plays a crucial role in regulating the analgesic effect and toxicity of aconitine by altering its pharmacokinetic characteristics[J], Toxicol. Appl.
Pharmacol. 320 (2017) 32–39. [11] L. Qiao, Y. Liu, X. Chen, et al., A HPLC-MS/MS method for determination of 6‴-feruloylspinosin in rat plasma and tissues: pharmacokinetics and tissue distribution study [J], J. Pharm. Biomed. Anal. 121 (2016) 77–83. [12] A. Zhao, Y. Zhang, X. Yang, Simultaneous determination and pharmacokinetics of sixteen Angelicae dahurica coumarins in vivo by LC–ESI-MS/MS following oral delivery in rats [J], Phytomedicine 23 (10) (2016) 1029–1036. [13] FDA Guidance for Industry: Bioanalytical Method Validation, http://www.fda.gov/ downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070107. pdf.
83
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
A novel xanthine oxidase inhibitor WSJ-557 study on pharmacokinetics and tissue distribution in rats by UPLC–MS/MS Donghu Zhang, Tian Yang, Jianyang Lin
T
⁎
Department of Pharmacy, the First Hospital of China Medical University, 155 Nanjing North Street, Shenyang 110001, Liaoning, China School of Pharmaceutical Science, China Medical University, 77 Puhe Road, Shenyang 110122, Liaoning, China
ARTICLE INFO
ABSTRACT
Keywords: WSJ-557 Pharmacokinetics Tissue distribution Bioavailability UPLC-MS/MS Rats
As a novel non-purine xanthine oxidase inhibitor, WSJ-557 is a potential drug for gout. To determine the WSJ557 concentration in plasma and various tissues of rats, a fast and sensitive method was first established by the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in this paper. The liquid–liquid extraction of ethyl acetate was adopted for the sample preparation, and carbamazepine was taken as the internal standard. In the process of chromatographic separation, MRM transitions for WSJ-557 and carbamazepine (internal standard, IS) were m/z 316.1 → 260.0 and m/z 237.0 → 194.0, correspondingly. The great linearity of WSJ-557 in all bio-samples was found in the corresponding concentration range (r > 0.99). The intra- and inter-day precision (RSD%) were below 9.5% in various tissues and plasma, whose accuracy (RE%) was within ± 9.2%. This method was resoundingly employed to the WSJ-557 study on rat pharmacokinetics and tissue distribution after the intravenous administration and oral administration. The average absolute bioavailability (F) of WSJ-557 was 6.48%. The highest distribution level of gastric and intestinal tissues indicated that WSJ-557 was first absorbed in the stomach and intestine. Moreover, this analytical method provides a significant approach for the further development and investigation of WSJ-557.
1. Introduction Gout is an abnormal metabolism resulted from the accumulation of urate crystals in joints or soft tissues, leading to an acute or no inflammatory response [1]. The long-term treatment of gout is concentrated on urate-lowering therapy (ULT), with which the sUA (serum uric acid) can be controlled in a sub-saturation range (usually < 6.0 mg·dL−1) [2,3]. To this end, two pharmacological approaches to ULT are adopted: the improvement of urinary excretion of uric acid and the inhibition of urate production with XO inhibitor [4,5]. XO inhibitors, such as febuxostat and allopurinol, have been currently approved for gout treatment. However, the drug-induced adverse reactions limit their long-term application [6]. Accordingly, new agents that possess potent XO inhibiting effects, lowered toxicity, and little adverse drug reactions are expected to prevent hyperuricemia or gout. WSJ-557, 2(4-(sec-butoxy)-3-cyanophenyl)-1-hydroxy-4-methyl-1H-imidazole-5carboxylic acid, is a novel non-purine XO inhibitor, which was synthesized by Shenyang Pharmaceutical University. It has a different structure from WSJ-537 [7] with a dominant XO inhibitory activity
⁎
(IC50 = 0.003 μM), which is much higher than febuxostat (IC50 = 0.01 μM) and WSJ-537 (IC50 = 0.005 μM) [8] in vitro pharmacological studies. Considering this remarkable activity, it is imperative to conduct a comprehensive study on vivo pharmacokinetics and distribution of WSJ-557 in the appropriate animal model. It is universally acknowledged that the study of pharmacokinetics and tissue distribution are considerable for the drug development [9], because it is helpful for prognosticating various issues related to drug efficacy and toxicity [10]. These issues were closely related to systemic concentration and exposure time. Previous LC-MS/MS methods have been achieved for WSJ-537, while WSJ-557 has a different structure and chromatographic performance from WSJ-537. Therefore, a new sensitive and reliable bio-analytical assays is essential for quantifying WSJ-557 concentrations biological matrices for further research. To our best acquaintance, there is no bioassay for the WSJ-557 determination in rat plasma and tissues. Thus, an fast, specific and sensitive UPLC-MS/MS method was first verified to investigate intravenous (i.v.), oral pharmacokinetics of WSJ-557 and tissue distribution after the oral administration in rats.
Corresponding author at: Department of Pharmacy, the First Hospital of China Medical University, 155 Nanjing North Street, Shenyang 110001, Liaoning, China. E-mail address: [email protected] (J. Lin).
https://doi.org/10.1016/j.jchromb.2019.03.013 Received 4 December 2018; Received in revised form 3 February 2019; Accepted 13 March 2019 Available online 15 March 2019 1570-0232/ © 2019 Elsevier B.V. All rights reserved.
D. Zhang, et al.
Journal of Chromatography B 1113 (2019) 77–83
2. Experimental process
2.5. Sample preparation
2.1. Animals
A biological sample of 100 μL aliquot (tissue sample or plasma sample) was transferred into a clean glass tube with 50 μL of IS solution (92 ng·mL−1) and 50 μL of methanol, respectively. The mixture was derived from 2.0 mL of ethyl acetate by 90 s vortex-mixing. After 10 min centrifugation at 3500 rpm, the supernatant was moved to another pure glass tube. Under the 40 °C nitrogen stream, the supernatant was dispersed. The remains were redissolved in 100 μL mobile phase by 30s vortex-mixing. Finally, 10 μL aliquot was transfused into UPLC-MS/ MS for the following analysis.
Before the experiment, Sprague-Dawley rats have been raised in SPF grade room of Shenyang Pharmaceutical University for a week, and then fasted for 12 h [11,12]. With the approval of Animal Ethics Committee of Shenyang Pharmaceutical University, this animal study was conducted in accordance with relevant regulations. 2.2. Reagents and chemicals WSJ-557 (purity 99.0%) was obtained from the pharmaceutical laboratory of Shenyang Pharmaceutical University. As an internal standard (IS), the purity 99.3% Carbamazepine was procured from National Institute for Food and Drug Control (Beijing, China). Ethyl acetate and methanol (HPLC grade), ammonium acetate (HPLC grade) were procured from Concord Technology Co., Ltd. (Tianjin, China) and Dikma (Richmond Hill, NY, USA), respectively. Ultrapure water was procured from Wahaha (Hangzhou, Jiangsu, China) in this experiment. Other chemicals and reagents were chromatographic grades and merchantable.
2.6. Verification procedures According to FDA guidelines [13] for verifying bio-analytical method, this method was validated in different properties. (1) Selectivity To study the selectivity of the proposed method, the chromatograms of blank rat plasma and liver tissue homogenate in 6 different batches were compared with those of QC (LLOQ) samples and biological samples subjected to the oral administration of 12 mg·kg−1 WSJ-557.
2.3. Equipments and operating conditions
(2) Linearity and sensitivity
2.3.1. Mass spectrometry In this experiment, triple-quadrupole tandem mass spectrometry was performed on the Micromass® Quattro micro™ API mass spectrometer (manufactured by Waters Corp., Milford, MA, USA) with an ESI source. Positive MRM modes were carried out in the quantification. MRM transitions for WSJ-557 and carbamazepine (IS) were m/z 316.1 → 260.0 and m/z 237.0 → 194.0, accordingly. Capillary voltage and cone voltage were 4.0 kV and 25 V, accordingly. Desolvation temperature and source temperature were maintained at 400 °C and 120 °C, correspondingly. Nitrogen was selected as the desolvation and cone gas, whose flowing rates were 450 and 30 L·h−1. The collision gas was highpurity argon. Optimized collision energy for WSJ-557 was set at 15 eV. All data were collected and analyzed by MassLynx™ NT 4.1 software.
To study the linearity of calibration curves, the method of weighted (1/x2) least-squares linear regression analysis, was used by plotting the peak area ratio of WSJ-557 to that of IS against the corresponding concentration of standard WSJ-557. Characterized as the lowest concentration on the standard curve, the LLOQ of the assay can be concluded with accuracy and precision. The accuracy of LLOQ should be within ± 20%, and the precision should not exceed 20%. (3) Accuracy and precision Six duplicates were assessed at the LLOQ and the 3 QC levels on the same day to obtain the intra-day precision and accuracy. The inter-day precision and accuracy were measured by assessing LLOQ and 3 QC levels within 3 consecutive days. In agreement with the guidance of FDA, the intra-day and inter-day precision were characterized as RSD within 15%, and the accuracy was characterized as the percentage variation between the calculated and the nominal concentration within 15%. LLOQ should be below 20% of precision and within ± 20% of accuracy.
2.3.2. Liquid chromatography The ACQUITY™ UPLC system was adopted with cooling auto-sampler and column oven in the chromatographic separation, guaranteeing the temperature control. The ACQUITY™ UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) was used as the analytical column, and the temperature was set at 40 °C. Methanol (A) and 5 mmol·L−1 ammonium acetate (B) constituted the mobile phase. This gradient elution program was operated as follows: 0–0.5 min, 50% A; 0.5–2.5 min, 80% A; 2.5–4.0 min, 50% A. Besides, flow rate was 0.20 mL·min−1, auto-sampler temperature was 4 °C, and injection volume was 10 μL.
(4) Extraction recovery and matrix effect Three QC concentrations (LQC, MQC and HQC) were all used to evaluate recoveries and matrix effects. Extraction recovery was assessed by contrasting the peak areas of extracted QC samples with unextracted QC samples. Matrix effects were estimated by contrasting peak areas of samples where blank samples were spiked with WSJ-557 after liquidliquid extraction to that where QC samples are spiked in the mobile phase.
2.4. Preparations of standard solutions and QC samples The stock solution of 50 μg·mL−1 WSJ-557 was provided by dissolving the certain amount of the chemical reference substance in methanol. A range of WSJ-557 working solutions were arranged by serially diluting stock solutions with methanol to the appropriate concentration. The 920 ng·mL−1 stock solution of carbamazepine was diluted with methanol for the preparation of IS working solution (92 ng·mL−1). The optimum temperature for storage of all solutions was 4 °C. To yield final concentrations of 5.0–20,000 ng·mL−1, the calibration standard was provided by spiking 100 μL of blank biological samples with appropriate amounts of working solutions. Quality control (QC) samples were served in the identical method with low, medium and high concentrations, correspondingly. The storage temperature for all standard solutions and QC samples was set at 4 °C.
(5) Stability The test on WSJ-557 stability in biological samples was carried out at two QC levels (LQC, HQC) under different conditions: after 4hours short-term storage at room temperature, after 6-hours posttreatment in auto-sampler at 4 °C, after 3 freeze-thaws cycles (from −20 °C to room temperature) and the long-term storage for 15 days at −20 °C. 78
D. Zhang, et al.
Journal of Chromatography B 1113 (2019) 77–83
2.7. Pharmacokinetic study
acetonitrile with different concentrations of ammonium acetate and formic acid. Because of shorter chromatographic running time and lower ground noise, methanol was selected as the organic portion of the mobile phase. 5 mmol·L−1 ammonium acetate was added to the mobile phase, so as to improve peak shapes without affecting the sensitivity of MS detection. Consequently, the mobile phase was composed of 5 mmol·L−1 ammonium acetate and methanol in water. Gradient elution was chosen for the better separation and symmetric peak shapes.
16 Male Sprague-Dawley (SD) rats (230 ± 20 g) were randomly branched into 2 groups (n = 8). The oral dose suspended in 0.5% CMCNa was employed for the first group by oral gavage, and the intravenous (i.v.) dose was employed for the second group through the caudal vein. The i.v. formulation was arranged by dissolving WSJ-557 in a mixture of castor oil: DMSO: ethanol in a ratio of 83:2:15. 0.3 mL of blood samples, which were gathered into heparinized tubes via the suborbital vein after oral administration of 12 mg·kg−1 WSJ-557 at 0, 0.333, 0.667, 1, 1.5, 2, 3, 4, 8, 12, 16 and 24 h, and after the intravenous administration of 12 mg·kg−1 WSJ-557 at 0, 0.017, 0.083, 0.17, 0.33, 0.5, 1, 2, 4, 6, 8 and 12 h. Blood samples were centrifuged at 13000 rpm for 10 min immediately. Before the analysis, all samples were reserved at −20 °C. All pharmacokinetic parameters were measured by Drug and Statistics, issued by Mathematical Pharmacology Professional Committee of China, Shanghai, China.
3.1.2. Improvement of sample preparations To extract the analyte and IS from tissue homogenate and plasma, protein precipitation and liquid–liquid extraction were investigated. In this study, liquid-liquid extraction was found to produce cleaner biological sample extracts with a high recovery and selectivity. Different extracting solvents were analyzed, including dichloromethane, ethyl ether, ethyl acetate, and MTBE (methyl tert-butyl ether). In the end, ethyl acetate was selected for its higher recovery and lower background absorption.
2.8. Tissue distribution studies To investigate the WSJ-557 tissue distribution, 32 SD rats (300 ± 30 g) were randomly classified into 4 groups, namely 8 rats in each group (female and male are equal in number). Subjected to 12 mg·kg−1 WSJ-557 oral administration, 8 rats were sacrificed at each time point (0.5, 4, 8 and 12 h). Their tissues were collected, including liver, heart, brain, spleen, kidney, lung, stomach, large intestinal, small intestinal, fat, muscle, ovary or testicle (the tissue of fat was get from the enterocoelia). Tissue samples from the rats were cleansed thoroughly, removing the blood or content by physiological saline. Then tissue samples were dried by filter papers, and deposited at −20 °C. 0.5 g tissue samples (if the total weight of the tissue is < 0.5 g, it is accepted totally and recorded the weight precisely) was accurately weighed, homogenized in 0.5 mL methanol by using tissue homogenizer, and stored at −20 °C for the following analysis.
3.2. Method verification (1) Selectivity The typical chromatograms of the blank rat plasma are shown in Fig. 2. WSJ-557 and IS were spiked with the blank rat plasma. After a single oral dose of 12 mg·kg−1 WSJ-557, real plasma sample was kept for 1 h and liver sample 0.5 h. No obvious interrupting endogenous peaks were observed within the retention time of WSJ-557 and IS. (2) Calibration curve and linearity A great linearity over the range of 10.0–20,000 ng·mL−1 in plasma and 5.0–10,000 ng·mL−1 for rat tissues was observed from the standard calibration curve. Table 1 presents typical calibration curves for different tissues and plasma. It should be noted that correlation coefficients (r) exceeded 0.99 completely.
2.9. Data analysis DAS 2.0 software, provided by the Pharmacological Society of China (Beijing, China), was used to calculate WSJ-557 pharmacokinetic parameters in rats. The oral absolute bioavailability (F) was calculated as [(AUCpo × Doseiv) / (AUCiv × Dosepo)] × 100%.
(3) Accuracy and precision Table 2 shows the intra- and inter-day precision and accuracy results of LLOQ and three levels of QC samples. In this study, the accuracy (RE) ranged from −5.8% to 9.2% in all plasma samples and liver homogenates, and the intra- and inter-day precision (RSD) were 3.0%–9.5% and 3.4%–9.5%.
3. Results and discussion 3.1. Method establishment 3.1.1. Improvement of mass conditions and chromatography Negative ion and positive ion modes of WSJ-557 ionization were analyzed on an ESI ion source, in order to determine the WSJ-557 accurately in the MS/MS method. The signal strength of the deprotonated molecule ion [M − H]− was weaker than that of the protonated molecule ion [M + H]+. Carbamazepine was chosen as the most appropriate IS owing to its identical mass spectrometric and chromatographic behaviors to WSJ-557 in the positive ion mode. As a result, the monitoring mode of the positive ion was selected for WSJ-557 and IS. Fig. 1 shows the spectrum of WSJ-557 and IS with chemical structures. After giving certain collision energy, the most plentiful and constant product ions for WSJ-557 and IS were monitored at m/z 260.0 and m/z 194.0, respectively. MRM transitions for WSJ-557 and IS were m/z 316.1 → 260.0 and m/z 237.0 → 194.0, which were employed for quantitative analysis. Parameters were improved by the highest intensity observed for product ions, including the source temperature, collision energy, cone, capillary voltage. To achieve the high sensitivity, symmetric peak and a short elution time in each WSJ-557 and IS analysis, the mobile phase optimization was obtained by investigating various proportion of methanol or
(4) Matrix effect and extraction recovery Table 2 also shows the extraction recovery and matrix effect of WSJ557 in plasma and liver homogenates. The RSD was within 12.9%, and the average WSJ-557 extraction recovery of QC samples at three levels was 77.1%–98.4% in plasma and 89.1%–97.2% in liver homogenates, indicating the reliability of this method. The RSD was within 6.3%, and values of matrix effect were 89.1%–112.4% in plasma and 93.7%–109.9% in liver homogenates, accordingly. The result shows that the liquid-liquid extraction efficiency was acceptable and no significant interference was found from the plasma or liver homogenates. (5) Stability The WSJ-557 stability in rat biological samples was performed from two QC levels (LQC, HQC) under different conditions. As shown in Table 3, WSJ-557 was proved to be steady in the rat plasma and liver homogenates during the storage and extraction. 79
Journal of Chromatography B 1113 (2019) 77–83
D. Zhang, et al.
Fig. 1. WSJ-557 and IS ion scanning mass spectrogram (A) WSJ-557 (B) IS.
Fig. 2. Representative MRM chromatograms for WSJ-557 and IS: (A) blank rat plasma; (B) blank rat plasma spiked with 10 ng·mL−1 of WSJ-557 and IS; (C) a plasma sample after 1 h administration of WSJ-557 to rat; (D) a liver homogenate sample after 0.5 h administration of WSJ-557 to rat.
80
Journal of Chromatography B 1113 (2019) 77–83
D. Zhang, et al.
Table 1 Calibration curve parameters of WSJ-557 in different biological samples. Tissues
Slope
Intercept
Plasma Liver Heart Brain Spleen Lung Kidney Stomach Small intestinal Large intestinal Fat Muscle Ovary Testicle
0.2359 0.2584 0.3819 0.3680 0.3614 0.4274 0.3966 0.3051 0.5278 0.2685 0.2518 0.3563 0.3346 0.3745
10.58 1.639 13.54 8.533 15.81 14.51 14.57 9.674 17.73 4.642 8.835 4.932 31.89 20.45
Table 4 Main pharmacokinetic parameters of WSJ-557 in rats after oral or i.v. administration (n = 8).
Linear ranges (ng·mL−1)
r 0.9990 0.9913 0.9912 0.9946 0.9918 0.9921 0.9906 0.9926 0.9940 0.9915 0.9902 0.9943 0.9947 0.9923
10.0–20,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000 5.0–10,000
Parameters
Unit
Cmax Tmax T1/2 AUC0−t AUC0−∞ MRT0−∞ CL Vd
ng·mL−1 h h ng·h·mL−1 ng·h·mL−1 h L·h−1·kg−1 L·kg−1
Oral administration
Intravenous administration
128.2 ± 34.7 3.38 ± 0.74 5.47 ± 1.08 1318.8 ± 296.5 1400.6 ± 287.9 9.65 ± 1.23 8.87 ± 1.68 71.09 ± 22.39
9519.4 ± 3600 0.017 3.76 ± 2.05 19,916 ± 12,331 21,632 ± 12,298 4.57 ± 2.70 0.70 ± 0.33 4.13 ± 3.21
plasma concentration-time curves (n = 8) of WSJ-557 in rats. Table 4 reveals the corresponding main pharmacokinetic parameters defied as mean ± SD. The average time of reaching the maximum concentration (Tmax) was 3.38 ± 0.74 h by oral administration, indicating the slow absorption of circulation in rats. The T1/2 values indicated that the elimination of WSJ-557 from plasma was a slow process. Compared with WSJ-537 [7], the longer Tmax and T1/2 contribute to a lasting effect of WSJ-557 on gout and may reduce the administration frequency so as to improve patients' compliance. The apparent distribution volume (Vd)
3.3. Pharmacokinetic research and bioavailability After the oral and i.v. administration at a single dose of 12 mg·kg−1, the UPLC-MS/MS method was resoundingly employed in the pharmacokinetic study of WSJ-557 in biological samples. Fig. 3 reveals average
Table 2 Precision, accuracy, recovery and matrix effect for the determination of WSJ-557 in rat biological samples (intra-day: n = 6; inter-day: n = 6 series per day, 3 days). Samples
Analyte
Plasma
WSJ-557
Liver
WSJ-557
Concentrations (ng·mL−1)
Precision (RSD, %)
Added
Found (mean ± SD)
Intra-day
Inter-day
10 25 800 16,000 5.0 12.5 333.3 8333
9.8 ± 0.9 25.4 ± 2.1 820.6 ± 63.4 15,065.6 ± 1036.5 5.1 ± 0.4 12.6 ± 1.1 331.6 ± 16.6 9097.4 ± 311.3
9.5 7.6 7.1 3.0 7.0 8.5 4.3 4.4
9.5 8.4 7.7 6.9 8.3 8.6 5.0 3.4
Accuracy (RE, %)
−2.4 1.6 2.6 −5.8 2.1 1.0 −0.5 9.2
Recovery
Matrix effect
Mean ± SD (%)
RSD (%)
Mean ± SD (%)
RSD (%)
– 77.1 ± 4.9 84.6 ± 10.9 98.4 ± 7.0 – 97.2 ± 5.3 89.5 ± 4.7 89.1 ± 4.8
– 6.4 12.9 7.1 – 5.4 5.2 5.4
– 112.4 ± 2.4 89.1 ± 5.3 94.9 ± 5.3 – 109.9 ± 5.3 96.2 ± 6.0 93.7 ± 3.6
– 2.2 6.0 5.6 – 4.8 6.3 3.8
Table 3 Stability of WSJ-557 in rat plasma and liver homogenate under various storage conditions (n = 3). Samples
Concentration (ng·mL−1)
4 h at room temperature
Three freeze-thaw cycles
6 h storage in the autosampler
15 days at −20 °C
Plasma
25 16,000 12.5 8333
99.3 ± 9.2 94.6 ± 7.9 107.8 ± 7.9 104.1 ± 3.6
97.0 ± 9.3 93.9 ± 8.4 101.2 ± 5.2 101.7 ± 4.2
96.0 ± 12.6 92.1 ± 7.9 109.1 ± 6.2 105.6 ± 0.7
103.6 ± 8.4 95.5 ± 11.0 104.4 ± 5.9 98.8 ± 2.6
Liver
Fig. 3. Concentration-time curve of WSJ-557 after oral (A) or i.v. (B) administration in rats (n = 8).
81
Journal of Chromatography B 1113 (2019) 77–83
D. Zhang, et al.
Fig. 4. The concentration–time profile of WSJ-557 in various tissue homogenates after the oral administration of WSJ-557 (12 mg·kg−1) to rats.
of WSJ-557 was 71.09 ± 22.39 L·kg−1, indicating that the drug distribution was extensive in tissues than that in plasma. The absolute bioavailability (F) of WSJ-557 was estimated as only 6.48% in rats. The low oral bioavailability of WSJ-557 was caused by the high intestinal bacterial biotransformation and first-pass metabolism. The new dosage forms will be used to improve the WSJ-557 bioavailability in future study.
highest. While WSJ-557 can hardly cross BBB in rat. The high distribution and low absolute oral bioavailability (6.48%) were observed in the intestine, suggesting the high first-pass metabolism and intestinal bacterial biotransformation probably. The parameters obtained from the study of WSJ-557 provide helpful information for further research and clinical references on rational use of drugs. Acknowledgments
3.4. Tissue distribution studies
This project is supported by National Natural Science Foundation of China under Grant 81302841 and University Outstanding Talent Support Plan Foundation of Liaoning Province under Grant LJQ2014086. The determination method of WSJ-557 in rat plasma by UPLC–MS/ MS in this report is the subject of a pending Chinese patent filed by The First Hospital of China Medical University in Shenyang City with application number CN201710052486.7.
Fig. 4 shows the WSJ-557 concentration in various tissues after the oral administration at different times. The drug was detected in most of the rat tissues at 0.5 h, indicating that it was extensively distributed in rat tissues after the oral administration. From the results, it was found that the concentration of WSJ-557 in stomach, small intestine and large intestine were much greater than other tissues, indicating that the drug was quickly absorbed from the gastrointestinal tract probably. The lower concentration of WSJ-557 was exhibited in the spleen, heart and kidney, indicating that blood flow and perfusion rate of the organ did not have an important function in the WSJ-557 distribution probably. In addition, the WSJ-557 was hardly detected in the rat brain, ovary or testicle, illustrating that WSJ-557 may not go through the blood-brain barrier (BBB).
References [1] G. Ragab, M. Elshahaly, T. Bardin, Gout: an old disease in new perspective – a review, J. Adv. Res. 8 (2017) 495–511. [2] H.R. Schumacher, M.A. Becker, E. Lloyd, P.A. MacDonald, C. Lademacher, Febuxostat in the treatment of gout: 5-yr findings of the FOCUS efficacy and safety study, Rheumatology 48 (2009) 188–194. [3] B.W. Coburn, K.A. Bendlin, H. Sayles, K.S. Hentzen, M.M. Hrdy, T.R. Mikuls, Target serum urate: do gout patients know their goal? Arthritis Care Res. 68 (2016) 1028–1035. [4] W. Zhang, M. Doherty, T. Bardin, E. Pascual, V. Barskova, P. Conaghan, J. Gerster, J. Jacobs, B. Leeb, F. Lioté, G. McCarthy, P. Netter, G. Nuki, F. Perez-Ruiz, A. Pignone, J. Pimentão, L. Punzi, E. Roddy, T. Uhlig, I. Zimmermann-Gòrska, EULAR evidence based recommendations for gout. Part II: management. Report of a task force of the EULAR Standing Committee for International Clinical Studies Including Therapeutics (ESCISIT), Ann. Rheum. Dis. 65 (2006) 1312–1324. [5] D. Khanna, P.P. Khanna, J.D. Fitzgerald, M.K. Singh, M. Kaldas, M. Gogia, F. Perez-ruiz, W. Taylor, F. Lioté, 2012 American college of rheumatology guidelines for management of gout part II: therapy and anti-inflammatory prophylaxis of acute gouty arthritis, Arthritis Care Res. 64 (2013) 1447–1461. [6] A.F. Rheumatology, P.K. Rheumatologist, The management of gout, Aust. Prescr. 39 (2016) 119–122.
4. Conclusions In this study, a fast, specific and sensitive UPLC-MS/MS method was first proposed to determine the WSJ-557 in plasma and tissue of rat. The pretreatment of the large number of plasma and tissue homogenates samples was obtained by the simple sample preparation procedure. The LLOQ was 5.0 ng·mL−1 in this method. Owing to the short running time of 4.0 min, the method will be useful for further preclinical studies of WSJ-557. After the oral administration of WSJ-557, it was widely spread in various tissues, where the gastrointestinal tract is
82
Journal of Chromatography B 1113 (2019) 77–83
D. Zhang, et al. [7] J. Lin, T. Yang, D. Zhang, Development of a selective and fast LC-MS/MS for determination of WSJ-537, an xanthine oxidase inhibitor, in rat plasma: application to a pharmacokinetic study, J. Chromatogr. B 1028 (2016) 51–55. [8] S. Chen, T. Zhang, J. Wang, F. Wang, H. Niu, C. Wu, S. Wang, Synthesis and evaluation of 1-hydroxy/methoxy-4-methyl-2-phenyl-1H-imidazole-5-carboxylic acid derivatives as non-purine xanthine oxidase inhibitors, Eur. J. Med. Chem. 103 (2015) 343–353. [9] E.A. Lakota, J.C. Bader, C.M. Rubino, Ensuring quality pharmacokinetic analyses in antimicrobial drug development programs[J], Curr. Opin. Pharmacol. 36 (2017) 139–145. [10] L. Zhu, J. Wu, M. Zhao, et al., Mdr1a plays a crucial role in regulating the analgesic effect and toxicity of aconitine by altering its pharmacokinetic characteristics[J], Toxicol. Appl.
Pharmacol. 320 (2017) 32–39. [11] L. Qiao, Y. Liu, X. Chen, et al., A HPLC-MS/MS method for determination of 6‴-feruloylspinosin in rat plasma and tissues: pharmacokinetics and tissue distribution study [J], J. Pharm. Biomed. Anal. 121 (2016) 77–83. [12] A. Zhao, Y. Zhang, X. Yang, Simultaneous determination and pharmacokinetics of sixteen Angelicae dahurica coumarins in vivo by LC–ESI-MS/MS following oral delivery in rats [J], Phytomedicine 23 (10) (2016) 1029–1036. [13] FDA Guidance for Industry: Bioanalytical Method Validation, http://www.fda.gov/ downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070107. pdf.
83