Pharmacokinetics and drug interactions of fraxin in rats

• Main Authors: Hui-ChunHong, 洪惠淳
• Other Authors: Chen-Hsi Chou
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/vu4a99

碩士 === 國立成功大學 === 臨床藥學與藥物科技研究所 === 104 === Pharmacokinetics and drug interactions of fraxin in rats Hui-Chun Hong Chen-Hsi Chou Institute of Clinical Pharmacy, Medical College, National Cheng Kung University Abstract Background. Cortex fraxini (named Qinpi in Chinese) is the dried bark of plants from the Oleaceae family, including Fraxinus rhynchophylla, F. chinensis, F. szaboana and F. stylosa. Cortex fraxini is commonly used as Chinese herbal drug, proven to be effective in the treatment of diarrhea and dysentery of intense heat type. Fraxin (7-hydroxy-6-methoxycoumarin 8-glucoside), a structurally derivative of coumarin glucoside, is one of the important bioactive components in Cortex fraxini. Fraxin is known to be responsible for the diuretic, anti-inflammatory, anti-oxidant, and anti-hyperuricemic effects. Following oral administration fraxin is extensively metabolized to the aglycone fraxetin (7,8-Dihydroxy-6-methoxycoumarin), which is also an effective constituent of Cortex fraxini and can be extensively metabolized to glucuronides by uridine diphosphate glucuronosyl transferases (UGTs). Glucuronidation mediated by uridine diphosphate glucuronosyl transferases (UGTs) is a common phase II metabolic pathway for various prescribed drugs. In view of these interactions, drugs with narrow therapeutic margins such as valproic acid should be carefully considered with respect to fraxin. Quantification and pharmacokinetics studies on constituents of Traditional Chinese Medicine in plasma are required to offer useful information in clinical application. Although fraxin has been used clinically for many years, its pharmacokinetic property still remains unknown so far due to the lack of quantification methods. Therefore, it is necessary to develop a suitable bioanalytical method for the determination of fraxin in plasma. Purpose. The aim of this study is to investigate the pharmacokinetics and drug interactions of fraxin in male Sprague-Dawley rats. To characterize the pharmacokinetic properties of fraxin, it is very necessary to develop an accurate and selective bioanalytical method for the determination of fraxin in rat plasma. Methods. Two sensitive high-performance liquid chromatography (HPLC) methods, one for simultaneous determination of fraxin and its conjugate metabolites using ferulic acid ((2E)-3-(4-hydroxy-3-methoxyphenyl) prop-2-enoic acid) as an internal standard, the other for quantification of fraxetin using piperonyl alcohol (2H-1,3-benzodioxol-5-ylmethanol) as an internal standard in rat plasma were developed. The analytes and internal standards were separated on Thermo Hypurity C18 column (4.6 mm × 250 mm, 5 μm) and protected by an ODS guard colum (10 mm × 4 mm, 5 μm). Fluorescence and ultraviolet detectors were used in this study. Since fraxin has fluorophoric properties, fluorescent detection (FD) was expected to provide an inexpensive, sensitive, and specific detection of fraxin in biological samples. The two methods have been fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery. The Sprague-Dawley rats (8 weeks; body weight: 230~250 g) received fraxin and fraxetin intravenously (iv) and orally at the dose level of 5 and 20 mg per kilogram, respectively. Kinetics of fraxetin following intravenous infusion at a rate of 150 μg per minutes for 20 minutes was also examined. Co-medication of fraxetin and valproate via iv and oral routes were employed to investigate potential drug-drug interaction. Serial blood samples (250 μL) were collected from the carotid intoheparinized 1.5 mL polythene tubes and centrifuged at 13,000 rpm for 10 min and stored frozen at -20 oC until analysis. The plasma concentrations of fraxin, fraxetin and the conjugate metabolites were determined by HPLC methods and the kinetics parameters were estimated by compartmental analysis. Results. The developed HPLC methods were found to be specific, precise and accurate. Calibration curves for fraxin was constructed over a range of 0.0014 – 27 μM and that of fraxetin was 0.24 – 120 μM. The lower limit of quantitation (LLOQ) for fraxin is 0.0014 μM ng/mL and that of fraxetin is 0.24 μM. The two methods were successfully applied to the pharmacokinetics of fraxin/fraxetin in rats. The disposition kinetics of fraxin in rats displayed two-compartmental characteristics, with a distribution half-life of 20 min and an elimination half-life of 120 minutes. The oral absorption of fraxin/fraxetin was rapid with a peak concentration occurred before 10 minutes. The estimated bioavailability of fraxin was 0.8% and that of fraxetin was 82 %. After iv bolus injection, plasma concentration of fraxetin declined rapidly with a short elimination half-life about 10 minutes, and two major metabolites, with high fluorescence intensity similar to fraxin, were generated. After co-administration with valproate intravenously, the AUC value of valproic acid, fraxetin and its conjugate metabolites were significantly increased. In the oral experiment group, the plasma levels of valproic acid and fraxetin were decreased and the major metabolites of fraxetin were decreased before 30 minutes. Conclusion. After oral administration of fraxin, it converts to fraxetin rapidly. And, fraxetin undergoes rapid and extensive conjugation metabolism to generate fraxetin glucuronide and fraxetin sulfate. Co-administration of the UGTs substrates fraxetin and valproate by intravenous route in rats resulted in a 1.5-fold increase of the AUC value of valproic acid, indicating that the elimination of valproic acid was affected by fraxetin. In the oral administration group, fraxetin significantly affected the absorption of valproic acid without altered its elimination. Keywords: fraxin，polyphenols，phase II reaction，glucuronidation，valproic acid