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The effects of Chuanxiong on the pharmacokinetics of warfarin in rats after biliary drainage
Journal of Ethnopharmacology 193 (2016) 117–124
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The effects of Chuanxiong on the pharmacokinetics of warfarin in rats after biliary drainage Haigang Li a,b, Chunhu Zhang a, Rong Fan a, Hua Sun c, Haitang Xie c, Jiekun Luo a, Yang Wang a,n, Huiying Lv d,nn, Tao Tang a,n a Department of Integrated Traditional Chinese and Western Medicine, Laboratory of Ethnopharmacology, Xiangya Hospital, Central South University, Changsha 410008, PR China b Department of Pharmacy, Changsha Medical University, Changsha 410219, PR China c Anhui Provincial Centre for Drug Clinical Evaluation, Yijishan Hospital of Wannan Medical College, Wuhu 241001, PR China d Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 April 2016 Received in revised form 13 July 2016 Accepted 3 August 2016 Available online 3 August 2016
Ethnopharmacological relevance: Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong Hort), known as Chuanxiong in Chinese, has been used for treating cardiovascular diseases for centuries. Chuanxiong is a classical activating blood circulation herb in the treatment of thromboembolism heart diseases. Warfarin often combines with herbal prescriptions containing Chuanxiong in China. Aim of the study: The herb-drug interaction involving enterohepatic circulation process remains unclear. This study aimed to elucidate the effects of Chuanxiong Rhizoma on the pharmacokinetics of warfarin in rats after biliary drainage. Materials and methods: Thirty-two rats were randomly divided into four groups: WN (healthy rats after the gastric-administration of 0.5 mg/kg warfarin sodium), WO (a rat model of biliary drainage after the gastric-administration of 0.5 mg/kg warfarin sodium), WCN (healthy rats after the gastric-administration of 0.5 mg/kg warfarin sodium and 10 g/kg Chuanxiong decoction), and WCO (a rat model of biliary drainage after the gastric-administration of 0.5 mg/kg warfarin sodium and 10 g/kg Chuanxiong decoction). The levels of warfarin and internal standard were quantified by LC-MS/MS. Comparisons between groups were performed according to the main pharmacokinetic parameters calculated by the DAS 2.1.1 software. Results: The established LC-MS/MS method was specific, precise and rapid. The pharmacokinetic parameters showed a significant difference between the WN and WO groups. There were significant differences in the area under the curve (AUC0 t), peak concentration (Cmax), total plasma clearance (CLz/F) and mean residence time (MRT0–t) between the WCO and WCN groups; the AUC0–t of warfarin in the WCN group was 2.42 times than that of the WN group (po 0.01); the WCO group displayed a decreased to 61.6% in the Cmax compared the WO group (p o0.01). Conclusion: Biliary drainage significantly influenced the disposition of warfarin, and Chuanxiong significantly affected the warfarin disposition in rat plasma. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Warfarin Chuanxiong Pharmacokinetics Enterohepatic circulation LC-MS/MS Biliary drainage
1. Introduction Warfarin is recommended most widely for the prevention of systemic embolism, stroke associated with atrial fibrillation, and
n Corresponding authors at: Department of Integrated Traditional Chinese and Western Medicine, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha 410008, PR China. nn Corresponding author at: Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, PR China. E-mail addresses: [email protected] (Y. Wang), [email protected] (H. Lv), [email protected] (T. Tang).
http://dx.doi.org/10.1016/j.jep.2016.08.005 0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
venous thromboembolism (Piatkov et al., 2010). The annual prescriptions of warfarin typically occur in 0.5–1.5% of the population (Johnson et al., 2011). Although novel oral anticoagulants (NOACs), such as apixaban, dabigatran, edoxaban and rivaroxaban, do not require more special laboratory monitoring or dose adjustment like warfarin does, warfarin has remained in use for over 60 years and is still the most common oral anticoagulant drug (Pirmohamed et al., 2015; Wadhera et al., 2014). This is mainly because warfarin can exert anticoagulant effects for several days after the subject stops taking it, while the NOACs do not have this merit, one missed dose results in low trough drug concentrations. In various countries and regions, especially in China, physicians
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prefer to combine herbal medicine with warfarin to achieve better effects and to prevent and reduce side effects (Lee et al., 2008). Certain herbs, including aloe, jalap, cascara, and rhubarb, have been found to affect the metabolism of warfarin via cytochrome P450s (CYP) and UDP-glucuronosyltransferases pathway (Zhou et al., 2012). Therefore, it is important to study the interactions between herbal medicines and warfarin so as to determine their optimal clinical applications. Warfarin can be detected in human bile after oral administration (Zeuzem, 2000). Some of the warfarin in the blood is absorbed from the intestine and secreted into the bile after entering liver cells, is then stored in the gallbladder through the bile duct, and finally is taken into the duodenum by gallbladder contraction (Erlinger, 1996; Gao et al., 2014). During enterohepatic circulation (EHC) process, the warfarin will be absorbed on two different occasions. The timing of the other peaks is related to eating and gallbladder emptying. Because of the irregularity of gallbladder emptying, the concentration-time (C-T) curve may present double peaks or multiple peaks. Unfortunately, previous studies about the pharmacokinetic effect of herb on warfarin failed to focus on influence of EHC process. Up to now, the herb-drug interaction involving enterohepatic circulation process remains unclear. Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong Hort), known as Chuanxiong in Chinese, a commonly used Chinese herbal medicine, has been used for treating cardiovascular diseases in China for centuries. Chuanxiong was used to relieve the pain induced by blood stagnation. According to traditional Chinese medicine (TCM) theory, the pharmacological action of Chuanxiong is “to activate the blood circulation and to promote qi”. Chuanxiong is a classical activating blood circulation herbs in China. In China and some regions such as Hong Kong, warfarin often combined with herbal prescriptions containing Chuanxiong, and these co-administration methods were recommended most widely for treatment the thromboembolism heart diseases in clinical practice (Feng et al., 2015; Zhang et al., 2016). But the effects of Chuanxiong on the pharmacokinetic of warfarin have not been reported. Scientists often use biliary drainage models to investigate the effects of enterohepatic circulation (Hellstern et al., 1990; Melnik et al., 1998; Lenzen et al., 1999). The protocol of the present study was planned to compare the pharmacokinetics of warfarin in rats with and without biliary drainage and to investigate the potential effects of Chuanxiong on the pharmacokinetics of warfarin in a biliary drainage model rats.
2. Materials and methods 2.1. Preparation of Chuanxiong extract Raw dried Chuanxiong Rhizoma was purchased from the pharmacy of Xiangya Hospital of Central South University, Changsha, China. The voucher specimen (no. 20140512, Chengdu, China) was deposited in the Laboratory of Ethnopharmacology, Xiangya Hospital of Central South University. Sixty gram raw Chuanxiong Rhizoma was thoroughly decocted in water (1:8, g/mL) for 1 h after soaking in water for half an hour, and then the gruffs decocted an hour with water (1:6, g/mL), filtered through gauze. The two filtrates were merged and evaporated by rotary evaporation under vacuum at 60 °C. The two filtrates were merged and evaporated under vacuum and concentrated to 60 mL with rotary evaporation at 60 °C, the final decoction was stored at 4 °C until use.
2.2. Chemicals and reagents Methyclothiazide (C9H11Cl2N3O4S2, CAS, 135-07-9, Lot, 101163201101; purity, 99.6%; internal standard, IS) and warfarin sodium (C19H15NaO4, CAS, 129-06-6, Lot, 101163-201001; purity, 92.3%) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Tetramethylpyrazine (C8H12N2, CAS, 112411-4, Lot, MUST-13022802; purity Z 98%), levistilide A (C24H28O4, CAS, 88182-33-6, Lot, MUST-13020211; purity Z98%) and senkyunolide I (C12H16O4, CAS, 94596-28-8, Lot, PA0804FA13; purity Z98%) were purchased from Chengdu Must Bio-technology Co., Ltd (Chengdu, China). Ferulic acid (C10H10O4, CAS, 1135-24-6, Lot, F1205050; purity, 99%) was purchased from Sigma-Aldrich (The Netherlands). Formic acid from Tianjing Gangfu Fine Chemical Reagent Factory (Tianjing, China). HPLC grade acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). Deionized water was purified with a TKA Samrt2pure water purification system (German) with a sensitivity of 18.2 MO. 2.3. Surgical procedures All animal procedures were conducted in strict compliance with our institutional protocols and were approved by the Animal Ethics Committee of Central South University. Thirty-two specific pathogen free (SPF) grade Sprague-Dawley (SD) male rats weighing 250–300 g were provided by Shanghai Laboratory Animal Center (SLAC). They were housed at temperature-controlled (21– 25 °C) and 40–70% relative humidity room in an SPF environment on 12 h day-night cycle with lights. All rats provided tap water ad libitum and were fed standard rodent chow, except for the 12-h fast before the pharmacokinetic study. The experimental rats were randomly divided into four groups, WN Group: healthy rats after a single gastric-administration of 0.5 mg/kg warfarin sodium; WO Group: rats subjected to biliary drainage after the gastric-administration of 0.5 mg/kg warfarin sodium; WCN Group: healthy rats after the gastric-administration of warfarin sodium and Chuanxiong Hort at doses of 0.5 mg/kg and 10 g/kg, respectively; and WCO Group: rats subjected to biliary drainage after the gastricadministration of warfarin sodium and Chuanxiong Hort at doses of 0.5 mg/kg and 10 g/kg, respectively. The dose of drugs we used in this study calculated according to the conventional dosage of clinical. The animals were fasted for food but not water overnight before the experiments. Bile drainage surgery was carried out under sterile conditions after anesthetized by intraperitoneal injection of 10% chloral hydrate at 0.4 mL/100 g body weight. A midline incision was made on the abdomen, the liver was opened up, and the common bile duct of rat was noted over the margin of the duodenal bulb. The distal end of the bile duct was ligated and a polyethylene tube (inner diameter was 0.8 mm and outer diameter 1.2 mm) was inserted into the proximal end of common bile duct. Yellow bile could be seen in the catheter either immediately or a few minutes after insertion, and the free end of the external biliary drainage catheter was fixed to the peritoneum and brought out of the body through a small hole made on the back of the rat's neck. 2.4. Sample collection and preparation On the second day after biliary drainage surgery, 500 μL of tail venous blood was collected into heparinized tubes prior to treatment and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h post-dose. The samples were centrifuged at 3000 rpm for 15 min, then the supernatants were stored at 80 °C until analysis. To a total of 200 μL of rat plasma were added with 20 μL of IS solution, 100 μL of 20% formic acid and 2 mL of methanol, which was followed by vortex mixing for 1 min and hyperacoustic mixing for 5 min. The
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samples were then centrifuged at 3000 rpm for 10 min. The supernatants were evaporated to dryness under a gentle stream of nitrogen at room temperature, followed by reconstitution with 200 μL mixture of acetonitrile and water (50:50). This was followed by vortex mixing for 1 min and hyperacoustic mixing for 5 min. The samples were then centrifuged at 15,000 rpm for 10 min, the supernatants were filtered through the 0.22-μm membrane, and 2.0 μL of the processed sample was injected for the analysis.
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2.5. Instrumentation and analytical conditions The sample analysis was performed on an UPLC-MS/MS system. The Acquity™ UPLC system (Waters Corporation, Milford, MA, USA) was constituted of an autosampler (set at 10 °C), a binary solvent delivery manager and a column oven (set at 35 °C). Chromatographic separation was acquired from a Waters Acquity BEH C18 column (2.1 mm 100 mm I.D., 1.7 mm, Waters, Wexford, Ireland). A mobile phase consisting of A (aqueous buffer containing 0.1% formic acid) and B (acetonitrile) was pumped at 0.3 mL/
Fig. 1. Representative MRM chromatograms of warfarin and methyclothiazide in rats: (A) blank plasma samples from healthy rats; (B) blank plasma samples spiked with warfarin and methyclothiazide; (C) plasma from healthy rats after a single administration of warfarin; (D) plasma from rats with biliary drainage after a single administration of warfarin; (E) plasma from healthy rats after the administration of warfarin and Chuanxiong; and (F) plasma from rats with biliary drainage after the administration of warfarin and Chuanxiong.
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min. The gradient elution was: 0 min, 10% B; 0–0.25 min, 10% B; 0.25–5.0 min, linear from 10% to 95% B; 5.0–8.0 min, holding at 95% B; then, between 8.0–8.2 min, an immediate decrease to the initial conditions (i.e., 10% B) for equilibration of the column. The typical injection volume was 2 μL. The detection system, a tandem quadrupole mass spectrometer (Waters Corporation, Manchester, UK), was operated using an electrospray ionization with the desolvation temperature was fixed at 365 °C, the source temperature was set at 110 °C, and the capillary voltage set at 2.5 kV in negative ion mode. Nitrogen was used for the desolvation gas flow (650 L/h)
and cone gas flow (50 L/h). For collision-induced dissociation, argon was used as the collision gas at a flow rate of 0.2 mL/min. The Masslynx 4.1 software program (Waters Corporation) was used for data processing and acquisition. The multiple-reaction monitoring (MRM) mode was selected for quantitation of IS and warfarin, the precursors of which for the production ion transitions were as follows: IS, 357.81-321.89; warfarin, 307.03-250.02. The data were collected and processed using the DAS 2.1.1 software.
Fig. 2. LC–MS/MS spectra of methyclothiazide, warfarin and four marker compounds of Chuanxiong. Warfarin and methyclothiazide can be ionized under negative ionization conditions. (M-H) predominated and was used as the precursor ion to obtain the ion spectra. The most sensitive mass transitions for methyclothiazide (A, the internal standard), were m/z 357.81-321.89, for warfarin (B) were 307.03-250.02. for tetramethylpyrazine (C) were 137.10-96.04, for levistilide A (D) were 381.09-190.92, for senkyunolide I (E) were 225.11-207.04 and for ferulic acid (F) were 192.98-133.96.
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2.6. Method validation The method was verified in terms of linearity, selectivity, precision, accuracy, extraction recovery, matrix effects and stability according to FDA guidelines for bioanalytical method validation. Calibration curve samples were prepared by spiking blank plasma. To evaluate linearity, duplicate calibration standards of warfarin at six concentration of blank plasma 5, 20, 100, 500, 2000 and 5000 ng/mL levels were prepared and analyzed. The linearity of the method was determined by analysis of standard plots associated with a six-point calibration curve. The overall intra- and inter-day variations all used five analytes.
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from 563.33 to 509.13 min, but this difference was not statistically significant. The clearance parameter (CLz/F) of the WO group significantly increased 1.61 times than the WN group. 3.4.2. The pharmacokinetics parameter comparations of WCO and WCN group The WCO group showed a decreased to 25.7% in the AUC0–t compared to the WCN group (Po 0.01). Compared with the WCN group, the Cmax of the WCO group was significantly decreased to 20.5% (P o0.01) and the MRT0–t also delayed by 30.2% (P o0.01). The CLz/F of the WCO group increased 2.04 times than the WCN group. The mean half-life (t1/2z) had a tendency to be extended from 625.04 to 1179.78 min, this difference was not statistically significant.
3. Results 3.1. Specificity No interference was displayed at the retention times of either warfarin or IS in plasma samples, which were used for the analysis, and the method showed good specificity. Typical MRM chromatograms are shown in Fig. 1, and the LC-MS/MS spectra of warfarin and methyclothiazide are presented in Fig. 2. 3.2. Linearity and LLOQ of warfarin The linear regression equation was y ¼0.0189x 0.0057 (r ¼0.999993), the LLOQ (lower limit of quantitation) was 5 ng/mL, and the linearity range was 5–5000 ng/mL, where y indicates the ratio of warfarin peak area to internal standard peak area, x refers to the warfarin concentration (ng/mL), and r is the correlation coefficient of the equation.
3.4.3. The pharmacokinetics parameter comparations of WN and WCN group Further, Table 2 revealed that the AUC0–t of warfarin in the WCN group was 2.42 times than that of the WN group (p o0.01), the Cmax in the WCN group was 2.35 times than the WN group (p o0.01), the WCN group showed a decreased to 79.9% in the t1/2z compared to the WN group (p o0.05), and the mean of CLz/F also decreased from 20.14 to 12.73 mL/h/kg (P o0.05). 3.4.4. The pharmacokinetics parameter comparations of WO and WCO group Chuanxiong obviously affected the disposition of warfarin after biliary drainage, because the WCO group displayed a decreased to 61.6% in the Cmax compared the WO group (p o0.01), the t1/2z of the WCO group was extended 2.15 times than the WO group (Po 0.01), and the MRT0–t also delayed by 22.1% (P o0.01).
3.3. Precision and accuracy
4. Discussion
Table 1 showed that the method had good precision and accuracy with good intra-day and inter-day precision. And all of the results were found to be within the accepted variable limits.
Most researches on warfarin have focused on dose and safety (Verhoef et al., 2014). As consequences of under- or over-anticoagulation, the incidence of thromboembolism or hemorrhage due to the use of warfarin ranged from 16% to 25% (Bejth et al., 2002; Fang et al., 2007). The above risks are most common during the initial period of treatment (Liu et al., 2015). Side effects of warfarin is associated with its narrow therapeutic range. What's more, different patients have different therapeutic ranges for warfarin. The reason for these issues is that genetic and multiple environmental factors can influence the metabolism and effects of warfarin in each patient, and it has been reported that there is an at least 20-fold inter-individual variability in warfarin sensitivity (Hirsh et al., 2003; Wittkowsky, 2005). It is therefore necessary to carefully monitor the warfarin concentration to avoid serious side effects and keep patients to be safe (Morais and De Caterina, 2016). The present study has shown that the biliary drainage operation and Chuanxiong altered significantly the pharmacokinetic profile of warfarin in rats plasma. There were significant differences in the pharmacokinetic parameters of warfarin between the WO and WN groups, including AUC0–t, Cmax, Tmax, t1/2z and CLz/F according to the above analysis. The notable decreases in the AUC
3.4. Pharmacokinetic study Fig. 3 shows the C-T curves for plasma warfarin in the four groups (n ¼8). The pharmacokinetic parameters (Table 2) of warfarin were calculated using non-compartmental modeling with the DAS2.1.1 software. 3.4.1. The pharmacokinetics parameter comparations of WO and WN group The WO group showed a decreased in the area under the curve (AUC0–t) to 74.4% compared to the WN group (P o0.01). The peak concentration (Cmax) of the WO group was significantly decreased by 21.4% compared to the WN group (Po 0.01), and the time taken to reach the Cmax (Tmax) in the WO group was 3.21 times slower than that in the WN group (Po0.05). The mean half-life (t1/2z) of the WO group was shortened 29.9% than the WN group (P o0.05). The mean residence time (MRT0–t) had a tendency to be shortened Table 1 The intra-day and inter-day precision of warfarin in rat plasma samples.
Warfarin
Spiked concentration (ng/mL)
Intra-day Measured (ng/mL)
RSD%
Measured (ng/mL)
Inter-day RSD%
50 500 5000
49.737 3.98 505.69 7 42.3 5093.92 7 461.1
8.00 8.36 9.05
50.26 7 3.29 496.79 738.7 5057.45 7 431.6
6.55 7.79 8.53
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Fig. 3. The mean 7SD plasma concentration-time curves for warfarin (n¼ 8). WN: healthy rats after a single administration of warfarin; WO: rats with biliary drainage after a single administration of warfarin; WCN: healthy rats after the administration of warfarin and Chuanxiong; WCO: rats with biliary drainage after the administration of warfarin and Chuanxiong.
Table 2 The pharmacokinetic parameters of warfarin (mean 7 SD, n ¼8). Group
AUC(0–t) (ng/mL min)
MRT(0–t) (min)
t1/2z (min)
CLz/F (mL/h/kg)
Tmax (min)
Cmax (ng/mL)
WN WO WCN WCO
1′102′028.69 7 151′173.74 819′455.317 172′906.63** 2′667′827.667 1′381′550.09** 685′879.94 7 189′529.01##
563.337 13.77 509.137 90.78 477.38 7 81.81 621.64 743.12##,※※
782.57 7108.38 548.88 7225.60* 625.04 7657.32* 1′179.78 7 450.16※※
20.14 72.43 32.43 7 12.09* 12.737 8.78* 25.94 7 5.11##
70.00 7118.23 225.007 100.14* 198.75 7 136.72 247.5 7 192.93
1′357.197 220.64 1′066.337138.14** 3′193.86 7 1304.34** 656.4 7198.23##,※※
WN: healthy rats after a single administration of warfarin; WO: rats with biliary drainage after a single administration of warfarin; WCN: healthy rats after the administration of warfarin and Chuanxiong Rhizoma; WCO: rats with biliary drainage after the administration of warfarin and Chuanxiong Rhizoma. **
Compared to the WN group p o 0.01. Compared to the WN group po 0.05. Compared to the WCN group p o0.01. ※※ Compared to the WO group po 0.01. *
##
and Cmax of the WO group caused by biliary drainage indicated that the operation decreased the bioavailability of warfarin, possibly due to the change in EHC. There were also significant differences in the pharmacokinetic parameters of warfarin between the WCO and WCN groups. These findings indicated that biliary drainage can affect the disposition of warfarin when it was coadministered with Chuanxiong in rats. In fact, EHC refers to the circulation of bile acid, substances or drugs from the liver to the bile, followed by re-absorption into the small intestine and transport back to the liver for systemic circulation. Intestinal reabsorption to complete the enterohepatic cycle might be determined by hydrolysis of a drug conjugate by gut
bacteria. The EHC process extends the drug residence time in the body. A drug undergoing EHC usually shows a multiple-peak phenomenon in terms of its plasma C-T curve and a prolonged elimination half-life (Roberts et al., 2002). EHC induced a second peak in the C-T profile of the WCN group (Fig. 3). However, the C-T curve of the WCO group did not show such double peaks due to blockade of EHC. Bile acid circulation therefore played an important role in the pharmacokinetics of warfarin combined with or without Chuanxiong, and we speculated that Chuanxiong influences the pharmacokinetics of warfarin by affecting its EHC. Ferulic acid is the main active constituent of Chuanxiong, and ferulic acid metabolism conjugation with glucuronic acid and/or
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sulfate, which takes place mainly in the liver, is the principal pathway in vivo. Ferulic acid is also metabolized into m-hydroxyphenylpropionic acid by the intestinal microflora through reduction, dehydroxylation, and demethylation (Zhao and Moghadasian, 2008). This molecular mechanism needs to be verified in additional studies. The pharmacokinetics study of warfarin in rats with biliary drainage is a very useful reference for warfarin research in clinical trials and applications. The significant differences in AUC0–t, Cmax, t1/2z and CLz/F between the WN and WCN group indicated that Chuanxiong affected the pharmacokinetics of warfarin in rat plasma and that Chuanxiong can promote the absorption rate of warfarin, possibly due to metabolic compete, and decreased systemic elimination or local interactions of warfarin with Chuanxiong in the intestine. The dose response variations of warfarin are remarkably influenced by pharmacokinetics aspects that depended on genetic, environmental and others as yet unknown factors. Polymorphisms in CYP2C9, CYP2C19, CYP1A1 and VKORC1 are the major determinants of the warfarin dosage requirement in human (Zhao and Moghadasian, 2008; Lee et al., 2009; Wadelius et al., 2009), whereas the corresponding enzymes in rats are CYP2C11, CYP2C6, CYP1A1 and CYP2B1 (Soucek and Gut, 1992). Genetic polymorphisms in CYP2C9 and VKORC1 account for approximately 50% of the variability in the pharmacologic response of warfarin in human (Bodin et al., 2005). Other genetic factors including vitamin K intake and recycling and warfarin transportation may also influence the stable maintenance dosage of warfarin. And CYP3A4, CYP2C9 and CYP1A2 are the major isoenzyme of in vitro metabolism of ligustilide, which is the major component of Chuanxiong (Qian et al., 2009). The compete of metabolism enzyme can explain the increase of the WCN group in the AUC compare to the WN group without biliary drainage in vivo. Physicians should be aware of the possibility of over-anticoagulation of drug interaction between warfarin and Chuanxiong owing to the increased AUC and Cmax. There is a dilemma in the opposite tendency of the AUC and Cmax between normal rats and rats subjected to biliary drainage. On one hand, we speculated that Chuanxiong must be participated in warfarin's EHC in vivo, Chuanxiong made Cmax and AUC0–t of warfarin rise 2.35 and 2.42 folds (P o0.01) in normal rats plasma, according to the comparations between the WN and WCN group, but Chuanxiong decreased the warfarin AUC and Cmax, according to the comparations between the WO and WCO group, in which the EHC process were interrupted by the operation of biliary drainage. On the other hand, the operation might take some complex factors in the disposition process in vivo, and we might further hypothesis that whether diseases conditions induce the similar changes just showing as in the present study. These corollaries provided a chance to investigate the EHC mechanism of warfarin. There is widespread undisclosed use of herbal medicine among Western doctors. Previous studies reported that the rate of herbal use was 75.4% to more than 90.0% in Singapore and Italy (Lim et al., 2005; Cuzzolin et al., 2007). The use of TCM is also significant in various countries and regions, with an estimated 58.0% and 23.5% of patients in the United Kingdom and Italy, respectively, co-ingesting warfarin and herb medicines (Cuzzolin et al., 2007; Ramsay et al., 2005; Wong et al., 2003). Although we have shown that Chuanxiong impacts the pharmacokinetics of warfarin in rats after biliary drainage, it is unclear how it exerts its effects. The identification and detailed quantitative assessment of the importance of various enzyme/transporter pathways in the elimination of Chuanxiong are still lacking, some previous studies on its main metabolism pharmacokinetics showed that these processes may involve CYPs, UDP-glucuronosyltransferases, sulfotransferases, and the monocarboxylic acid transporter (Poquet
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et al., 2008; Rondini et al., 2002).
5. Conclusion Biliary drainage significantly affected the disposition of warfarin in rats. Chuanxiong co-administration significantly influenced the disposition of warfarin in rat plasma in both normal rats and rats subjected to biliary drainage. The present study can provide clinic guidance for the administration of warfarin.
Acknowledgments This work was supported by the Science Project of the Education Department of Hunan Province (Grant No. 13C1126), the Natural Science Foundation of Hunan Province (Grant No. 14JJ2024), the National Natural Science Foundation for Young Scientists of China (Grant Nos. 81303074 and 81403259) and the National Natural Science Foundation (Grant No. 81473573).
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The effects of Chuanxiong on the pharmacokinetics of warfarin in rats after biliary drainage Haigang Li a,b, Chunhu Zhang a, Rong Fan a, Hua Sun c, Haitang Xie c, Jiekun Luo a, Yang Wang a,n, Huiying Lv d,nn, Tao Tang a,n a Department of Integrated Traditional Chinese and Western Medicine, Laboratory of Ethnopharmacology, Xiangya Hospital, Central South University, Changsha 410008, PR China b Department of Pharmacy, Changsha Medical University, Changsha 410219, PR China c Anhui Provincial Centre for Drug Clinical Evaluation, Yijishan Hospital of Wannan Medical College, Wuhu 241001, PR China d Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 April 2016 Received in revised form 13 July 2016 Accepted 3 August 2016 Available online 3 August 2016
Ethnopharmacological relevance: Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong Hort), known as Chuanxiong in Chinese, has been used for treating cardiovascular diseases for centuries. Chuanxiong is a classical activating blood circulation herb in the treatment of thromboembolism heart diseases. Warfarin often combines with herbal prescriptions containing Chuanxiong in China. Aim of the study: The herb-drug interaction involving enterohepatic circulation process remains unclear. This study aimed to elucidate the effects of Chuanxiong Rhizoma on the pharmacokinetics of warfarin in rats after biliary drainage. Materials and methods: Thirty-two rats were randomly divided into four groups: WN (healthy rats after the gastric-administration of 0.5 mg/kg warfarin sodium), WO (a rat model of biliary drainage after the gastric-administration of 0.5 mg/kg warfarin sodium), WCN (healthy rats after the gastric-administration of 0.5 mg/kg warfarin sodium and 10 g/kg Chuanxiong decoction), and WCO (a rat model of biliary drainage after the gastric-administration of 0.5 mg/kg warfarin sodium and 10 g/kg Chuanxiong decoction). The levels of warfarin and internal standard were quantified by LC-MS/MS. Comparisons between groups were performed according to the main pharmacokinetic parameters calculated by the DAS 2.1.1 software. Results: The established LC-MS/MS method was specific, precise and rapid. The pharmacokinetic parameters showed a significant difference between the WN and WO groups. There were significant differences in the area under the curve (AUC0 t), peak concentration (Cmax), total plasma clearance (CLz/F) and mean residence time (MRT0–t) between the WCO and WCN groups; the AUC0–t of warfarin in the WCN group was 2.42 times than that of the WN group (po 0.01); the WCO group displayed a decreased to 61.6% in the Cmax compared the WO group (p o0.01). Conclusion: Biliary drainage significantly influenced the disposition of warfarin, and Chuanxiong significantly affected the warfarin disposition in rat plasma. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Warfarin Chuanxiong Pharmacokinetics Enterohepatic circulation LC-MS/MS Biliary drainage
1. Introduction Warfarin is recommended most widely for the prevention of systemic embolism, stroke associated with atrial fibrillation, and
n Corresponding authors at: Department of Integrated Traditional Chinese and Western Medicine, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha 410008, PR China. nn Corresponding author at: Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, PR China. E-mail addresses: [email protected] (Y. Wang), [email protected] (H. Lv), [email protected] (T. Tang).
http://dx.doi.org/10.1016/j.jep.2016.08.005 0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
venous thromboembolism (Piatkov et al., 2010). The annual prescriptions of warfarin typically occur in 0.5–1.5% of the population (Johnson et al., 2011). Although novel oral anticoagulants (NOACs), such as apixaban, dabigatran, edoxaban and rivaroxaban, do not require more special laboratory monitoring or dose adjustment like warfarin does, warfarin has remained in use for over 60 years and is still the most common oral anticoagulant drug (Pirmohamed et al., 2015; Wadhera et al., 2014). This is mainly because warfarin can exert anticoagulant effects for several days after the subject stops taking it, while the NOACs do not have this merit, one missed dose results in low trough drug concentrations. In various countries and regions, especially in China, physicians
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prefer to combine herbal medicine with warfarin to achieve better effects and to prevent and reduce side effects (Lee et al., 2008). Certain herbs, including aloe, jalap, cascara, and rhubarb, have been found to affect the metabolism of warfarin via cytochrome P450s (CYP) and UDP-glucuronosyltransferases pathway (Zhou et al., 2012). Therefore, it is important to study the interactions between herbal medicines and warfarin so as to determine their optimal clinical applications. Warfarin can be detected in human bile after oral administration (Zeuzem, 2000). Some of the warfarin in the blood is absorbed from the intestine and secreted into the bile after entering liver cells, is then stored in the gallbladder through the bile duct, and finally is taken into the duodenum by gallbladder contraction (Erlinger, 1996; Gao et al., 2014). During enterohepatic circulation (EHC) process, the warfarin will be absorbed on two different occasions. The timing of the other peaks is related to eating and gallbladder emptying. Because of the irregularity of gallbladder emptying, the concentration-time (C-T) curve may present double peaks or multiple peaks. Unfortunately, previous studies about the pharmacokinetic effect of herb on warfarin failed to focus on influence of EHC process. Up to now, the herb-drug interaction involving enterohepatic circulation process remains unclear. Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong Hort), known as Chuanxiong in Chinese, a commonly used Chinese herbal medicine, has been used for treating cardiovascular diseases in China for centuries. Chuanxiong was used to relieve the pain induced by blood stagnation. According to traditional Chinese medicine (TCM) theory, the pharmacological action of Chuanxiong is “to activate the blood circulation and to promote qi”. Chuanxiong is a classical activating blood circulation herbs in China. In China and some regions such as Hong Kong, warfarin often combined with herbal prescriptions containing Chuanxiong, and these co-administration methods were recommended most widely for treatment the thromboembolism heart diseases in clinical practice (Feng et al., 2015; Zhang et al., 2016). But the effects of Chuanxiong on the pharmacokinetic of warfarin have not been reported. Scientists often use biliary drainage models to investigate the effects of enterohepatic circulation (Hellstern et al., 1990; Melnik et al., 1998; Lenzen et al., 1999). The protocol of the present study was planned to compare the pharmacokinetics of warfarin in rats with and without biliary drainage and to investigate the potential effects of Chuanxiong on the pharmacokinetics of warfarin in a biliary drainage model rats.
2. Materials and methods 2.1. Preparation of Chuanxiong extract Raw dried Chuanxiong Rhizoma was purchased from the pharmacy of Xiangya Hospital of Central South University, Changsha, China. The voucher specimen (no. 20140512, Chengdu, China) was deposited in the Laboratory of Ethnopharmacology, Xiangya Hospital of Central South University. Sixty gram raw Chuanxiong Rhizoma was thoroughly decocted in water (1:8, g/mL) for 1 h after soaking in water for half an hour, and then the gruffs decocted an hour with water (1:6, g/mL), filtered through gauze. The two filtrates were merged and evaporated by rotary evaporation under vacuum at 60 °C. The two filtrates were merged and evaporated under vacuum and concentrated to 60 mL with rotary evaporation at 60 °C, the final decoction was stored at 4 °C until use.
2.2. Chemicals and reagents Methyclothiazide (C9H11Cl2N3O4S2, CAS, 135-07-9, Lot, 101163201101; purity, 99.6%; internal standard, IS) and warfarin sodium (C19H15NaO4, CAS, 129-06-6, Lot, 101163-201001; purity, 92.3%) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Tetramethylpyrazine (C8H12N2, CAS, 112411-4, Lot, MUST-13022802; purity Z 98%), levistilide A (C24H28O4, CAS, 88182-33-6, Lot, MUST-13020211; purity Z98%) and senkyunolide I (C12H16O4, CAS, 94596-28-8, Lot, PA0804FA13; purity Z98%) were purchased from Chengdu Must Bio-technology Co., Ltd (Chengdu, China). Ferulic acid (C10H10O4, CAS, 1135-24-6, Lot, F1205050; purity, 99%) was purchased from Sigma-Aldrich (The Netherlands). Formic acid from Tianjing Gangfu Fine Chemical Reagent Factory (Tianjing, China). HPLC grade acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). Deionized water was purified with a TKA Samrt2pure water purification system (German) with a sensitivity of 18.2 MO. 2.3. Surgical procedures All animal procedures were conducted in strict compliance with our institutional protocols and were approved by the Animal Ethics Committee of Central South University. Thirty-two specific pathogen free (SPF) grade Sprague-Dawley (SD) male rats weighing 250–300 g were provided by Shanghai Laboratory Animal Center (SLAC). They were housed at temperature-controlled (21– 25 °C) and 40–70% relative humidity room in an SPF environment on 12 h day-night cycle with lights. All rats provided tap water ad libitum and were fed standard rodent chow, except for the 12-h fast before the pharmacokinetic study. The experimental rats were randomly divided into four groups, WN Group: healthy rats after a single gastric-administration of 0.5 mg/kg warfarin sodium; WO Group: rats subjected to biliary drainage after the gastric-administration of 0.5 mg/kg warfarin sodium; WCN Group: healthy rats after the gastric-administration of warfarin sodium and Chuanxiong Hort at doses of 0.5 mg/kg and 10 g/kg, respectively; and WCO Group: rats subjected to biliary drainage after the gastricadministration of warfarin sodium and Chuanxiong Hort at doses of 0.5 mg/kg and 10 g/kg, respectively. The dose of drugs we used in this study calculated according to the conventional dosage of clinical. The animals were fasted for food but not water overnight before the experiments. Bile drainage surgery was carried out under sterile conditions after anesthetized by intraperitoneal injection of 10% chloral hydrate at 0.4 mL/100 g body weight. A midline incision was made on the abdomen, the liver was opened up, and the common bile duct of rat was noted over the margin of the duodenal bulb. The distal end of the bile duct was ligated and a polyethylene tube (inner diameter was 0.8 mm and outer diameter 1.2 mm) was inserted into the proximal end of common bile duct. Yellow bile could be seen in the catheter either immediately or a few minutes after insertion, and the free end of the external biliary drainage catheter was fixed to the peritoneum and brought out of the body through a small hole made on the back of the rat's neck. 2.4. Sample collection and preparation On the second day after biliary drainage surgery, 500 μL of tail venous blood was collected into heparinized tubes prior to treatment and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h post-dose. The samples were centrifuged at 3000 rpm for 15 min, then the supernatants were stored at 80 °C until analysis. To a total of 200 μL of rat plasma were added with 20 μL of IS solution, 100 μL of 20% formic acid and 2 mL of methanol, which was followed by vortex mixing for 1 min and hyperacoustic mixing for 5 min. The
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samples were then centrifuged at 3000 rpm for 10 min. The supernatants were evaporated to dryness under a gentle stream of nitrogen at room temperature, followed by reconstitution with 200 μL mixture of acetonitrile and water (50:50). This was followed by vortex mixing for 1 min and hyperacoustic mixing for 5 min. The samples were then centrifuged at 15,000 rpm for 10 min, the supernatants were filtered through the 0.22-μm membrane, and 2.0 μL of the processed sample was injected for the analysis.
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2.5. Instrumentation and analytical conditions The sample analysis was performed on an UPLC-MS/MS system. The Acquity™ UPLC system (Waters Corporation, Milford, MA, USA) was constituted of an autosampler (set at 10 °C), a binary solvent delivery manager and a column oven (set at 35 °C). Chromatographic separation was acquired from a Waters Acquity BEH C18 column (2.1 mm 100 mm I.D., 1.7 mm, Waters, Wexford, Ireland). A mobile phase consisting of A (aqueous buffer containing 0.1% formic acid) and B (acetonitrile) was pumped at 0.3 mL/
Fig. 1. Representative MRM chromatograms of warfarin and methyclothiazide in rats: (A) blank plasma samples from healthy rats; (B) blank plasma samples spiked with warfarin and methyclothiazide; (C) plasma from healthy rats after a single administration of warfarin; (D) plasma from rats with biliary drainage after a single administration of warfarin; (E) plasma from healthy rats after the administration of warfarin and Chuanxiong; and (F) plasma from rats with biliary drainage after the administration of warfarin and Chuanxiong.
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min. The gradient elution was: 0 min, 10% B; 0–0.25 min, 10% B; 0.25–5.0 min, linear from 10% to 95% B; 5.0–8.0 min, holding at 95% B; then, between 8.0–8.2 min, an immediate decrease to the initial conditions (i.e., 10% B) for equilibration of the column. The typical injection volume was 2 μL. The detection system, a tandem quadrupole mass spectrometer (Waters Corporation, Manchester, UK), was operated using an electrospray ionization with the desolvation temperature was fixed at 365 °C, the source temperature was set at 110 °C, and the capillary voltage set at 2.5 kV in negative ion mode. Nitrogen was used for the desolvation gas flow (650 L/h)
and cone gas flow (50 L/h). For collision-induced dissociation, argon was used as the collision gas at a flow rate of 0.2 mL/min. The Masslynx 4.1 software program (Waters Corporation) was used for data processing and acquisition. The multiple-reaction monitoring (MRM) mode was selected for quantitation of IS and warfarin, the precursors of which for the production ion transitions were as follows: IS, 357.81-321.89; warfarin, 307.03-250.02. The data were collected and processed using the DAS 2.1.1 software.
Fig. 2. LC–MS/MS spectra of methyclothiazide, warfarin and four marker compounds of Chuanxiong. Warfarin and methyclothiazide can be ionized under negative ionization conditions. (M-H) predominated and was used as the precursor ion to obtain the ion spectra. The most sensitive mass transitions for methyclothiazide (A, the internal standard), were m/z 357.81-321.89, for warfarin (B) were 307.03-250.02. for tetramethylpyrazine (C) were 137.10-96.04, for levistilide A (D) were 381.09-190.92, for senkyunolide I (E) were 225.11-207.04 and for ferulic acid (F) were 192.98-133.96.
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2.6. Method validation The method was verified in terms of linearity, selectivity, precision, accuracy, extraction recovery, matrix effects and stability according to FDA guidelines for bioanalytical method validation. Calibration curve samples were prepared by spiking blank plasma. To evaluate linearity, duplicate calibration standards of warfarin at six concentration of blank plasma 5, 20, 100, 500, 2000 and 5000 ng/mL levels were prepared and analyzed. The linearity of the method was determined by analysis of standard plots associated with a six-point calibration curve. The overall intra- and inter-day variations all used five analytes.
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from 563.33 to 509.13 min, but this difference was not statistically significant. The clearance parameter (CLz/F) of the WO group significantly increased 1.61 times than the WN group. 3.4.2. The pharmacokinetics parameter comparations of WCO and WCN group The WCO group showed a decreased to 25.7% in the AUC0–t compared to the WCN group (Po 0.01). Compared with the WCN group, the Cmax of the WCO group was significantly decreased to 20.5% (P o0.01) and the MRT0–t also delayed by 30.2% (P o0.01). The CLz/F of the WCO group increased 2.04 times than the WCN group. The mean half-life (t1/2z) had a tendency to be extended from 625.04 to 1179.78 min, this difference was not statistically significant.
3. Results 3.1. Specificity No interference was displayed at the retention times of either warfarin or IS in plasma samples, which were used for the analysis, and the method showed good specificity. Typical MRM chromatograms are shown in Fig. 1, and the LC-MS/MS spectra of warfarin and methyclothiazide are presented in Fig. 2. 3.2. Linearity and LLOQ of warfarin The linear regression equation was y ¼0.0189x 0.0057 (r ¼0.999993), the LLOQ (lower limit of quantitation) was 5 ng/mL, and the linearity range was 5–5000 ng/mL, where y indicates the ratio of warfarin peak area to internal standard peak area, x refers to the warfarin concentration (ng/mL), and r is the correlation coefficient of the equation.
3.4.3. The pharmacokinetics parameter comparations of WN and WCN group Further, Table 2 revealed that the AUC0–t of warfarin in the WCN group was 2.42 times than that of the WN group (p o0.01), the Cmax in the WCN group was 2.35 times than the WN group (p o0.01), the WCN group showed a decreased to 79.9% in the t1/2z compared to the WN group (p o0.05), and the mean of CLz/F also decreased from 20.14 to 12.73 mL/h/kg (P o0.05). 3.4.4. The pharmacokinetics parameter comparations of WO and WCO group Chuanxiong obviously affected the disposition of warfarin after biliary drainage, because the WCO group displayed a decreased to 61.6% in the Cmax compared the WO group (p o0.01), the t1/2z of the WCO group was extended 2.15 times than the WO group (Po 0.01), and the MRT0–t also delayed by 22.1% (P o0.01).
3.3. Precision and accuracy
4. Discussion
Table 1 showed that the method had good precision and accuracy with good intra-day and inter-day precision. And all of the results were found to be within the accepted variable limits.
Most researches on warfarin have focused on dose and safety (Verhoef et al., 2014). As consequences of under- or over-anticoagulation, the incidence of thromboembolism or hemorrhage due to the use of warfarin ranged from 16% to 25% (Bejth et al., 2002; Fang et al., 2007). The above risks are most common during the initial period of treatment (Liu et al., 2015). Side effects of warfarin is associated with its narrow therapeutic range. What's more, different patients have different therapeutic ranges for warfarin. The reason for these issues is that genetic and multiple environmental factors can influence the metabolism and effects of warfarin in each patient, and it has been reported that there is an at least 20-fold inter-individual variability in warfarin sensitivity (Hirsh et al., 2003; Wittkowsky, 2005). It is therefore necessary to carefully monitor the warfarin concentration to avoid serious side effects and keep patients to be safe (Morais and De Caterina, 2016). The present study has shown that the biliary drainage operation and Chuanxiong altered significantly the pharmacokinetic profile of warfarin in rats plasma. There were significant differences in the pharmacokinetic parameters of warfarin between the WO and WN groups, including AUC0–t, Cmax, Tmax, t1/2z and CLz/F according to the above analysis. The notable decreases in the AUC
3.4. Pharmacokinetic study Fig. 3 shows the C-T curves for plasma warfarin in the four groups (n ¼8). The pharmacokinetic parameters (Table 2) of warfarin were calculated using non-compartmental modeling with the DAS2.1.1 software. 3.4.1. The pharmacokinetics parameter comparations of WO and WN group The WO group showed a decreased in the area under the curve (AUC0–t) to 74.4% compared to the WN group (P o0.01). The peak concentration (Cmax) of the WO group was significantly decreased by 21.4% compared to the WN group (Po 0.01), and the time taken to reach the Cmax (Tmax) in the WO group was 3.21 times slower than that in the WN group (Po0.05). The mean half-life (t1/2z) of the WO group was shortened 29.9% than the WN group (P o0.05). The mean residence time (MRT0–t) had a tendency to be shortened Table 1 The intra-day and inter-day precision of warfarin in rat plasma samples.
Warfarin
Spiked concentration (ng/mL)
Intra-day Measured (ng/mL)
RSD%
Measured (ng/mL)
Inter-day RSD%
50 500 5000
49.737 3.98 505.69 7 42.3 5093.92 7 461.1
8.00 8.36 9.05
50.26 7 3.29 496.79 738.7 5057.45 7 431.6
6.55 7.79 8.53
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Fig. 3. The mean 7SD plasma concentration-time curves for warfarin (n¼ 8). WN: healthy rats after a single administration of warfarin; WO: rats with biliary drainage after a single administration of warfarin; WCN: healthy rats after the administration of warfarin and Chuanxiong; WCO: rats with biliary drainage after the administration of warfarin and Chuanxiong.
Table 2 The pharmacokinetic parameters of warfarin (mean 7 SD, n ¼8). Group
AUC(0–t) (ng/mL min)
MRT(0–t) (min)
t1/2z (min)
CLz/F (mL/h/kg)
Tmax (min)
Cmax (ng/mL)
WN WO WCN WCO
1′102′028.69 7 151′173.74 819′455.317 172′906.63** 2′667′827.667 1′381′550.09** 685′879.94 7 189′529.01##
563.337 13.77 509.137 90.78 477.38 7 81.81 621.64 743.12##,※※
782.57 7108.38 548.88 7225.60* 625.04 7657.32* 1′179.78 7 450.16※※
20.14 72.43 32.43 7 12.09* 12.737 8.78* 25.94 7 5.11##
70.00 7118.23 225.007 100.14* 198.75 7 136.72 247.5 7 192.93
1′357.197 220.64 1′066.337138.14** 3′193.86 7 1304.34** 656.4 7198.23##,※※
WN: healthy rats after a single administration of warfarin; WO: rats with biliary drainage after a single administration of warfarin; WCN: healthy rats after the administration of warfarin and Chuanxiong Rhizoma; WCO: rats with biliary drainage after the administration of warfarin and Chuanxiong Rhizoma. **
Compared to the WN group p o 0.01. Compared to the WN group po 0.05. Compared to the WCN group p o0.01. ※※ Compared to the WO group po 0.01. *
##
and Cmax of the WO group caused by biliary drainage indicated that the operation decreased the bioavailability of warfarin, possibly due to the change in EHC. There were also significant differences in the pharmacokinetic parameters of warfarin between the WCO and WCN groups. These findings indicated that biliary drainage can affect the disposition of warfarin when it was coadministered with Chuanxiong in rats. In fact, EHC refers to the circulation of bile acid, substances or drugs from the liver to the bile, followed by re-absorption into the small intestine and transport back to the liver for systemic circulation. Intestinal reabsorption to complete the enterohepatic cycle might be determined by hydrolysis of a drug conjugate by gut
bacteria. The EHC process extends the drug residence time in the body. A drug undergoing EHC usually shows a multiple-peak phenomenon in terms of its plasma C-T curve and a prolonged elimination half-life (Roberts et al., 2002). EHC induced a second peak in the C-T profile of the WCN group (Fig. 3). However, the C-T curve of the WCO group did not show such double peaks due to blockade of EHC. Bile acid circulation therefore played an important role in the pharmacokinetics of warfarin combined with or without Chuanxiong, and we speculated that Chuanxiong influences the pharmacokinetics of warfarin by affecting its EHC. Ferulic acid is the main active constituent of Chuanxiong, and ferulic acid metabolism conjugation with glucuronic acid and/or
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sulfate, which takes place mainly in the liver, is the principal pathway in vivo. Ferulic acid is also metabolized into m-hydroxyphenylpropionic acid by the intestinal microflora through reduction, dehydroxylation, and demethylation (Zhao and Moghadasian, 2008). This molecular mechanism needs to be verified in additional studies. The pharmacokinetics study of warfarin in rats with biliary drainage is a very useful reference for warfarin research in clinical trials and applications. The significant differences in AUC0–t, Cmax, t1/2z and CLz/F between the WN and WCN group indicated that Chuanxiong affected the pharmacokinetics of warfarin in rat plasma and that Chuanxiong can promote the absorption rate of warfarin, possibly due to metabolic compete, and decreased systemic elimination or local interactions of warfarin with Chuanxiong in the intestine. The dose response variations of warfarin are remarkably influenced by pharmacokinetics aspects that depended on genetic, environmental and others as yet unknown factors. Polymorphisms in CYP2C9, CYP2C19, CYP1A1 and VKORC1 are the major determinants of the warfarin dosage requirement in human (Zhao and Moghadasian, 2008; Lee et al., 2009; Wadelius et al., 2009), whereas the corresponding enzymes in rats are CYP2C11, CYP2C6, CYP1A1 and CYP2B1 (Soucek and Gut, 1992). Genetic polymorphisms in CYP2C9 and VKORC1 account for approximately 50% of the variability in the pharmacologic response of warfarin in human (Bodin et al., 2005). Other genetic factors including vitamin K intake and recycling and warfarin transportation may also influence the stable maintenance dosage of warfarin. And CYP3A4, CYP2C9 and CYP1A2 are the major isoenzyme of in vitro metabolism of ligustilide, which is the major component of Chuanxiong (Qian et al., 2009). The compete of metabolism enzyme can explain the increase of the WCN group in the AUC compare to the WN group without biliary drainage in vivo. Physicians should be aware of the possibility of over-anticoagulation of drug interaction between warfarin and Chuanxiong owing to the increased AUC and Cmax. There is a dilemma in the opposite tendency of the AUC and Cmax between normal rats and rats subjected to biliary drainage. On one hand, we speculated that Chuanxiong must be participated in warfarin's EHC in vivo, Chuanxiong made Cmax and AUC0–t of warfarin rise 2.35 and 2.42 folds (P o0.01) in normal rats plasma, according to the comparations between the WN and WCN group, but Chuanxiong decreased the warfarin AUC and Cmax, according to the comparations between the WO and WCO group, in which the EHC process were interrupted by the operation of biliary drainage. On the other hand, the operation might take some complex factors in the disposition process in vivo, and we might further hypothesis that whether diseases conditions induce the similar changes just showing as in the present study. These corollaries provided a chance to investigate the EHC mechanism of warfarin. There is widespread undisclosed use of herbal medicine among Western doctors. Previous studies reported that the rate of herbal use was 75.4% to more than 90.0% in Singapore and Italy (Lim et al., 2005; Cuzzolin et al., 2007). The use of TCM is also significant in various countries and regions, with an estimated 58.0% and 23.5% of patients in the United Kingdom and Italy, respectively, co-ingesting warfarin and herb medicines (Cuzzolin et al., 2007; Ramsay et al., 2005; Wong et al., 2003). Although we have shown that Chuanxiong impacts the pharmacokinetics of warfarin in rats after biliary drainage, it is unclear how it exerts its effects. The identification and detailed quantitative assessment of the importance of various enzyme/transporter pathways in the elimination of Chuanxiong are still lacking, some previous studies on its main metabolism pharmacokinetics showed that these processes may involve CYPs, UDP-glucuronosyltransferases, sulfotransferases, and the monocarboxylic acid transporter (Poquet
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et al., 2008; Rondini et al., 2002).
5. Conclusion Biliary drainage significantly affected the disposition of warfarin in rats. Chuanxiong co-administration significantly influenced the disposition of warfarin in rat plasma in both normal rats and rats subjected to biliary drainage. The present study can provide clinic guidance for the administration of warfarin.
Acknowledgments This work was supported by the Science Project of the Education Department of Hunan Province (Grant No. 13C1126), the Natural Science Foundation of Hunan Province (Grant No. 14JJ2024), the National Natural Science Foundation for Young Scientists of China (Grant Nos. 81303074 and 81403259) and the National Natural Science Foundation (Grant No. 81473573).
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