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The effect of Compound Danshen Dripping Pills, a Chinese herb medicine, on the pharmacokinetics and pharmacodynamics of warfarin in rats
Journal of Ethnopharmacology 137 (2011) 1457–1461
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
The effect of Compound Danshen Dripping Pills, a Chinese herb medicine, on the pharmacokinetics and pharmacodynamics of warfarin in rats Yang Chu a , Ling Zhang a,b , Xiang-yang Wang a , Jia-hua Guo a , Zhi-xin Guo a,b , Xiao-hui Ma a,∗ a b
Department of Pharmacology, Tasly R&D Institute, Tianjin Tasly Group Co., Ltd., Pujihe East Road (No. 2), BeiChen District, Tianjin 300410, China Tianjin Key Laboratory of Chinese Medicine Pharmacology, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
a r t i c l e
i n f o
Article history: Received 2 June 2011 Received in revised form 9 August 2011 Accepted 14 August 2011 Available online 29 August 2011 Keywords: Compound Danshen Dripping Pills Warfarin Pharmacokinetics Pharmacodynamics Interaction
a b s t r a c t Aim of the study: Significant pharmacokinetic/pharmacodynamic (PK/PD) interactions between various herbal products and warfarin have recently been reported. The present study was conducted to determine whether Compound Danshen Dripping Pills (CDDP), a Chinese herb medicine used for the treatment of cardiovascular diseases, interacts with warfarin when administered concomitantly. Materials and methods: Each day for 7 days two groups of rats were treated orally with CDDP (50 mg/kg and 250 mg/kg, twice daily), and the control group received similar treatment with appropriate volumes of water only. Sixty minutes after the final daily administration of CDDP or water, an aqueous solution of warfarin (0.2 mg/mL) was given to each rat at a dose of 1.0 mg/kg, and blood samples were collected at 0, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h after warfarin-treatment. The concentration of warfarin in blood plasma was determined by high performance liquid chromatography (HPLC). Prothrombin time (PT) in blood plasma was measured using thromboplastin reagent. Results: Excellent linearity was found between 0.05 and 10 g/mL with a lower limit of quantitation (LLOQ) of 0.05 ng/mL (r > 0.999); moreover, all the validation data including accuracy and precision (intra- and inter-day), were within the required limits. No significant differences were found in PTmax and AUCPT 0–∞ between the two CDDP-treated groups and the control. Besides, there was little alteration in any of the pharmacokinetic parameters of warfarin between the two CDDP-treated groups and the control. Conclusion: The concomitant application of CDDP and warfarin did not give rise to significant effect on the pharmacodynamics of warfarin, and practically no effect on its pharmacokinetics. It was speculated that the PK/PD interactions between CDDP and warfarin was likely to be negligible as long as the patients took CDDP at a normal dose. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Compound Danshen Dripping Pills (CDDP, Chinese name Fufang Danshen Diwan), consisting of Radix salviae miltiorrhizae (Labiatae sp. plant, Chinese name Danshen), Radix notoginseng (Araliaceae plant, Chinese name Sanqi), and Borneolum (Chinese name Bingpian), are an herbal medicine recognized in the official Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2005). The drug is widely used for the prevention and treatment of coronary arteriosclerosis, angina pectoris and hyperlipaemia in China. The annual sales volume of CDDP has exceeded 140 million dollar since 2002. This herbal medicine is also available as a dietary supplement or drug (Prescription/OTC) in countries including USA,
∗ Corresponding author. Tel.: +86 22 26736372; fax: +86 22 26736376. E-mail address: [email protected] (X.-h. Ma). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.08.035
Singapore, Republic of Korea, India, UAE, Russia, Cuba, and South Africa. Warfarin, a coumarin anticoagulant, is widely used in the prophylaxis and treatment of a number of thromboembolic disorders, including transient ischemic attacks (Mirsen and Hachinski, 1998), atrial fibrillation (Albers et al., 1991), and thromboembolic complications that occur with prosthetic heart valves (Stein and Kantrowitz, 1989). However, coumarin anticoagulants, such as warfarin, exhibit high protein binding, cytochrome P450dependent metabolism and a narrow therapeutic window, all of which can contribute to the potential for adverse anticoagulant interactions following co-administration with a wide range of drugs, including cardiovascular and antilipidemic agents (Harder and Thurmann, 1996). These interactions can result in enhancement of the hypothrombinemic effect of warfarin and can lead to bleeding complications and disruption in warfarin metabolism. So far, a number of publications have reported significant PK/PD interactions between herbal products and warfarin (Janetzky and
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0.35
2
AU
0.30 0.25 0.20 0.15
1 8
0.10 0.05
3 4
0.00 0.0
1.0
2.0
3.0
5
6
4.0
7 5.0
6.0
7.0
8.0
Time (min) Fig. 1. UPLC chromatogram of phenolic acid components in CDDP. (1) TSL; (2) PCA; (3 and 4) two new phenolic acid components; (5) SAD; (6) RMA; (7) SAB; (8) SAA.
Marreale, 1997; Foster et al., 1999; Zhu et al., 1999; Chan, 2001; Makino et al., 2002; Hovhannisyan et al., 2006). Since there are some cases in which patients taking warfarin desire to take CDDP additionally, in order to receive a holistic treatment for cardiovascular diseases, the information about the interactions between CDDP and warfarin are in considerable demand. In this present study, the effects of CDDP on the pharmacokinetics and pharmacodynamics of warfarin have been firstly investigated in rats.
Milford, MA, USA) and the UPLC condition for phenolic acid components were as follows: column, WATERS ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m); mobile phase, ACN/H2 O/AcOH 2:98:0.5 (0 min) → 90:10:0.5 (8 min), linear gradient; flow rate, 0.6 mL/min; column temperature, 40 ◦ C; detectable wavelength: 280 nm; the UPLC condition for triterpene saponin components were as follows: column, WATERS ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m); mobile phase, ACN/H2 O 15:85 (0 min) → 20:80 (7.5 min) → 30:70 (9 min) → 40:60 (14 min); flow rate, 0.5 mL/min; column temperature, 40 ◦ C; detectable wavelength: 203 nm. The UPLC chromatogram of phenolic acid and triterpene saponin components in CDDP was shown in Figs. 1 and 2, respectively.
2. Materials and methods 2.1. Chemicals and reagents
2.3. Animals and experimental design
CDDP (25 mg/pill, lot 100605) was supplied by Tianjin Tasly Pharmaceutical Co., Ltd. Warfarin sodium salt (purity > 98%) was obtained from Wuhan Yuancheng Technology Development Co. Ltd. (Wuhan, China). Diazapam (purity > 99%), which was used as the internal standard (IS), was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile (HPLC grade) was purchased from Fisher Scientific (Pittsburgh, PA, USA), while all other chemicals were analytical grade and used without further purification. The distilled water, prepared from demineralized water, was used throughout the study. All solvents were filtered through 0.45 m filter membranes before injecting into HPLC.
18 male Wistar rats, weighing 220 ± 20 g, were provided by Vital River Lab Animal Technology Co. Ltd (Beijing, China) and housed with a 12 h light/12 h night cycle at ambient temperature (about 23 ± 2 ◦ C) and 50 ± 5% relative humidity, and the air was changed 1–2 times/h. All the rats were kept separately in wire bottom cages (55 cm × 35 cm × 25 cm) with lattice framed steel lids, and woodsawdust was used as bedding. Free access to food and water was allowed at all times except for fasting 12 h before the experiment. All the animal experiment procedures were approved by the Animal Ethics Committee of Tianjin Tasly Institute. 18 study animals were divided into three groups. Group 1 animals, constituting the control group, were treated by oral gavage with water (10 mL/kg twice per day) for 7 days: 60 min after the final administration of water, an aqueous solution of warfarin (containing 0.2 mg/mL) was administered orally to each animal at a dose of 1.0 mg/kg, and blood samples (approximately 0.5 mL) were collected by tail vein at 0, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h after warfarin-treatment. Group 2 and 3 animals, were treated by oral gavage with CDDP (50 mg/kg and 250 mg/kg, twice per day) for 7 days, respectively: 60 min after the final administration of CDDP, an aqueous solution of warfarin (containing 0.2 mg/mL) was administered orally to each animal at a dose of 1.0 mg/kg, and blood samples (approximately 0.5 mL) were collected by tail vein at 0, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h after warfarin-treatment.
2.2. The main constituents in CDDP
AU
In CDDP, the main constituents of Radix Salviae miltiorrhizae are phenolic acids, including tanshinol (TSL), protocatechuic aldehyde (PCA), rosmarinic acid (RMA), salvianolic acids A, B, and D (SAA, SAB, and SAD), and the constituents of Radix Notoginseng are triterpene saponins, including ginsenosides Rb1 , Re, Rg1 , and notoginsenoside R1 ; and the constituent of Borneolum is borneol. 12 pills CDDP were dissolved in 25 mL of methanol under the ultrasonic treatment for 10 min. After vortexing for 1 min, the samples were centrifuged at 2000 rpm for 5 min, and then 10 L of supernatant fluid was injected into UPLC for analysis. Chromatography was performed on an Acquity UPLC system (Waters Corp., 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.0
4
2
1 1.0
2.0
3.0
4.0
5.0
6.0
7.0
3 8.0
9.0
10.0
11.0 12.0
13.0 14.0
Time (min) Fig. 2. UPLC chromatogram of triterpene saponin components in CDDP. (1) Notoginsenoside R1 ; (2) Ginsenoside Rg1 ; (3) Ginsenoside Re; (4) Ginsenoside Rb1 .
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2.4. Preparation of blood samples Each blood sample was separated into two 1.5 mL Eppendorf tubes, which were prepared by heparin and sodium citrate, respectively. Blood samples in heparinized tubes were centrifuged at 1200 × g for 15 min in order to obtain plasma samples for the determination of warfarin. Citrated blood samples (0.03 mL of 109 mmol/L sodium citrate and 0.27 mL of blood) were centrifuged at 1200 × g for 15 min in order to obtain plasma samples for the measurement of prothrombin time. 2.5. Determination of warfarin in rat plasma by HPLC The plasma samples were analyzed for warfarin concentration by the Agilent 1200 series liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA), equipped with a quaternary pump, an autosampler, thermostatically controlled column apartment, and a DAD detector. Sample pretreatment involved in one-step protein precipitation with acetonitrile. 100 L IS solution (5.0 g/mL diazepam, prepared in acetonitrile) and 100 L acetonitrile were added to the 100 L plasma sample. After vortexing for 30 s, the sample was centrifuged at 6500 × g for 10 min and the supernatant was transferred in a vial, 20 L was injected into the HPLC system for analysis. The HPLC conditions were as follows: column, Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm i.d.; particle size 3.5 m) protected by a C18 guard column (Phemomenex, USA); mobile phase, acetonitrile/water/formic acid (50:50:0.2, v/v/v); flow rate, 0.8 mL/min; column temperature, 30 ◦ C; detection wavelength, 305 nm. 2.6. Measurement of prothrombin time Reconstituted thromboplastin reagent (Beijing Steellex Instrument Co. Ltd., Beijing, China) and the citrated plasma sample (0.1 mL) were incubated separately at 37 ◦ C for 2 min prior to mixing. The time taken for a clot to form following introduction of the thromboplastin reagent into the plasma was recorded. 2.7. Statistical analysis Pharmacokinetic parameters of warfarin were calculated using the Topfit 2.0 program by the non-compartmental method. The elimination rate constant (ke ) was obtained as the slope of the linear regression of the log-transformed concentration values versus time date in the terminal phase. The elimination half-life (T1/2 ) was calculated as 0.693/ke . The peak plasma concentration (Cmax ) and the corresponding time (Tmax ) were directly obtained from the raw data. The area under the curve to the last measurable concentration (AUC 0–t ) was calculated by the linear trapezoidal rule. The area under the curve to infinity (AUC 0–∞ ) was calculated as AUC 0–∞ = AUC 0–t + Ct /ke , where Ct is the last measurable concentration. PTmax (s) was maximum prothrombin time and AUC PT0–∞ (s/h) was area under the curve prothrombin time versus time curve extrapolated to infinity. Data are expressed as mean values ± standard error. Statistical analysis was carried out using Student’s unpaired two-tail t-test with significance at the 95% confidence interval. 3. Results and discussion 3.1. Method validation The chromatograms of warfarin in rat were shown in Fig. 3, and no interference was observed at the retention time of the warfarin 6.58 min and diazepam (IS) 7.72 min due to endogenous substances
Fig. 3. Chromatograms of blank plasma (A); blank plasma spiked with warfarin (at the LLOQ, 0.05 g/mL) and diazapam (B) and plasma sample 2 h after oral administration of warfarin (C). Peak I, warfarin; Peak II, diazapam.
in blank rat plasma. The peak area ratios of warfarin to IS were plotted with plasma concentrations to give the correlation coefficient and linear regression equation after weighed linear regression. Excellent linearity was found between 0.05 and 10 g/mL with a lower limit of quantitation (LLOQ) of 0.05 g/mL (r > 0.999). Validation experiments demonstrated that the accuracy was in the range 91.5–109.8%, the precision (intra- and inter-day) was within 12.0%, and the recovery was more than 85%. In conclusion, the high sensitivity and selectivity and relatively short analytical time of the HPLC assay make it suitable for the pharmacokinetic study of warfarin. 3.2. The effect of CDDP on the PK/PD of warfarin The aim of the study was to find possible PK/PD interactions associated with the simultaneous application of CDDP and warfarin. The recommended daily human dose of CDDP is 12.5 mg/kg. In terms of pharmacological activity and toxicity with respect to the rat model, this would translate on an empirical basis to an equivalent daily dose of about 80 mg/kg of CDDP. In the present study, CDDP was applied to the treatment animals at a daily dose of 0.1 and 0.5 g/kg (50 mg/kg and 250 mg/kg, twice daily), respectively, about 8-fold and 40-fold greater than the recommended human
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Control
Concentration (µg/mL)
10.0
CDDP, 0.1 g/kg CDDP, 0.5 g/kg
8.0 6.0 4.0 2.0 0.0 0
15
30
45
60
Time (h) Fig. 4. The plasma concentration versus time profile of warfarin determined after application of a single oral dose (1 mg/kg) of warfarin to control rats and to those that had received daily treatment with CDDP (n = 6).
Control
Prothrombin time (s)
80.0
CDDP, 0.1 g/kg CDDP, 0.5 g/kg
60.0 40.0 20.0 0.0
0
15
30
45
60
Time (h) Fig. 5. The prothrombin time versus time profile of warfarin determined after application of a single oral dose (1 mg/kg) of warfarin to control rats and to those that had received daily treatment with CDDP (n = 6).
dose and about 1.25 and 6.25 times higher than the equivalent dose in rats. Figs. 4 and 5 show the plasma concentration versus time profile of warfarin and the prothrombin time versus time profile of warfarin following a single oral dose (1.0 mg/kg) of warfarin to control rats and CDDP-treated rats, respectively. The main pharmacokinetic and pharmacodynamic parameters of warfarin in each group were summarized in Table 1. It is obtained that there was no significant difference between the PK/PD parameters of warfarin in control group and those of warfarin in group treated with CDDP,
Table 1 Effect of Compound Danshen Dripping Pills (CDDP) on pharmacokinetic and pharmacodynamic (PK/PD) parameters of warfarin (n = 6). Parameters
Control
Cmax (g/mL) Tmax (h) T1/2 (h) ke (1/h) AUC 0–t (g h/mL) AUC 0–∞ (g h/mL) MRT (h) CL (mL/min) Vd (L) PTmax (s) AUC PT0–∞ (s/h)
6.16 3.33 14.8 0.047 106.26 117.38 14.3 0.15 0.19 55.1 1519.9
CDDP-treatment (twice daily) 50 mg/kg ± ± ± ± ± ± ± ± ± ± ±
1.59 2.42 1.79 0.007 21.05 25.79 1.62 0.04 0.03 8.7 239.3
6.76 3.17 14.8 0.047 110.65 122.48 14.7 0.14 0.18 55.1 1572.2
± ± ± ± ± ± ± ± ± ± ±
250 mg/kg 2.93 2.84 1.59 0.006 12.19 12.98 0.98 0.01 0.03 9.3 192.6
5.41 4.42 13.7 0.051 91.96 99.63 14.3 0.17 0.20 53.2 1487.9
± ± ± ± ± ± ± ± ± ± ±
1.03 3.07 1.44 0.006 12.79 15.20 1.43 0.02 0.03 6.3 128.6
Cmax and Tmax , the peak plasma concentration and the corresponding time; T1/2 , elimination half-life; ke , elimination rate constant; AUC 0–t , the area under the curve to the last measurable concentration; AUC 0–∞ , the area under the curve to infinity; MRT, mean residence time; CL, clearance; Vd , volume of distribution; PTmax , maximum prothrombin time; AUC PT0–∞ , the area under the curve prothrombin time versus time curve extrapolated to infinity.
following statistical analysis by Student’s unpaired two-tail t-test (P > 0.05). Warfarin is a widely used oral anticoagulant that requires a tight control on dosage. This drug is easily metabolized in the organism by CYP450 (mainly hydroxylation). The PK/PD interactions between herbal products and warfarin are of particular concern when perturbations to absorption, metabolism, or protein binding are suspected. Wu and Yeung (2010) and Kuo et al. (2006) reported aqueous extract of S. miltiorrhiza, the main components of CDDP, had no effect on warfarin being metabolized by CYP450 to form various hydroxylation products. Lo et al. (1997) and Chan et al. (1995) studied the PK/PD interactions of warfarin and S. miltiorrhiza extract in rats. Three days pretreatment with the extract (5.0 g/kg per day i.p.) in rat increased the absorption rate, Cmax , AUC, T1/2 , and decreased the Vd and CL of a single oral treatment of warfarin. Besides, S. miltiorrhiza extract also prolonged the prothrombin time further than its extension by warfarin. Considering that they applied intraperitoneal injection of S. miltiorrhiza extract, the dosage of CDDP in our study was quite smaller than that used in their experiments, and concomitant treatment with warfarin and CDDP did not give rise to significant effects on the pharmacokinetics and pharmacodynamic of warfarin. Furthermore, the dosage of CDDP-treated groups in the present study, which was 8 times and 40 times the amount of the human daily dose, did not exhibit pharmacokinetic or pharmacodynamic interactions. It was speculated that the PK/PD interactions between CDDP and warfarin was likely to be negligible as long as the patients took CDDP at a normal dose. However, the results of the present study were obtained by single treatment with warfarin, so we could not exclude the possibility that warfarin might be accumulated and prothrombin time might be increased if patients who were taking daily warfarin also took CDDP. Currently, the research in this area is dealt with in our laboratory. Acknowledgement This study was partly supported by National Key Special Project of Science and Technology for Innovation Drugs of China (grant no. 2008ZX09401-006). References Albers, G.W., Atwood, J.E., Hirsh, J., Sherman, D.G., Hughes, R.A., Connolly, S.J., 1991. Stroke prevention in nonvalvular atrial fibrillation. Annals of Internal Medicine 115, 727–736. Chan, K., Lo, A.C., Yeung, J.H., Woo, K.S., 1995. The effects of Danshen (Salvia miltiorrhiza) on warfarin pharmacodynamics and pharmacokinetics of warfarin enantiomers in rats. The Journal of Pharmacy and Pharmacology 47, 402–406. Chan, T.Y., 2001. Interaction between warfarin and Danshen (Salvia miltiorrhiza). The Annals of Pharmacotherapy 35, 501–504. Foster, B.C., Vandenhoek, S., Hanna, J., Akhtar, M.H., Krantis, A., 1999. Effect of natural health products on cytochrome P-450 drug metabolism. The Canadian Journal of Infectious Diseases 10B, 150. Harder, S., Thurmann, P., 1996. Clinically important drug interactions with anticoagulants: an update. Clinical Pharmacokinetics 30, 416–444. Hovhannisyan, A.S., Abrahamyan, H., Gabrielyan, E.S., Panossian, A.G., 2006. The effect of Kan Jang extract on the pharmacokinetics and pharmacodynamics of warfarin in rats. Phytomedicine 13, 318–323. Janetzky, K., Marreale, A.P., 1997. Probable interaction between warfarin and Ginseng. American Journal of Health-System Pharmacy 54, 692–693. Kuo, Y.H., Lin, Y.L., Don, M.J., Chen, R.M., Ueng, Y.F., 2006. Induction of cytochrome P450-dependent monooxygenase by extracts of the medicinal herb Salvia miltiorrhiza. Journal of Pharmacy and Pharmacology 58, 521–527. Lo, A.C., Chan, K., Yeung, J.H., Woo, K.S., 1997. The effects of Danshen (Salvia miltiorrhiza) on pharmacokinetics and pharmacodynamics of warfarin in rats. European Journal of Drug Metabolism and Pharmacokinetics 17, 257–262. Makino, T., Wakushima, H., Okamoto, T., Okukubo, Y., Deguchi, Y., Kano, Y., 2002. Pharmacokinetic interactions between warfarin and kangen-karyu, a Chinese traditional herbal medicine, and their synergistic action. Journal of Ethnopharmacology 82, 35–40. Mirsen, T.R., Hachinski, V.C., 1998. Transient ischemic attacks and stroke. Canadian Medical Association Journal 138, 1099–1105.
Y. Chu et al. / Journal of Ethnopharmacology 137 (2011) 1457–1461 Stein, P.D., Kantrowitz, A., 1989. Antithrombotic therapy in mechanical and biological prosthetic heart valves and saphenous vein bypass grafts. Chest 95, 107s–117s. The State Pharmacopoeia Commission of China, 2005. Chinese Pharmacopoeia, 528. Wu, W.W., Yeung, J.H., 2010. Inhibition of warfarin hydroxylation by major tan-
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shinones of Danshen (Salvia miltiorrhiza) in the rat in vitro and in vivo. Phytomedicine 17, 219–226. Zhu, M., Chan, K.W., Ng, L.S., Chang, Q., Chang, S., Li, R., 1999. Possible influences of Ginseng on the pharmacokinetics and pharmacodynamics of warfarin in rats. The Journal of Pharmacy and Pharmacology 51, 175–180.
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
The effect of Compound Danshen Dripping Pills, a Chinese herb medicine, on the pharmacokinetics and pharmacodynamics of warfarin in rats Yang Chu a , Ling Zhang a,b , Xiang-yang Wang a , Jia-hua Guo a , Zhi-xin Guo a,b , Xiao-hui Ma a,∗ a b
Department of Pharmacology, Tasly R&D Institute, Tianjin Tasly Group Co., Ltd., Pujihe East Road (No. 2), BeiChen District, Tianjin 300410, China Tianjin Key Laboratory of Chinese Medicine Pharmacology, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
a r t i c l e
i n f o
Article history: Received 2 June 2011 Received in revised form 9 August 2011 Accepted 14 August 2011 Available online 29 August 2011 Keywords: Compound Danshen Dripping Pills Warfarin Pharmacokinetics Pharmacodynamics Interaction
a b s t r a c t Aim of the study: Significant pharmacokinetic/pharmacodynamic (PK/PD) interactions between various herbal products and warfarin have recently been reported. The present study was conducted to determine whether Compound Danshen Dripping Pills (CDDP), a Chinese herb medicine used for the treatment of cardiovascular diseases, interacts with warfarin when administered concomitantly. Materials and methods: Each day for 7 days two groups of rats were treated orally with CDDP (50 mg/kg and 250 mg/kg, twice daily), and the control group received similar treatment with appropriate volumes of water only. Sixty minutes after the final daily administration of CDDP or water, an aqueous solution of warfarin (0.2 mg/mL) was given to each rat at a dose of 1.0 mg/kg, and blood samples were collected at 0, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h after warfarin-treatment. The concentration of warfarin in blood plasma was determined by high performance liquid chromatography (HPLC). Prothrombin time (PT) in blood plasma was measured using thromboplastin reagent. Results: Excellent linearity was found between 0.05 and 10 g/mL with a lower limit of quantitation (LLOQ) of 0.05 ng/mL (r > 0.999); moreover, all the validation data including accuracy and precision (intra- and inter-day), were within the required limits. No significant differences were found in PTmax and AUCPT 0–∞ between the two CDDP-treated groups and the control. Besides, there was little alteration in any of the pharmacokinetic parameters of warfarin between the two CDDP-treated groups and the control. Conclusion: The concomitant application of CDDP and warfarin did not give rise to significant effect on the pharmacodynamics of warfarin, and practically no effect on its pharmacokinetics. It was speculated that the PK/PD interactions between CDDP and warfarin was likely to be negligible as long as the patients took CDDP at a normal dose. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Compound Danshen Dripping Pills (CDDP, Chinese name Fufang Danshen Diwan), consisting of Radix salviae miltiorrhizae (Labiatae sp. plant, Chinese name Danshen), Radix notoginseng (Araliaceae plant, Chinese name Sanqi), and Borneolum (Chinese name Bingpian), are an herbal medicine recognized in the official Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2005). The drug is widely used for the prevention and treatment of coronary arteriosclerosis, angina pectoris and hyperlipaemia in China. The annual sales volume of CDDP has exceeded 140 million dollar since 2002. This herbal medicine is also available as a dietary supplement or drug (Prescription/OTC) in countries including USA,
∗ Corresponding author. Tel.: +86 22 26736372; fax: +86 22 26736376. E-mail address: [email protected] (X.-h. Ma). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.08.035
Singapore, Republic of Korea, India, UAE, Russia, Cuba, and South Africa. Warfarin, a coumarin anticoagulant, is widely used in the prophylaxis and treatment of a number of thromboembolic disorders, including transient ischemic attacks (Mirsen and Hachinski, 1998), atrial fibrillation (Albers et al., 1991), and thromboembolic complications that occur with prosthetic heart valves (Stein and Kantrowitz, 1989). However, coumarin anticoagulants, such as warfarin, exhibit high protein binding, cytochrome P450dependent metabolism and a narrow therapeutic window, all of which can contribute to the potential for adverse anticoagulant interactions following co-administration with a wide range of drugs, including cardiovascular and antilipidemic agents (Harder and Thurmann, 1996). These interactions can result in enhancement of the hypothrombinemic effect of warfarin and can lead to bleeding complications and disruption in warfarin metabolism. So far, a number of publications have reported significant PK/PD interactions between herbal products and warfarin (Janetzky and
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Y. Chu et al. / Journal of Ethnopharmacology 137 (2011) 1457–1461
0.35
2
AU
0.30 0.25 0.20 0.15
1 8
0.10 0.05
3 4
0.00 0.0
1.0
2.0
3.0
5
6
4.0
7 5.0
6.0
7.0
8.0
Time (min) Fig. 1. UPLC chromatogram of phenolic acid components in CDDP. (1) TSL; (2) PCA; (3 and 4) two new phenolic acid components; (5) SAD; (6) RMA; (7) SAB; (8) SAA.
Marreale, 1997; Foster et al., 1999; Zhu et al., 1999; Chan, 2001; Makino et al., 2002; Hovhannisyan et al., 2006). Since there are some cases in which patients taking warfarin desire to take CDDP additionally, in order to receive a holistic treatment for cardiovascular diseases, the information about the interactions between CDDP and warfarin are in considerable demand. In this present study, the effects of CDDP on the pharmacokinetics and pharmacodynamics of warfarin have been firstly investigated in rats.
Milford, MA, USA) and the UPLC condition for phenolic acid components were as follows: column, WATERS ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m); mobile phase, ACN/H2 O/AcOH 2:98:0.5 (0 min) → 90:10:0.5 (8 min), linear gradient; flow rate, 0.6 mL/min; column temperature, 40 ◦ C; detectable wavelength: 280 nm; the UPLC condition for triterpene saponin components were as follows: column, WATERS ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m); mobile phase, ACN/H2 O 15:85 (0 min) → 20:80 (7.5 min) → 30:70 (9 min) → 40:60 (14 min); flow rate, 0.5 mL/min; column temperature, 40 ◦ C; detectable wavelength: 203 nm. The UPLC chromatogram of phenolic acid and triterpene saponin components in CDDP was shown in Figs. 1 and 2, respectively.
2. Materials and methods 2.1. Chemicals and reagents
2.3. Animals and experimental design
CDDP (25 mg/pill, lot 100605) was supplied by Tianjin Tasly Pharmaceutical Co., Ltd. Warfarin sodium salt (purity > 98%) was obtained from Wuhan Yuancheng Technology Development Co. Ltd. (Wuhan, China). Diazapam (purity > 99%), which was used as the internal standard (IS), was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile (HPLC grade) was purchased from Fisher Scientific (Pittsburgh, PA, USA), while all other chemicals were analytical grade and used without further purification. The distilled water, prepared from demineralized water, was used throughout the study. All solvents were filtered through 0.45 m filter membranes before injecting into HPLC.
18 male Wistar rats, weighing 220 ± 20 g, were provided by Vital River Lab Animal Technology Co. Ltd (Beijing, China) and housed with a 12 h light/12 h night cycle at ambient temperature (about 23 ± 2 ◦ C) and 50 ± 5% relative humidity, and the air was changed 1–2 times/h. All the rats were kept separately in wire bottom cages (55 cm × 35 cm × 25 cm) with lattice framed steel lids, and woodsawdust was used as bedding. Free access to food and water was allowed at all times except for fasting 12 h before the experiment. All the animal experiment procedures were approved by the Animal Ethics Committee of Tianjin Tasly Institute. 18 study animals were divided into three groups. Group 1 animals, constituting the control group, were treated by oral gavage with water (10 mL/kg twice per day) for 7 days: 60 min after the final administration of water, an aqueous solution of warfarin (containing 0.2 mg/mL) was administered orally to each animal at a dose of 1.0 mg/kg, and blood samples (approximately 0.5 mL) were collected by tail vein at 0, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h after warfarin-treatment. Group 2 and 3 animals, were treated by oral gavage with CDDP (50 mg/kg and 250 mg/kg, twice per day) for 7 days, respectively: 60 min after the final administration of CDDP, an aqueous solution of warfarin (containing 0.2 mg/mL) was administered orally to each animal at a dose of 1.0 mg/kg, and blood samples (approximately 0.5 mL) were collected by tail vein at 0, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h after warfarin-treatment.
2.2. The main constituents in CDDP
AU
In CDDP, the main constituents of Radix Salviae miltiorrhizae are phenolic acids, including tanshinol (TSL), protocatechuic aldehyde (PCA), rosmarinic acid (RMA), salvianolic acids A, B, and D (SAA, SAB, and SAD), and the constituents of Radix Notoginseng are triterpene saponins, including ginsenosides Rb1 , Re, Rg1 , and notoginsenoside R1 ; and the constituent of Borneolum is borneol. 12 pills CDDP were dissolved in 25 mL of methanol under the ultrasonic treatment for 10 min. After vortexing for 1 min, the samples were centrifuged at 2000 rpm for 5 min, and then 10 L of supernatant fluid was injected into UPLC for analysis. Chromatography was performed on an Acquity UPLC system (Waters Corp., 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.0
4
2
1 1.0
2.0
3.0
4.0
5.0
6.0
7.0
3 8.0
9.0
10.0
11.0 12.0
13.0 14.0
Time (min) Fig. 2. UPLC chromatogram of triterpene saponin components in CDDP. (1) Notoginsenoside R1 ; (2) Ginsenoside Rg1 ; (3) Ginsenoside Re; (4) Ginsenoside Rb1 .
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2.4. Preparation of blood samples Each blood sample was separated into two 1.5 mL Eppendorf tubes, which were prepared by heparin and sodium citrate, respectively. Blood samples in heparinized tubes were centrifuged at 1200 × g for 15 min in order to obtain plasma samples for the determination of warfarin. Citrated blood samples (0.03 mL of 109 mmol/L sodium citrate and 0.27 mL of blood) were centrifuged at 1200 × g for 15 min in order to obtain plasma samples for the measurement of prothrombin time. 2.5. Determination of warfarin in rat plasma by HPLC The plasma samples were analyzed for warfarin concentration by the Agilent 1200 series liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA), equipped with a quaternary pump, an autosampler, thermostatically controlled column apartment, and a DAD detector. Sample pretreatment involved in one-step protein precipitation with acetonitrile. 100 L IS solution (5.0 g/mL diazepam, prepared in acetonitrile) and 100 L acetonitrile were added to the 100 L plasma sample. After vortexing for 30 s, the sample was centrifuged at 6500 × g for 10 min and the supernatant was transferred in a vial, 20 L was injected into the HPLC system for analysis. The HPLC conditions were as follows: column, Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm i.d.; particle size 3.5 m) protected by a C18 guard column (Phemomenex, USA); mobile phase, acetonitrile/water/formic acid (50:50:0.2, v/v/v); flow rate, 0.8 mL/min; column temperature, 30 ◦ C; detection wavelength, 305 nm. 2.6. Measurement of prothrombin time Reconstituted thromboplastin reagent (Beijing Steellex Instrument Co. Ltd., Beijing, China) and the citrated plasma sample (0.1 mL) were incubated separately at 37 ◦ C for 2 min prior to mixing. The time taken for a clot to form following introduction of the thromboplastin reagent into the plasma was recorded. 2.7. Statistical analysis Pharmacokinetic parameters of warfarin were calculated using the Topfit 2.0 program by the non-compartmental method. The elimination rate constant (ke ) was obtained as the slope of the linear regression of the log-transformed concentration values versus time date in the terminal phase. The elimination half-life (T1/2 ) was calculated as 0.693/ke . The peak plasma concentration (Cmax ) and the corresponding time (Tmax ) were directly obtained from the raw data. The area under the curve to the last measurable concentration (AUC 0–t ) was calculated by the linear trapezoidal rule. The area under the curve to infinity (AUC 0–∞ ) was calculated as AUC 0–∞ = AUC 0–t + Ct /ke , where Ct is the last measurable concentration. PTmax (s) was maximum prothrombin time and AUC PT0–∞ (s/h) was area under the curve prothrombin time versus time curve extrapolated to infinity. Data are expressed as mean values ± standard error. Statistical analysis was carried out using Student’s unpaired two-tail t-test with significance at the 95% confidence interval. 3. Results and discussion 3.1. Method validation The chromatograms of warfarin in rat were shown in Fig. 3, and no interference was observed at the retention time of the warfarin 6.58 min and diazepam (IS) 7.72 min due to endogenous substances
Fig. 3. Chromatograms of blank plasma (A); blank plasma spiked with warfarin (at the LLOQ, 0.05 g/mL) and diazapam (B) and plasma sample 2 h after oral administration of warfarin (C). Peak I, warfarin; Peak II, diazapam.
in blank rat plasma. The peak area ratios of warfarin to IS were plotted with plasma concentrations to give the correlation coefficient and linear regression equation after weighed linear regression. Excellent linearity was found between 0.05 and 10 g/mL with a lower limit of quantitation (LLOQ) of 0.05 g/mL (r > 0.999). Validation experiments demonstrated that the accuracy was in the range 91.5–109.8%, the precision (intra- and inter-day) was within 12.0%, and the recovery was more than 85%. In conclusion, the high sensitivity and selectivity and relatively short analytical time of the HPLC assay make it suitable for the pharmacokinetic study of warfarin. 3.2. The effect of CDDP on the PK/PD of warfarin The aim of the study was to find possible PK/PD interactions associated with the simultaneous application of CDDP and warfarin. The recommended daily human dose of CDDP is 12.5 mg/kg. In terms of pharmacological activity and toxicity with respect to the rat model, this would translate on an empirical basis to an equivalent daily dose of about 80 mg/kg of CDDP. In the present study, CDDP was applied to the treatment animals at a daily dose of 0.1 and 0.5 g/kg (50 mg/kg and 250 mg/kg, twice daily), respectively, about 8-fold and 40-fold greater than the recommended human
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Control
Concentration (µg/mL)
10.0
CDDP, 0.1 g/kg CDDP, 0.5 g/kg
8.0 6.0 4.0 2.0 0.0 0
15
30
45
60
Time (h) Fig. 4. The plasma concentration versus time profile of warfarin determined after application of a single oral dose (1 mg/kg) of warfarin to control rats and to those that had received daily treatment with CDDP (n = 6).
Control
Prothrombin time (s)
80.0
CDDP, 0.1 g/kg CDDP, 0.5 g/kg
60.0 40.0 20.0 0.0
0
15
30
45
60
Time (h) Fig. 5. The prothrombin time versus time profile of warfarin determined after application of a single oral dose (1 mg/kg) of warfarin to control rats and to those that had received daily treatment with CDDP (n = 6).
dose and about 1.25 and 6.25 times higher than the equivalent dose in rats. Figs. 4 and 5 show the plasma concentration versus time profile of warfarin and the prothrombin time versus time profile of warfarin following a single oral dose (1.0 mg/kg) of warfarin to control rats and CDDP-treated rats, respectively. The main pharmacokinetic and pharmacodynamic parameters of warfarin in each group were summarized in Table 1. It is obtained that there was no significant difference between the PK/PD parameters of warfarin in control group and those of warfarin in group treated with CDDP,
Table 1 Effect of Compound Danshen Dripping Pills (CDDP) on pharmacokinetic and pharmacodynamic (PK/PD) parameters of warfarin (n = 6). Parameters
Control
Cmax (g/mL) Tmax (h) T1/2 (h) ke (1/h) AUC 0–t (g h/mL) AUC 0–∞ (g h/mL) MRT (h) CL (mL/min) Vd (L) PTmax (s) AUC PT0–∞ (s/h)
6.16 3.33 14.8 0.047 106.26 117.38 14.3 0.15 0.19 55.1 1519.9
CDDP-treatment (twice daily) 50 mg/kg ± ± ± ± ± ± ± ± ± ± ±
1.59 2.42 1.79 0.007 21.05 25.79 1.62 0.04 0.03 8.7 239.3
6.76 3.17 14.8 0.047 110.65 122.48 14.7 0.14 0.18 55.1 1572.2
± ± ± ± ± ± ± ± ± ± ±
250 mg/kg 2.93 2.84 1.59 0.006 12.19 12.98 0.98 0.01 0.03 9.3 192.6
5.41 4.42 13.7 0.051 91.96 99.63 14.3 0.17 0.20 53.2 1487.9
± ± ± ± ± ± ± ± ± ± ±
1.03 3.07 1.44 0.006 12.79 15.20 1.43 0.02 0.03 6.3 128.6
Cmax and Tmax , the peak plasma concentration and the corresponding time; T1/2 , elimination half-life; ke , elimination rate constant; AUC 0–t , the area under the curve to the last measurable concentration; AUC 0–∞ , the area under the curve to infinity; MRT, mean residence time; CL, clearance; Vd , volume of distribution; PTmax , maximum prothrombin time; AUC PT0–∞ , the area under the curve prothrombin time versus time curve extrapolated to infinity.
following statistical analysis by Student’s unpaired two-tail t-test (P > 0.05). Warfarin is a widely used oral anticoagulant that requires a tight control on dosage. This drug is easily metabolized in the organism by CYP450 (mainly hydroxylation). The PK/PD interactions between herbal products and warfarin are of particular concern when perturbations to absorption, metabolism, or protein binding are suspected. Wu and Yeung (2010) and Kuo et al. (2006) reported aqueous extract of S. miltiorrhiza, the main components of CDDP, had no effect on warfarin being metabolized by CYP450 to form various hydroxylation products. Lo et al. (1997) and Chan et al. (1995) studied the PK/PD interactions of warfarin and S. miltiorrhiza extract in rats. Three days pretreatment with the extract (5.0 g/kg per day i.p.) in rat increased the absorption rate, Cmax , AUC, T1/2 , and decreased the Vd and CL of a single oral treatment of warfarin. Besides, S. miltiorrhiza extract also prolonged the prothrombin time further than its extension by warfarin. Considering that they applied intraperitoneal injection of S. miltiorrhiza extract, the dosage of CDDP in our study was quite smaller than that used in their experiments, and concomitant treatment with warfarin and CDDP did not give rise to significant effects on the pharmacokinetics and pharmacodynamic of warfarin. Furthermore, the dosage of CDDP-treated groups in the present study, which was 8 times and 40 times the amount of the human daily dose, did not exhibit pharmacokinetic or pharmacodynamic interactions. It was speculated that the PK/PD interactions between CDDP and warfarin was likely to be negligible as long as the patients took CDDP at a normal dose. However, the results of the present study were obtained by single treatment with warfarin, so we could not exclude the possibility that warfarin might be accumulated and prothrombin time might be increased if patients who were taking daily warfarin also took CDDP. Currently, the research in this area is dealt with in our laboratory. Acknowledgement This study was partly supported by National Key Special Project of Science and Technology for Innovation Drugs of China (grant no. 2008ZX09401-006). References Albers, G.W., Atwood, J.E., Hirsh, J., Sherman, D.G., Hughes, R.A., Connolly, S.J., 1991. Stroke prevention in nonvalvular atrial fibrillation. Annals of Internal Medicine 115, 727–736. Chan, K., Lo, A.C., Yeung, J.H., Woo, K.S., 1995. The effects of Danshen (Salvia miltiorrhiza) on warfarin pharmacodynamics and pharmacokinetics of warfarin enantiomers in rats. The Journal of Pharmacy and Pharmacology 47, 402–406. Chan, T.Y., 2001. Interaction between warfarin and Danshen (Salvia miltiorrhiza). The Annals of Pharmacotherapy 35, 501–504. Foster, B.C., Vandenhoek, S., Hanna, J., Akhtar, M.H., Krantis, A., 1999. Effect of natural health products on cytochrome P-450 drug metabolism. The Canadian Journal of Infectious Diseases 10B, 150. Harder, S., Thurmann, P., 1996. Clinically important drug interactions with anticoagulants: an update. Clinical Pharmacokinetics 30, 416–444. Hovhannisyan, A.S., Abrahamyan, H., Gabrielyan, E.S., Panossian, A.G., 2006. The effect of Kan Jang extract on the pharmacokinetics and pharmacodynamics of warfarin in rats. Phytomedicine 13, 318–323. Janetzky, K., Marreale, A.P., 1997. Probable interaction between warfarin and Ginseng. American Journal of Health-System Pharmacy 54, 692–693. Kuo, Y.H., Lin, Y.L., Don, M.J., Chen, R.M., Ueng, Y.F., 2006. Induction of cytochrome P450-dependent monooxygenase by extracts of the medicinal herb Salvia miltiorrhiza. Journal of Pharmacy and Pharmacology 58, 521–527. Lo, A.C., Chan, K., Yeung, J.H., Woo, K.S., 1997. The effects of Danshen (Salvia miltiorrhiza) on pharmacokinetics and pharmacodynamics of warfarin in rats. European Journal of Drug Metabolism and Pharmacokinetics 17, 257–262. Makino, T., Wakushima, H., Okamoto, T., Okukubo, Y., Deguchi, Y., Kano, Y., 2002. Pharmacokinetic interactions between warfarin and kangen-karyu, a Chinese traditional herbal medicine, and their synergistic action. Journal of Ethnopharmacology 82, 35–40. Mirsen, T.R., Hachinski, V.C., 1998. Transient ischemic attacks and stroke. Canadian Medical Association Journal 138, 1099–1105.
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