Curcumin Alters the Pharmacokinetics of Warfarin and Clopidogrel in Wistar Rats but Has No Effect on Anticoagulation or Antiplatelet Aggregation

• Introduction
• Materials and Methods
• Chemicals
• Animals
• Pharmacokinetic interactions
• Liquid chromatography tandem mass spectrometry (LC-MS/MS)
• Warfarin analysis
• Clopidogrel analysis
• Pharmacodynamic interactions
• Data analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

This study examined the effects of curcumin on the pharmacokinetic and pharmacodynamic properties of warfarin and clopidogrel in Wistar rats. Results showed that oral administration of curcumin at 25 mg/kg, 50 mg/kg, and 100 mg/kg for 7 days had no substantial effects on the pharmacodynamics of warfarin and clopidogrel in this animal model. However, oral administration of 100 mg/kg curcumin for 7 days significantly increased the AUC_0-∞ and C_max of the two drugs (by × 1.6 and × 1.5, respectively, for warfarin, and × 1.61 and × 1.81, respectively, for clopidogrel carboxylic acid). However, compared to warfarin alone, different doses of curcumin combined with warfarin had no effects on the prothrombin time in rats. Similarly, a combination of curcumin and clopidogrel had no significant effect on the maximum platelet aggregation rate of rats compared with the use of clopidogrel alone. This work demonstrated that preadministration of 100 mg/kg curcumin affected the pharmacokinetics of warfarin and clopidogrel but had no effect on pharmacodynamic parameters such as anticoagulation rate and antiplatelet aggregation.

Introduction

Botanicals found in Chinese herbal medicines have been widely used as drugs or healthcare products, and many botanical substances have been proven beneficial to health. It has also been reported that approximately 14–31 % of patients taking prescription drugs simultaneously use botanical products [1], [2], [3], [4], [5], [6]. Although botanicals are generally thought to be safe, recent reports of herb-drug interactions between botanicals and other medicines have raised safety concerns about their use in combination. These interactions mainly occur in patients with chronic diseases such as cardiovascular and cerebrovascular disorders or where the drug has a narrow therapeutic window.

Curcumin ([Fig. 1 A]) is the primary active component of tubers of Curcuma longa Linn, a member of the ginger family (Zingiberaceae). Curcumin is also the principle component of turmeric powder, ginger, and curry, which have widely been used as spices or food additives throughout the world. The major biological effects of curcumin have been shown to be antioxidative, anti-inflammatory, and antitumor activities [7], [8], [9]. Phase I and phase II clinical trials of curcumin have been carried out in the UK [10], Taiwan [11], and US [12]. A large number of in vitro and in vivo studies have shown that simultaneous use of curcumin and certain drugs may cause herb-drug interactions. For example, curcumin can significantly improve AUC and C_max of loratadine and etoposide, which are both substrates of CYP3A4 and P-glycoprotein (P-gp). This improvement might be mainly attributed to curcumin inhibiting intestinal CYP3A4 and P-gp expression [13], [14]. Our previous study also showed that preadministration of 100 mg/kg curcumin improved the plasma concentrations of the antihypertensive drug losartan, as well as its active metabolite EXP3174 in rats [15].

Warfarin ([Fig. 1 B]) is an important oral anticoagulant for antithrombotic treatment, which inhibits the liver vitamin K epoxide reductase (VKOR) to reduce clotting factors, further preventing thrombus formation [16]. Warfarin interacts with many drugs [17], [18] and exhibits a narrow therapeutic window. Consequently, drug interactions often cause fatal bleeding or thrombosis episodes in patients taking warfarin therapy.

The antiplatelet drug clopidogrel ([Fig. 1 C]) belongs to the thienopyridine class of antiplatelet drugs and has been shown to reduce the incidence of major ischaemic cardiovascular events. It has no inherent antiplatelet aggregation activity but generates metabolites in vivo via two pathways, including the carboxylation pathway for generating non-active metabolite(s) and a cytochrome P450 (CYP)-mediated pathway for generating active metabolite(s) [19], [20]. Only after conversion to active metabolite via the CYP-mediated pathway does clopidogrel irreversibly inhibit the antiplatelet activity of the P2Y12 adenosine diphosphate receptor. Consequently, influence on the activity of CYP-subtypes involved in clopidogrel metabolism will alter the production of the active metabolite, further affecting the pharmacodynamics of clopidogrel, making it prone to metabolic interactions with other drugs [21], [22]. The occurrence of herb-drug interactions may therefore affect the antiplatelet activity of clopidogrel, which is potentially dangerous to patients taking this medication.

Curcumin is commonly found in many diets worldwide and in recent years has been shown to have a protective effect on ischemic cardiovascular and cerebrovascular diseases [23], [24], [25] while also being considered as a potential drug for treatment of cardiac-cerebral vascular diseases. While curcumin can potentially be used in combination with warfarin or clopidogrel for treatment of relevant diseases, it has been shown that it can inhibit drug-metabolizing enzymes such as CYP2C9 and CYP3A4 [26]. Several in vivo tests also confirmed that curcumin inhibits activity, protein expression, and mRNA levels of P-gp [27], [28]. In vivo oxidation of warfarin is mediated by CYP1A2, CYP2C9, and CYP3A4 [29], whereas clopidogrel is metabolized to the active metabolite by CYP2C19 and CYP3A4 before inhibition of platelet aggregation begins [19], [20]. In addition, clopidogrel and warfarin are both P-gp substrates [29], [30]. Theoretically, the combination of curcumin and warfarin or clopidogrel may lead to herb-drug interactions. However, due to the complex in vivo environment of organisms, the drugs are influenced by multiple factors after being absorbed into the body. Whether in vitro experimental data are applicable to the whole-animal level needs to be validated by relevant animal experiments.

Materials and Methods

Chemicals

Curcumin was purchased from Sigma (95 %, Fluka); warfarin tablets and warfarin reference material were purchased from Qilu Pharmaceutical Co., Ltd.; clopidogrel tablets were supplied by Sanofi-Aventis Minsheng Pharmaceutical Co., Ltd; clopidogrel carboxylic acid was obtained from Toronto Research Chemicals, Inc.; loratadine and genistein were purchased from the National Institutes for Food and Drug Control (Beijing, China); methanol and formic acid were purchased from Tedia Company, Inc.; deionized water was prepared using a Millipore Milli-Q gradient water purification system.

Animals

Six- to eight-week-old male Wistar rats (180–220 g) were purchased from Lukang Pharmaceutical Co., Ltd. The animal laboratory temperature was maintained at 25 ± 2 °C, with a humidity of 40–70 %. The rats were provided with standard diet and free drinking water, adapted to the environment 7 days prior to the experiment and submitted to fasting with free access to water 12 h prior to the experiment. The animal experiments complied with the requirements of the National Act on the Use of Experimental Animals of the Peopleʼs Republic of China (approval of the Ethics Committee of Qilu Hospital of the Shandong University for animal experiments, No. 2011003).

Pharmacokinetic interactions

Curcumin and warfarin: Twenty-four male rats were randomly divided into 4 groups (n = 6 for each group). Oral administrations of curcumin suspension (25 mg/kg, 50 mg/kg, and 100 mg/kg; suspended in 0.5 % CMC-Na) and the same volume of 0.5 % CMC-Na were performed for 7 continuous days. Fasting was carried out with free access to water on day 6, and intragastric administration of 0.2 mg/kg warfarin (suspended in 0.5 % CMC-Na) was conducted 30 min after curcumin on day 7. Then, 0.3 mL of blood obtained from the subclavian vein was collected at 0 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h. The samples were injected into centrifuge tubes with heparin and centrifuged at 4000 rpm for 15 min. The plasma was collected and frozen at − 80 °C prior to analysis.

Curcumin and clopidogrel: Rat grouping and dosing of curcumin/clopidogrel were the same as described for the curcumin/warfarin test. Fasting was performed on the evening of day 6 with free access to water. Oral administration of 30 mg/kg clopidogrel (suspended in 0.5 % CMC-Na) was carried out 30 min after curcumin on day 7. Approx. 0.3 mL of blood was collected at 5, 10, 20, 40, 60, 120, 240, 480, 720, 1440, and 2880 min, and centrifuged to obtain plasma. All samples were frozen at − 80 °C before analysis.

Liquid chromatography tandem mass spectrometry (LC-MS/MS)

LC-MS/MS was performed using an Agilent 1200 series LC system with an Agilent G6410 B triple quadrupole mass spectrometer, which was equipped with electrospray ionization (ESI) operated by Agilent Mass Hunter Workstation B.01.03. The chromatographic column was a ProntoSil C18 column (150 mm × 3 mm, 3 µm) (Bischoff), with a C18 guard column (4.0 mm × 3.0 mm i. d.) (Phenomenex).

Warfarin analysis

For warfarin analysis, 25 µL plasma was transferred to a 1.5-mL centrifuge tube, and 25 µL of internal standard solution (genistein, 2 µg/mL) was added. The mixture was vortexed for 10 s and then 75 µL acetonitrile was added. This was then vortexed for another 1 min and centrifuged at 11 000 r/min for 5 min. An aliquot of the supernatant (20 µL) was analyzed using LC-MS/MS.

The mobile phase consisted of 0.1 % formic acid-methanol: 0.1 % formic acid (60 : 40, v : v). Flow rates were 0.4 mL/min (0–1.0 min) and 0.6 mL/min (1.1–2.5 min).

The ESI(+) was selected as an ionization source, and the detection was conducted using a multiple reaction mode (MRM). The selected detection ion to warfarin was 309.2 → 163.1, and genistein (IS) was 271.1 → 215.1. High-purity N₂ was used as the carrier gas (325 °C and 10 L/min). Other parameters were as follows: spray gas pressure 350 Pa; nebulizer 35 psi; capillary 4000 (−) and 4000 (+); dwell time 200 ms; fragment electric voltage, 110 V for warfarin and 155 V for genistein; collision energy, 10 eV for warfarin and 25 eV for genistein.

Clopidogrel analysis

Due to the difficulty in quantification of clopidogrel and its metabolites in blood, this study determined the concentration of clopidogrel–clopidogrel carboxylic acid (an inactive metabolite of clopidogrel) to reflect pharmacokinetic processes of clopidogrel in rat.

Twenty-five microliters of internal standard solution (loratadine, 1000 ng/mL) was added to 25 µL of blank plasma, followed by 25 µL of standard serial solutions of clopidogrel carboxylic acid. To this 75 µL of methanol was added for precipitation of protein, the mixture vortexed for 1 min and then centrifuged at 11 000 rpm for 5 min. An aliquot of the supernatant (20 µL) was then analyzed.

The mobile phase consisted of 0.1 % formic acid-methanol: 0.1 % formic acid (95 : 5, v : v), at a flow rate of 0.6 mL/min.

The settings of MS detector were the same as those used for warfarin analysis. The selected detection ion to clopidogrel was 308.1 → 152.0, and loratadine was 383.2 → 337.2. The fragment electric voltage was 100 V for clopidogrel and 130 V for loratadine. The collision energy was 10 eV for clopidogrel and 25 eV for loratadine.

Pharmacodynamic interactions

Curcumin and warfarin: The rats were randomly divided into 8 groups (n = 6) as follows, the blank control group, three curcumin-only groups (dosed at 25 mg/kg, 50 mg/kg, and 100 mg/kg), the warfarin-only group (0.2 mg/kg), and three combined curcumin-warfarin groups (curcumin at 25 mg/kg, 50 mg/kg, and 100 mg/kg, each combined with warfarin at 0.2 mg/kg). The rats were subjected to daily oral administration of the respective regimes for 7 days continuously; the blank control group received an equal volume of solvent. Two hours after the last dose, 1.8 mL of blood was sampled from the rat jugular sinus and placed in a tube containing anticoagulant (sodium citrate, 0.109 mol/L). The samples were centrifuged at 3500 rpm for 10 min, and the plasma was collected for analyses of prothrombin time (PT), activated partial thromboplastin time (APPT), thrombin time (TT), and fibrinogen levels (FIB) using an ACL-8000 fully-automated coagulation/fibrinolysis analyzer (Backman Coulter).

Curcumin and clopidogrel: Rats were randomly divided into the same groupings described above, warfarin being substituted by clopidogrel (7 mg/kg). The rats were subjected to daily oral administration of the respective regimes for 7 days continuously; the blank control group received an equal volume of the solvent. Two hours after the last dose, 1.8 mL of blood was added to a tube containing anticoagulant (sodium citrate, 0.109 mol/L). The mixture was centrifuged at 1000 rpm for 10 min, and the upper platelet-rich plasma (PRP) was collected. The PRP was centrifuged at 3000 rpm for 30 min to collect the upper platelet-poor plasma (PPP). The PPP was analyzed for platelet aggregation within 5 min stimulated by 20 µM ADP using a PACK-4 eight-channel platelet aggregation analyzer. Maximum aggregation rate was calculated as follows:

Maximum aggregation rate = (post-aggregation PRP light transmittance – pre-aggregation PRP light transmittance)/(PPP light transmittance – pre-aggregation PRP light transmittance) × 100 %.

Data analysis

Data are presented as the mean values ± standard deviation (SD). Pharmacokinetic parameters were calculated using the non-compartmental model with DAS 2.0, and PT, activated partial thromboplastin time (APPT), thrombin time (TT), and fibrinogen levels (FIB), as well as the maximum platelet aggregation rate (MAPR) directly obtained from the readings of the instrument. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by comparison of different test groups using the Tukey test. P < 0.05 was considered a statistically significant difference.

Results

The drug concentration of warfarin or clopidogrel was determined using a highly sensitive and specific LC-MS/MS method. The retention times of warfarin and genistein were 1.8 and 1.1 min, respectively, whereas those of clopidogrel carboxylic acid and loratadine were 2.7 and 3.1 min, respectively. The mean recoveries of warfarin and clopidogrel were both between 94 to 107 %, and the intraday and interday precision were both less than 10 %. Besides, warfarin and clopidogrel in analyzed samples were both stable for 24 h at room temperature, 30 days at − 20 °C and three freeze-thaw cycles. All validation experiments of these methods met the requirements of the Guidance for Industry Bioanalytical Method Validation Document of the American Food and Drug Administration (FDA). For warfarin, the calibration curve was linear over the concentration range of 1–30 000 ng/mL, and the regression equation was Y = 0.0316X + 0.0041 with the mean correlation coefficient of 0.9938. The concentration range for clopidogrel carboxylic acid was 20–50 000 ng/mL, and the regression equation Y = 0.0001X + 0.00001 with the mean correlation coefficient of 0.9964.

The mean concentration-time curve of warfarin combined with or without different doses of curcumin is shown in [Fig. 2], and associated pharmacokinetic parameters are shown in [Table 1]. Compared with the warfarin-only group, the group pre-administered with 100 mg/kg curcumin had AUC_0-∞ and C_max that were × 1.6 and × 1.5 higher, respectively, and the plasma clearance (CL) decreased by 57.14 % (p < 0.05). There were no significant differences in relevant parameters of the 25 or 50 mg/kg curcumin group and control group. These results indicate that preadministration of 100 mg/kg curcumin substantially changed the pharmacokinetics of warfarin in rats, leading to the elevation of the warfarin plasma concentration and C_max, and the simultaneous reduction of plasma clearance.

The mean concentration-time curve of clopidogrel combined with or without different doses of curcumin is shown in [Fig. 3], and associated pharmacokinetic parameters are shown in [Table 2]. Compared with the clopidogrel group, the preadministered 100 mg/kg curcumin group showed AUC_0-∞ and C_max for clopidogrel carboxylic acid × 1.61 and × 1.81 higher, respectively, and CL decreased by 58.33 % (p < 0.05). There were no substantial differences in relevant parameters between the 25 or 50 mg/kg curcumin group and control group. Results from the pharmacokinetic experiment showed that preadministration of 100 mg/kg curcumin significantly influenced the pharmacokinetics of clopidogrel in rats, leading to the elevation of the clopidogrel carboxylic acid plasma concentration and C_max and the reduction of CL.

The PT, APTT, TT, and FIB were determined for rats subjected to oral blank solvent and curcumin (25, 50, and 100 mg/kg) for 7 days. Results showed that oral administration of 25, 50, and 100 mg/kg curcumin had no substantial effects on coagulation in rats. There were no significant differences in relevant parameters between the dosed and blank groups ([Fig. 4]).

The PT was determined for rats subjected to oral administration of 0.2 mg/kg warfarin combined with 7-day oral blank solvent (control) or curcumin (25, 50, and 100 mg/kg). As compared with the use of warfarin alone, the combined use of 25, 50, or 100 mg/kg curcumin and warfarin had no substantial effects on coagulation in rats ([Fig. 5]).

The ADP-induced maximum platelet aggregation rate was determined for rats subjected to 7-day oral blank solvent (control) and curcumin (25, 50, and 100 mg/kg). As shown in [Table 3], the 7-day oral administration of 25, 50, or 100 mg/kg curcumin had no substantial effect on ADP-induced platelet aggregation in rats. There were no significant differences in relevant parameters between the blank control and dosed groups.

The ADP-induced maximum platelet aggregation rate was determined for rats subjected to oral administration of 7 mg/kg clopidogrel combined with or without 7 days curcumin (25, 50, and 100 mg/kg). Compared to clopidogrel alone, the 7-day oral administration of 25, 50, or 100 mg/kg curcumin combined with clopidogrel had no substantial effect on ADP-induced platelet aggregation in rats ([Table 3]).

Discussion

In this study, small (25 mg/kg) and medium doses of curcumin (50 mg/kg) had no substantial inhibition effect on pharmacokinetics of warfarin in rats. However, AUC and C_max of rats in the large dose curcumin group (100 mg/kg) were significantly higher, suggesting that relevant pharmacokinetics of warfarin were affected. The pharmacokinetics of drugs includes four distinct stages, namely, absorption, distribution, metabolism, and excretion, and any effects on these will alter the way in which the drug acts upon the body. According to the pharmacokinetic measurements, the rats in the 100 mg/kg curcumin preadministration group had no substantial changes in T_1/2 when compared with the control group. We propose that curcumin and warfarin interactions occurred mainly because curcumin inhibited P-gp, further improving the adsorption of warfarin. This was confirmed in [Fig. 2 C], which showed that the warfarin plasma concentration of the preadministration group was significantly improved compared with that of the control group.

Although the concentration of warfarin was substantially increased in the 100 mg/kg curcumin preadministration group compared with the control group, such variations were not observed in the pharmacodynamic test. In the anticoagulation test, coagulation parameters showed no significant differences between the control and preadministration groups. This was probably due to the AUC of warfarin only being increased by × 1.6 compared to the control group. It has been reported that in the presence of an inhibitor, only × 2 or greater changes in AUC can cause substantial pharmacodynamic changes. Otherwise, variations caused by individual differences [31] may explain this finding. As a result, the inhibition effect of curcumin on warfarin did not reach the degree required to change the anticoagulant properties of warfarin in the present study.

There are two metabolic pathways for clopidogrel in vivo: 85 % is metabolized by liver carboxylesterase to form the inactive metabolite clopidogrel carboxylic acid, and 15 % is metabolized by CYP2C19 and CYP3A4 to form the active metabolite capable of platelet aggregation [19], [20]. After oral administration of clopidogrel, approximately 50 % of the dose can be absorbed by the gastrointestinal tract. Its low bioavailability is a consequence of clopidogrel being a substrate of P-gp [30]. Theoretically, P-gp inhibitors can therefore improve the absorption of clopidogrel, increasing its bioavailability.

Owing to the low concentrations of clopidogrel and its active metabolite in blood, many pharmacokinetic experiments primarily use the non-active metabolite – clopidogrel carboxylic acid – to reflect the pharmacokinetic properties of clopidogrel [32]. In pharmacodynamic tests, to use the ADP-induced platelet aggregation rate in rats as an indicator has been recognized as the gold standard for evaluation of clopidogrel efficacy [33].

In the small (25 mg/kg) and medium dose (50 mg/kg) curcumin groups, curcumin had no significant effects on pharmacokinetic processes of clopidogrel in rats. However, in the large dose curcumin group, AUC and C_max values of clopidogrel carboxylic acid were substantially improved, suggesting changes in a relevant intracorporal process of clopidogrel. Inhibition of P-gp or other relevant CYP subtypes involved in the metabolization process can change the pharmacokinetics of clopidogrel.

Although clopidogrel concentrations were changed in the 100 mg/kg curcumin preadministration group compared with the control group, such variations were not shown in the pharmacodynamic test. In the ADP-induced platelet aggregation test, no significant changes were observed in the maximum aggregation rate between the control and the preadministration groups.

The contradiction between pharmacokinetic and pharmacodynamic data could be attributed to the metabolic pathways of clopidogrel. Clopidogrel has two metabolic pathways, of which carboxylation is the major biotransformation pathway that results in production of inactive metabolites. The CYP-mediated metabolism accounts for approximately 15 % of the total clopidogrel metabolism. Hence, the effect of curcumin is expected to be negligible, which has been confirmed by the pharmacokinetic parameters T_1/2 observed in the present study. Our results showed that T_1/2 had no significant changes, suggesting that curcumin mainly affects the pharmacokinetics of clopidogrel via its absorption. In addition, the increased concentration of clopidogrel carboxylic acid reflected the change in plasma concentration of clopidogrel in vivo. This change was of significance to the carboxylation pathway that accounts for 85 % of clopidogrel metabolism but was negligible to P450-induced oxidation reaction. This probably explains the lack of substantial increases in the production of active metabolites needed to induce platelet aggregation.

In summary, this study found that curcumin slightly changed the pharmacokinetic parameters of warfarin and clopidogrel but did not affect their pharmacodynamic properties in Wistar rats. These indicated that curcumin is safe when used in combination with warfarin and clopidogrel. However, at levels of 100 mg/kg, curcumin was found to alter the concentrations of both warfarin and clopidogrel in rats in the present study. Although no pharmacodynamic interaction between curcumin and warfarin or clopidogrel was observed in the present study, the clinical significance of the present results is unclear. Therefore, the possibility of an increased therapeutic effect of the two drugs, such as unexpected bleeding and hemorrhage, should be considered when prescribing a combination therapy. We propose therefore that when warfarin or clopidogrel is used in combination with high doses of curcumin in patients with cardiovascular and cerebrovascular diseases (particularly elderly patients), the variations in international normalized ratio (INR) or maximum platelet aggregation rate of the patients should be monitored to avoid treatment failure and enhanced drug toxicity.

Acknowledgements

This work was supported by the Natural Science Foundation of China (No. 31101241) and the Independent Innovation Foundation of Shandong University (IIFSDU, Grant No. 2012TS163).

Conflict of Interest

The authors declare that they have no conflict of interest.

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• 28 Zhang W, Tan TM, Lim LY. Impact of curcumin-induced changes in P-glycoprotein and CYP3A expression on the pharmacokinetics of peroral celiprolol and midazolam in rats. Drug Metab Dispos 2007; 35: 110-115
  CrossrefPubMedSearch in Google Scholar
• 29 Wadelius M, Sorlin K, Wallerman O, Karlsson J, Yue QY, Magnusson PK, Wadelius C, Melhus H. Warfarin sensitivity related to CYP2C9, CYP3A5, ABCB1 (MDR1) and other factors. Pharmacogenomics J 2004; 4: 40-48
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• 30 Taubert D, von Beckerath N, Grimberg G, Lazar A, Jung N, Goeser T, Kastrati A, Schomig A, Schomig E. Impact of P-glycoprotein on clopidogrel absorption. Clin Pharmacol Ther 2006; 80: 486-501
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• 31 Williams JA, Hyland R, Jones BC, Smith DA, Hurst S, Goosen TC, Peterkin V, Koup JR, Ball SE. Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab Dispos 2004; 32: 1201-1208
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• 32 Mullangi R, Srinivas NR. Clopidogrel: review of bioanalytical methods, pharmacokinetics/pharmacodynamics, and update on recent trends in drug-drug interaction studies. Biomed Chromatogr 2009; 23: 26-41
  CrossrefPubMedSearch in Google Scholar
• 33 Nguyen TA, Diodati JG, Pharand C. Resistance to clopidogrel: a review of the evidence. J Am Coll Cardiol 2005; 45: 1157-1164
  CrossrefPubMedSearch in Google Scholar

Correspondence

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