Effects of co-administraton of neferine and doxorubicin on the pharmacokinetics of doxorubicin             
Qingdan Xue, Aixia Ju, Yuhong Kang, Chunyu Zheng, Qiuhong Li*
Heilongjiang University of Chinese Medicine, Harbin 150040, China 


Abstract: Doxorubicin is one of the anthracycline anti-neoplastic drugs widely used to treat leukemia, liver, breast, and ovarian cancers and other solid tumors. However, its clinical applications have been limited by its serious cardio-cytotoxic effects. The aim of this study was to investigate the effect of neferine, a bisbenzylisoquinoline alkaloid extracted from the green embryo in the mature lotus seed, on the pharmacokinetics of doxorubicin. The levels of doxorubicin in plasma and tissues were measured using the high performance liquid chromatography (HPLC) method. The chromatographic separation was completed on a reversed-phase C18 column using acetonitrilephosphate buffer (30:70, v/v) as the mobile phase at a flow rate of 1 mL/min and ultraviolet detectionwave length was set at 233 nm. The pharmacokinetic study found that the co-administration of neferine and doxorubicin significantly affected the pharmacokinetics of doxorubicin. There were evident changes in area under the curve (AUC), clearance (CL) and t1/2β in group of pretreatment neferine as compared with those in group treated with doxorubicin alone. Tissue distribution analysis showed that the concentrations of doxorubicin distributed to heart, liver and kidney were statistically significant higher in group of pretreatment with neferine plus doxorubicin than those in the doxorubicin alone-group at 0.5 h and 2 h after drug administration, respectively.While the doxorubicin concentrations in spleen and lung drug were slightly increased in the group of pretreatment with neferine plus doxorubicin as compared to that of group of doxorubicin alone, the difference between two groups were not statistically significant. Therefore, the dose of doxorubicin needs to be taken into consideration when it is administrated in combination with neferine.                            
Keywords: Neferine, Doxorubicin, Pharmacokinetics, HPLC method, Anthracycline anti-neoplastic drugs
CLC number: R969.1                Document code: A                 Article ID: 10031057(2015)422506
 
 
1. Introduction
Doxorubicin is one of the anthracycline anti-neoplastic drugs and can inhibit the synthesis of RNA and DNA, especially RNA. It belongs to a class of non-specific drugs with a broad-spectrum of anticancer activities and is widely used for the treatment of leukemia, liver, breast, ovarian cancers and other solid cancers. It is one of the most potent chemotherapeutic agents[1].However, like other anti-neoplastic agents, doxorubicin has a large number of side effects, including bone marrow suppression, alopecia, and gastrointestinal reactions etc. These symptoms gradually disappear after drug withdrawal, and generally do not affect the clinical use of drugs and anti-neoplastic function. However, due to its severe cardiatoxicity, administration of doxorubicin often causes cardiac dysfunction. The common therapeutic method does not work for doxorubicin-induced cardiomyopathy and heart failure[2,3]. Thereby, the advantages of powerful anti-cancer activity of doxorubicin and broad spectrum of anti-cancer effects are limited by its cardiotoxicity. On the other hand, the process of cancer chemotherapy may lead to multidrug resistance and reduction in the efficacy of doxorubicin[4], the two key factors which limit the application of doxorubicin. Therefore, it is particularly important to seek methods for reversing multidrug resistance and for the protection of doxorubicin-induced myocardial injury. A new study suggested that doxorubicin-induced myocardial injury was mainly related to free radical damage, calcium overload, apoptosis, and mitochondrial damage etc[5]. Based on the above reasons, co-administration of doxorubicin in combination with other drug such as vitamin E[6], coenzyme Q10[7], erythropoietin[8] and captopril[9] has been reported to protect against doxorubicin-induced myocardial injury. 
Neferine is a bisbenzylisoquinoline alkaloid extracted from the green embryo in the mature seed of lotus of the Nymphaeaceae. Numerous studies have shown that neferine has a lot of pharmacological actions including chemo-sensitization[10,11], antioxidant[12], antiarrhythmia[13] and the demonstrated activity in the treatment of arrhythmia[13] and platelet aggregation[14]. Many studies have shown that neferine improved the chemotherapic effect and reduced the poisonous side effects of other drugs. Several studies found that neferine, which itself has no significant cytotoxicity, can enhance the cytotoxic effects of doxorubicin on human breast cancer cells[15], and improved the inhibitory effects of doxorubicin on proliferation of human acute myelogenous leukemia cells[16] and human osteosarcoma cells[17] suggesting that neferine has a chemosensitization effect.
Doxorubicin is mainly metabolized in the liver. The principal metabolism pathways of doxorubicin is catalyzedby the cytosolic NADPH-dependent aldehyde-keto reductase, which metabolizes doxorubicinol, the secondary alcohol metabolite, and by the NADPH-dependent cytochrome P450 reductase to a sequence of hydroxylatedor deoxy aglycone[18]. Previous studies have shown that doxorubicin is metabolized by cytochrome P450 isoenzymes CYP3A4 and CYP2D6[19]. Neferine metabolism is mediated by cytochrome P450 isoenzymes CYP3A4and CYP2D6[20], and neferine is metabolized by CYP2D6 to liensinine[21]. However, whether neferine affects the pharmacokinetics and tissue distribution of doxorubicin is not clear and remains to be investigated.
2. Materials and methods
2.1. Chemicals and reagents
Both doxorubicin hydrochloride and daunomycin hydrochloride (approximately 98% pure, HPLC grade) were purchased from Nanjing Zelang Medical Technology Co., Ltd (Nanjing, Jiangsu, China). Doxorubicin hydrochloride injection was purchased from Pfizer (New York, USA). Neferine (purity>95%) was offered by the Department of Pharmacology’s Laboratory, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, Hubei, China). Phosphoric acid was obtained from Tianli Chemical Reagent Co., Ltd (Tianjin, China). Ammonium dihydrogen phosphate was purchased from Bodi Chemical Co., Ltd (Tianjin, China). All other chemicals were of analytical grade and all solvents used were of HPLC grade.
2.2. Study design and drug administration
Sprague-Dawley (SD) rats (260300 g) and Kunming (KM) mice (18–20 g) were purchased from the Laboratory Animal Center of Heilongjiang University of Chinese Medicine (Harbin, Heilongjiang, China). The rats and mice were maintained under a 12 h light/12 h dark cycle at temperature of 21–27 ºC and relative humidity of 40%–70%. All animal-related studies were conducted with the approval of the Animal Care and Use Committee of the Heilongjiang University of Chinese Medicine.
To explore the effect of neferine on the pharmacokinetics of doxorubicin, rats were randomly divided into two groups with six animals in each group and treated as the follows: group 1 was given doxorubicin via a tail vein at a dose level of 4 mg/kg body weight (bw); group 2 was administered doxorubicin via a tail vein at a dose level of 4 mg/kg bw and administered 10 mg/kg of neferine via an intragastric administration. Blood samples were obtained at 0, 5, 15, 30, 45 min and at 1, 2, 4, 6, 8, 12 and 24 h after drug administration for analysis of doxorubicin. The blood samples were centrifuged at 3500 r/min for 5 min to obtain the plasma.
To investigate the effect of neferine on the tissue distribution of doxorubicin, mice were randomly divided into two groups with thirty animals in each group and treated as the follows: group 1 was administered with doxorubicin at 4 mg/kg via a tail vein of mice; group 2 was administered with doxorubicin at 4 mg/kg via a tailvein of mice and with neferine at 10 mg/kg via an intragastric administration of mice. The heart, liver, spleen, lung and kidney samples were collected at 0.5, 2, 4, 8, 12 and 24 h after drug administration for tissue distribution analysis. The tissue samples were homogenized with a Xinzhi homogenizer (Kinematica, Ningbo, Zhejiang, China), resulting in final concentrations of approximately 0.4 mg/kg. All the biological samples were stored at –20 ºC until analysis.
2.3. Analysis of doxorubicin with high performance liquid chromatography
The concentrations of doxorubicin in plasma and tissues were determined by high performance liquid chromatography (HPLC) spectrometry with the ShimadzuLC-2010AHT system. TopsilTMC18 (250 mm×4.6 mm, 5 μm) columns were used for separation. The mobile phase was made up of 10 mmol/L ammonium dihydrogen phosphate, adjusted to pH 3.0 with phosphoric acid (A) and acetonitrile (B) at a flow rate of 1.0 mL/min and the ratio of A:B of 70:30 (v/v). Daunorubicin was used as the internal standard (IS). The signal was detected using an ultraviolet detector at 233 nm for doxorubicin and the column temperature was maintained at 30 ºC. Sample homogenate (20 μL) was injected onto the column.
2.4. Statistical analysis
All the data were presented as means±standard deviations (SD). Statistical analysis was conducted usingSPSS 18.0. The difference between groups with a P-valueof less than 0.05 was considered as statistically significant.
3. Results
3.1. Altered pharmacokinetics of doxorubicin in combination with neferine
The HPLC method was used to determine the plasma concentration of doxorubicin after single administration with or without the co-administration of neferine. The mean plasma concentration-time profiles of doxorubicinalone and in combination with neferine were characterized in rats and illustrated in Figure 1. The mean values of the pharmacokinetic parameters of doxorubicin were summarized in Table 1.


Figure 1. Mean plasma concentration- and time-dependent profiles of doxorubicin following an intravenous administration of doxorubicin (4 mg/kg) to rats with or without neferine (10 mg/kg) (mean±SD, n = 6). Note: filled triangles, doxorubicin alone; filled diamonds, pretreatment with neferine at 10 mg/kg.


Table 1.
Pharmacokinetic parameters of doxorubicin after an intragastric administration of neferine (10 mg/kg) to rats (mean±SD, n = 6)

*
P<0.05, **P<0.01 compared with those of control. 

As shown in Table 1, the pretreatment of rats with neferine (10 mg/kg) followed by administration of doxorubicin significant altered the pharmacokinetic profiles of doxorubicin as compared to those in rats treated with doxorubicin alone. There were obvious changes in AUC,t1/2β, and CL. However, there was no difference in t1/2α. These result revealed that neferine significantly affected the pharmacokinetics properties of doxorubicin.
3.2. Altered tissue distribution of doxorubicin in combination with neferine
The HPLC method was used to determine the concentrations of doxorubicin among tissues after a single intragastric administration with or without the co-administration of neferine in mice at 0.5, 2, 4, 8, 12, and 24 h after drug administration. The results presented in Figure 2 revealed that the doxorubicin had a rapid and wide distribution among tissues. The highest concentration was found in the liver, followed by those in kidney, heart, spleen and lung.  





Figure 2.
Tissue distribution profiles of doxorubicin following an intravenous administration of doxorubicin (4 mg/kg) to mice pretreated with or without neferine (10 mg/kg). Panels AE showed the concentrations of doxorubicin distributed to heart, liver, kidney, spleen and lung (mean±SD, n = 5), respectively. *P<0.05, **P<0.01 compared with that of the control. 
4. Discussion
Doxorubicin belongs to a class of non-specific anthracycline drugs with broad-spectrum of anti-canceractivities and has been used for the treatment of leukemia, liver, breast, and ovarian cancers and solid tumors. It is one of the most potent chemotherapeutic agents[1]. However, its clinical potential has been seriously limited by dose-dependent myocardial injury in clinical application. At the same time, since tumor cells can acquire multidrug resistance and the clinical efficacy of doxorubicin has been limited, both of which together limit the application of doxorubicin. It has been demonstrated that multi-drug resistance is attribuated to different mechanisms including decreased uptake of drugs, the changes that affect the capacity of pharmacokinetics properties or cytotoxic drugs to kill cells, such as reduced apoptosis and increased drug detoxification; increased energy-dependent efflux of drugs, the ATP-dependent binding cassette transporters, such as P-glycoprotein, and multidrug resistance-related proteins[2224].
In the present study, we conducted the experiments to investigate two important aspects of the pharmacokinetic profiles of doxorubicin: First, we divided experimental rats into two groups to study the effect of co-administration of neferine on the pharmacokinetic properties of the administrated doxorubicin and found that co-administration of neferine significantly affected the pharmacokinetics of doxorubicin. There were significant changes in AUC, CL and t1/2β when the group of pretreatment neferine plus doxorubicin was compared with the group treated with doxorubicin alone; Second, in this study, we also examined the effect of the co-administration of neferine on the tissue distribution of the administered doxorubicin and found that the co-administration of neferine significantly affected the tissue distribution profiles of doxorubicin. Comparing pretreatment neferine with doxorubicin alone, the concentrations of doxorubic in heart, liver and kidney at 0.5 and 2 h post administration were statistically significant higher than those in the doxorubicin alone group, respectively. However, the concentrations of doxorubinsin in spleen and lung of mice in pretreatmentneferine group were slightly higher than those in doxorubicin alone but the difference was not statistically significant. These results imply that the distribution of doxorubicin could depend on the perfusion rate of the particular organ or blood flow rate, because the entry rate of a drug into a tissue depends on the blood flow rate to the tissue, tissue binding capacity, tissue mass, and the partition characteristics between blood and tissues[25].
One of the major alcohol metabolites of doxorubicin is doxorubicinol, which is further metabolized in cytoplasm of cells by aldehyde-ketone reductase and detoxified most likely by NADPH cytochrome P450 reductase-catalyzed may be the reason that the metabolism properties of doxorubicin are changed after the pretreatment with neferine. The effect of neferine on the metabolism property
of doxorubicin is likely mediated by the metabolism mediated by the same cytochrome P450 enzymes, resulting in the effect of competitive metabolism on doxorubicin pharmacokinetics, which, in turn, affects doxorubicin pharmacokinetics in vivo[20]. This may be one of the mechanisms by which neferine can reverse the multidrug resistance of neoplasms.
5. Conclusions
This study has suggested that intragastric administration of animals with neferine can increase plasma concentration of doxorubicin and decrease the rate of plasma clearance to doxorubicin. In addition, co-administration with neferine significantly increased the distribution of doxorubicin into heart, liver and kidney. Therefore, the dosage of doxorubicin needs to be taken into consideration when it is administrated in combination with neferine.
Acknowledgements
This work was supported by the Scientific Research Fund Project of Heilongjiang University of Chinese Medicine (Grant No.201420).
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甲基莲心碱对阿霉素药动学影响
薛清丹, 鞠爱霞, 康宇红, 郑春雨, 李秋红*
黑龙江中医药大学, 黑龙江哈尔滨150040       
摘要: 阿霉素是一种蒽环类广谱抗肿瘤药物, 用于治疗白血病、肝癌, 乳腺癌等实体肿瘤。本研究的目的是探讨甲基莲心碱对阿霉素药动学影响。通过高效液相色谱法测定阿霉素的血浆和组织中的药物浓度, 运用C18反相色谱柱进行分离, 流动相是乙腈: 磷酸水溶液比例为30:70, 流速为1 mL/min, 检测波长为233 nm。药代动力学研究发现给予甲基莲心碱明显影响阿霉素的药动学。与对照组比较甲基莲心碱预处理组的AUC, CL t1/2β明显改变。组织分布表明: 心脏、肝脏和肾脏中的药物浓度在0.52小时明显高于阿霉素单独组药物浓度。甲基莲心碱预处理组与阿霉素组比较脾脏和肺脏药物浓度轻微的增加, 但没有显著性差异。因此, 当阿霉素与甲基莲心碱联用时应该考虑阿霉素的给药剂量。 
关键词: 甲基莲心碱; 阿霉素; 药代动力学; 高效液相色谱法; 蒽环类广谱抗肿瘤药物
 

 
Received: 2015-01-03, Revised: 2015-02-12, Accepted: 2015-02-27.
Foundation item: The Scientific Research Fund Project of Heilongjiang University of Chinese Medicine (Grant No.201420).
*Corresponding author. Tel.: 86-451-87266902, E-mail: liqiuhong64@163.com