莲必治注射液安全性再评价研究
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摘要
中药注射剂是中药现代化和进步的重要标志,它从诞生到至今,在医疗实践中发挥着重要的作用。但随着在临床的广泛应用,中药注射剂的安全性问题日益引起人们的关注,并制约着中药注射剂的发展。为了发展中药注射剂这一特色中药剂型,2009年国家食品药品监督管理局(SFDA)要求对现有中药注射剂在安全性、有效性、合并用药等方面进行再评价,控制中药注射剂安全风险,确保中药注射液质量的稳定和临床用药的安全有效。
莲必治注射液是我国70年代研制出的一种中药单体注射剂(成分为亚硫酸氢钠穿心莲内酯),主要用于细菌性痢疾、肺炎、急性扁桃体炎、腮腺炎、喉炎及上呼吸道感染等疾病。2008年,莲必治注射液的市场销售量占有率4.43%,医院销售额为8700多万元。随着临床用药量增加,其有关不良反应的报道也在日益增多,其中以急性肾损伤和过敏反应尤为引起人们的关注,国内已开始研究莲必治注射液引起的肾毒性,但现有研究未在动物中复制出临床上毒性特征,与临床出现不良反应的相关性不紧密,所以莲必治注射液引起的不良反应还有待进一步定论。藉此,本课题依据SFDA通知的指导精神,参考莲必治注射液临床报道的不良反应情况对其进行实验验证,找出发生原因,客观地对莲必治注射液进行安全性再评价研究。根据发生的不良反应类型进行实验设计,通过对两种纯度不同的莲必治注射液制剂(莲必治注射液A中亚硫酸氢钠穿心莲内酯含量235.5mg/ml,相关物质含量1.3%;莲必治注射液B中亚硫酸氢钠穿心莲内酯含量117.4mg/ml,相关物质含量50.9%)急性毒性试验(LD50)、单次静注毒性试验、30天重复给药试验和过敏试验等一般毒性试验,首次复制出临床不良反应症状特征的动物模型,通过对不同时间段尿液中敏感指标的测定,比较这两种莲必治注射液毒性大小,观察和测定药物对机体的损害和影响,探究其引起不良反应物质基础,为莲必治注射液的临床应用提供安全性再评价资料;其次利用现代技术代谢组学、体外细胞毒性试验及与卡那霉素联合用药试验,探究莲必治注射液引发急性肾损伤的可能机制,为莲必治注射液安全用药及中药安全性评价提供依据。一般毒性试验研究如下:
1.用上下法急性毒性试验检测得到莲必治注射液A对SD大鼠的LD50为2101mg/kg(2000～2350mg/kg),莲必治注射液B的LD50为612.4mg/kg(500～750mg/kg),按毒性分级均为低毒。动物染毒后立即出现中毒症状,死亡一般发生在染毒后0～30min之间,与临床报道的不良反应发病迅速短相符;莲必治注射液B的毒性明显大于莲必治注射液A,两个受试物的毒性大小差异可能与药物中相关物质含量有关。
2.一次给药毒性试验研究:SPF级雄性SD大鼠随机分成7组,每组10只。其中3组分别给予莲必治注射液A 400、800和1600mg/kg,3组分别给予莲必治注射液B 50、100和200mg/kg;另1组为空白对照组,给予生理盐水。每组按10ml/kg给药体积尾静脉单次注射。给药后观察大鼠活动和中毒表现。连续收集给药后0～6、7～12、13～18和19～24h各组大鼠尿液,进行尿常规,尿素氮(BUN)、尿肌酐(Crea)、尿酶测定。结果发现莲必治注射液在高浓度时能引起SD大鼠尿中KET、ERY、BUN、Crea、ALP、LDH和NAG增加,推测莲必治注射液高浓度时可能影响了肾小球滤过率,肾小管受损导致重吸收障碍。尿酶多在给药后0～12h升高显著,随着给药后时间的延长,尿酶变化恢复正常。这表明莲必治注射液引起尿酶改变是一过性的,与文献报道莲必治引起急性肾功能损害发病时间短相符。在观察尿酶增加与亚硫酸氢钠穿心莲内酯和相关物质含量关系时,发现NAG和β2-MG水平升高与相关物质呈剂量效应关系,表明莲必治注射液中所含的相关物质可加剧其对肾脏的损伤作用。
3. 30天重复试验研究:两种莲必治注射液高、中、低3个剂量组(莲必治注射液A,450,150,50mg/kg;B,150,50,25mg/kg)和生理盐水对照组,尾静脉注射每天给药1次,连续给药30天。观察包括SD大鼠一般行为、体重和摄食量、尿常规、血液学、血液生化学以及肾脏组织病理学等指标。试验结果:给药期间莲必治注射液A高剂量(450mg/kg)有一只动物死亡,莲必治注射液B高剂量(150mg/kg)组有4只只动物死亡。莲必治注射液A高剂量(450mg/kg)组和莲必治注射液B高剂量(150mg/kg)组能够引起动物摄食量减少和体重降低,尿中红细胞(ERY)增加,BUN、Crea和β2-MG水平显著增高,推测莲必治注射液可能影响了肾小球滤过率和肾小管功能,毒性作用与其纯度和相关物质可能相关。
4.按照《中药、天然药物免疫毒性(过敏性、光变态反应)研究的技术指导原则》的试脸方法对两种不同纯度的莲必治注射液进行被动皮肤过敏试验和全身主动过敏试验研究。过敏试验研究结果表明,在被动皮肤过敏试验中莲必治A和B的低剂量组中50%的动物出现阳性反应,莲必治注射液A和B的高剂量组中SD大鼠均出现阳性反应;而在全身主动过敏试验中,莲必治A和B的低剂量组均未出现阳性反应症状,莲必治A高剂量组只有33.3%动物出现阳性反应,而莲必治B高剂量组77.8%动物出现阳性反应。说明莲必治注射液在亚硫酸氢钠穿心莲内酯高剂量(大鼠200mg/kg,相当于成人临床常用剂量的等效剂量10mg/kg/次的20倍)均能引起过敏反应,从两种注射液过敏反应的差异推测相关物质可以增加莲必治注射液的过敏反应几率。
莲必治注射液引起急性肾损伤机制研究如下:
1.代谢组学技术:25只雄性SD大鼠,随机分为莲必治注射液A低剂量组(400mg/kg)、高剂量组(1600mg/kg);莲必治注射液B低剂量组(100mg/kg)、高剂量组(400mg/kg)和空白对照组(生理盐水),每组5只。各组动物均按10ml/kg单次尾静脉给药。收集各组大鼠给药前12h及给药后0～6、7～12、13～24和25～48h各时间段尿液,测定其核磁共振氢谱(~1H NMR),利用代谢组学技术研究动物尿样的~1H NMR谱变化情况,发现大鼠尿样的内源性代谢物图谱出现先偏离、再回归对照组的变化轨迹,反映出动物机体受到莲必治注射液的影响。每一个样本在主成分(PC)图上的位置由它的代谢反应决定,在给药后0～12h大鼠尿液1HNMR代谢谱变化与莲必治毒性作用强度密切相关,对于毒性越明显的剂量点,代谢谱偏离对照组也越远。给药组尿样中三甲胺(TMA)(δ2.70)和二甲基甘氨酸(DMG)(δ2.94)不同程度升高,柠檬酸(δ2.54,δ2.66)和α-酮戊二酸(δ2.46,δ3.02)降低。由此推测莲必治注射液在高浓度时能引起肾脏损伤作用,其机制可能是高浓度的莲必治影响肾髓质渗透压和干扰肾细胞中线粒体相关酶的活性,影响线粒体能量代谢,改变线粒体功能,破坏膜的完整性,最终引发肾细胞损伤,影响肾脏对原尿的浓缩和稀释作用。
2.亚硫酸氢钠穿心莲内酯对HK-2细胞毒性机制研究:通过倒置显微镜观察莲必治注射液A的原料药亚硫酸氢钠穿心莲内酯对HK-2细胞形态改变,应用噻唑蓝(MTT)比色法和流式细胞术法检测其对HK-2细胞的抑制率,细胞周期改变、凋亡率、线粒体膜电位(MMP)和活性氧(ROS),并用Western blot技术检测凋亡的相关蛋白(细胞色素c(CytC)、pro-Caspase-3、Bcl-2、Bax),探讨亚硫酸氢钠穿心莲内酯对HK-2细胞毒性机制。其研究表明,亚硫酸氢钠穿心莲内酯呈剂量依赖性抑制HK-2细胞增殖,作用24h和48h后IC50值分别为(19.12±3.55)和(11.56±3.88)mg/ml。细胞形态变化:细胞收缩变圆,黏附性变差,部分脱落;LDH漏出率显著增加,线粒体膜电位(MMP)下降,细胞出现明显凋亡,阻滞HK-2细胞周期于G2/M期,细胞内的ROS和上清液中的MDA水平明显增加,GSH、SOD和总ATP酶活性降低;Westernblot印迹分析凋亡相关蛋白显示,亚硫酸氢钠穿心莲内酯使HK-2细胞内细胞色素c(CytC)、pro-Caspase-3、Bcl-2和Bax蛋白水平均升高,并且呈现明显的剂量和时间效应关系,但Bcl-2和Bax比值不变,说明这两类蛋白的对细胞的作用处于相对平衡的状态。推测亚硫酸氢钠穿心莲酯对HK-2细胞的损伤作用可能改变HK-2细胞内氧化还原状态,耗竭细胞内GSH、SOD,导致细胞内ROS大量堆积,引发脂质过氧化作用,线粒体MMP下降,破坏细胞膜的通透性,使细胞损伤,凋亡诱导因子(AIF)从线粒体释放,包括CytC的释放、Caspase-3酶原激活、Bcl基因家族成员的介入等,最终导致细胞周期阻滞和细胞凋亡或坏死。
3.以HK-2细胞为模型,比较亚硫酸氢钠穿心莲内酯和卡那霉素对HK-2细胞毒性作用的差异。研究结果表明,亚硫酸氢钠穿心莲内酯和硫酸卡那霉素对HK-2细胞的形态、细胞周期、MMP和ROS的影响不同,推测亚硫酸氢钠穿心莲内酯和硫酸卡那霉素以不同的途径对HK-2细胞产生毒性作用,分别阻滞细胞周期于不同时期。它们联合作用,加重对肾脏损害作用。
总之,我们根据临床不良反应报道的特征,从一般毒性试验对两种莲必治注射液进行安全性再评价,首次在动物模型上复制出莲必治注射引起肾损伤和过敏反应等不良反应症状特征,发现引起这些不良反应与莲必治注射液的制剂质量有很大关系,因此通过对莲必治注射液的安全性再评价,需要制药企业提高中药注射剂的质量,控制制剂中相关物质的含量,减少莲必治注射液引发不良反应的风险。在临床应用中注意剂量和适应症,监测患者尿中KET、ERY、BUN、Crea、ALP、LDH和NAG水平,减少莲必治注射液引起急性肾损伤的发生率。
体内和体外机制研究证实,莲必治在高浓度时对肾脏有潜在的毒性作用,尿中三甲胺、二甲基甘氨酸、柠檬酸和α-酮戊二酸的变化情况可作为监测莲必治注射液肾损伤的指标。莲必治引起肾损伤的机制可能是通过影响肾髓质渗透压,干扰肾细胞线粒体中相关酶的活性,干扰线粒体能量代谢,改变细胞内氧化还原状态,产生过多的ROS,破坏细胞膜的通透性,线粒体MMP下降,线粒体功能紊乱,导致CytC等凋亡因子从线粒体释放,进而引发细胞凋亡或坏死。
莲必治注射液在大剂量使用和不合理的联合用药用会出现毒性反应,因此在临床上用药不要超过10mg/kg/次,用药期间要注意监测其肾功能,肾病患者应用时需要调整剂量。临床上禁止与卡那霉素等氨基糖苷类抗生素合用。制剂中相关物质对莲必治注射液的安全性影响很大,因此,应该建立质量标准,明确相关物质中的毒性成分,控制毒性成份的含量,加强不良反应监测。随着代谢组学技术和细胞生物技术的发展,这些新技术将会越来越多的用于中草药毒性毒理的研究,对于实现中药现代化具有重要的意义。
Traditional Chinese Medicine Injections (TCMIs) is an important landmark of modernization and progress of Traditional Chinese Medicine (TCM), and have played an important role in medical practice. However, with the widespread clinical applications, the safety issues of TCMI increasingly became the main obstacle for the development of TCMI. In order to develop these featured TCM formulations, the State Food and Drug Administration (SFDA) required in 2009 the reassessment of all existing TCMIs on aspects such as safety, effectiveness and drug combination, to control the safety risks of TCMI and ensure the safety, stability and effectiveness.
Lianbizhi Injection was developed as a single component of TCM injections in 1970s, with the main constituent being Andrographolidi Natrii Bisulfis. Lianbizhi Injections is mainly used for bacterial dysentery, pneumonia, acute tonsillitis, mumps, laryngitis and upper respiratory tract infections. The market share of Lianbizhi Injections within all TCMIs was 4.43% and the hospital sales had reached more than 8700 million in 2008.With the increased clinical applications, the relevant reports of adverse reactions were increased, in which acute renal injury and allergic reactions were particular cause for concerns. Nephrotoxicity caused by Lianbizhi injections has already been studied in China, but the current study did not replicate its clinical toxicity features, with little correlation with clinical adverse reactions. Therefore, it needs to further investigate the adverse reactions caused by Lianbizhi injections. Therefore, we carried out safety re-evaluation of the Lianbizhi Injection in this program, based on the guiding spirit of the SFDA note for the industry, and on the adverse reactions of Lianbizhi Injections reported in clinicals. In this study, firstly, the general toxicity tests such as acute toxicity test, single dose kidney toxicity test, 30-day repeated dose toxicity test and allergy test had been conducted to explore the toxicity of two kinds of formulations of Lianbizhi Injections (the concentrations of Andrographolidi Natrii Bisulfis for Lianbizhi Injections A and B were 235.5 and 117.4mg/ml, respectively, with relevant impurities contents being 1.3% and 50.9%, respectively), which first replicated the clinical adverse reactions in animal models, to compare the toxicity of these two kinds of formulations by detecting the sensitive indices in urine at different time point, to observe the biological damage to animals, to explored the potential toxicity biomarkers and the material fundation of the clinical adverse reactions, and to provide toxicological informations for its clinical applications. Secondly, the nephrotoxicity mechanisms are explored, by using modern metabonomic technologies, in vitro cytotoxicity test and the combination effect study with of kanamycin, we aim to provide some evidences on safe administration of Lianbizhi injections as well as the safe evaluation of TCM.
Results of the general toxicity test are as follows:
1. By the up and down acute toxicity test, the LD50 of Lianbizhi Injections A and B in male SD rats were determined to be 2101mg/kg (95% Confidential Interval, CI, 2000 to 2350 mg/kg) and 612.4 mg/kg (CI, 500 to 750mg/kg), respectively, with both formulations being low toxic. Toxic signs appeared immediately after dosing, with animal death usually evidented within 30 minutes after dosing, similar to the clinical adverse reaction symptoms induced by Lianbizhi Injections. The toxicity differences of these two kinds of formulations may be related with the concentrations of relevant impurities in the formulations.
2. Studies of the single-dose kidney toxicity test are as follows: SPF male SD rats were assigned into seven groups at random: the first three groups were intravenously (i.v.) injected with 400, 800, 1600mg/kg of Lianbizhi Injections A, the second three groups were i.v. injected with 50, 100, 200mg/kg of Lianbizhi Injections B and the control animals were injected with saline (10 rats in each group). The dosing volume is 10ml/kg via caudal vein. Activities and intoxication signs of animals were observed after dosing, and urine were collected in the intervals of 0-6h, 7-12h, 13-18h and 19-24 h after dosing, for urine analysis, and the urea nitrogen (BUN), urine creatinine (Crea) and urine enzymes measurement. Then blood clinical chemistry and histopathology examination were also performed. The results showed that the levels of KET, ERY, BUN, Crea, ALP, LDH, and NAG in urine were increased in the high dose Lianbizhi Injections treated rats, suggested that Lianbizhi Injections may affect the glomerular filtration rate and cause the renal tubular reabsorption dysfunction in at high dose level. Urine enzyme level increased significantly in 0-12h after dosing, while urine enzyme changes had no significant difference compared with the control group with the extension of time after administration, which showed that a transient change of urine enzymes caused by the Lianbizhi Injections was close to the clinical reported fact that Lianbizhi Injections caused acute renal injury in a short time after administration. Meanwhile inspecting the relationship between the changes of urine enzymes and the concentration of andrographolide sodium bisulfite and related substances, the increase in levels of NAG andβ2-MG was associated with the amount of related substances, indicating the amount of related substance in injection potential intensify the nephrotoxic effects of Lianbizhi Injections.
3. The methods of the chronic toxicity test for thirty days: the male SD rats were administered intravenously with 50, 150, and 450mg/kg of Lianbizhi Injections A, 25, 50, and 150mg/kg of Lianbizhi Injections B, and the physiological saline for 30 days, respectively. The clinical signs, body weight gain and the food consumption, examine the haematology (ERY), blood chemistry, the change of routine urine and urine enzymess were measured and necropsy and histopathology were recorded. One male in high dose (450mg/kg) Lianbizhi A group and four males in high dose (150mg/kg) Lianbizhi B group died during the administration. Clinical signs of toxicity in SD rats of the high dose Lianbizhi A and B group included: reduced food intake, slow body weight gain, significant increases the levels of urine ERY, BUN, Crea, andβ2-MG, et al. From these results, the Lianbizhi Injections were estimated to influence the glomerular filtration rate and tubular reabsorption and its potential nephnotoxic effects on rats is related to its purity and dosage.
4. In accordance with the traditional Chinese medicine, natural medicine, immunity toxicity (allergic, photoallergic) of the technical guidelines, the passive cutaneous anaphylaxis (PCA) and active systemic anaphylaxis (ASA) tests were carried out on two different purity of lianbizhi injections. The results of the allergy testing showed that positive symptoms in SD rat of the high dose Lianbizhi A and B group all occurred, and 50 percent animals in the low dose Lianbizhi A and B group showed the positive symptoms in PCA toxicity study. While in ASA toxicity study, 33.3 percent animals appeared weak positive in high dose Lianbizhi A group and 77.8 percent animals appeared positive in in high dose Lianbizhi B group and there are no anminal in the low dose Lianbizhi A and B group occurring the positive symptoms. It is demonstrated that Lianbizhi Injections in high concentrations of Andrographolidi Natrii Bisulfis (200mg·Kg-1 in SD rats, which is approximately equal to 20 times of adult clinical dosage) can cause allergic reactions. From the allergic differences of two kinds of formulations, the probability of allergic reaction would be increased by the concentration of non- Andrographolidi Natrii Bisulfis.
Main studies on the mechanisms of acute kidney injury induced by Lianbizhi Injections are as follow:
1. Technique on Metabonomics:Twenty five male SD rats were randomly divided into the five groups: the low-dose (400 mg/kg) and the high-dose(1600 mg/kg)Lianbizhi A groups, the low-dose (100 mg/kg) and the high-dose(400 mg/kg)Lianbizhi B groups, and the blank control group (normal saline). Each group comprised 5 rats. All rats received respectively a single intravenous injection of Lianbizhi 10 ml/kg via caudal vein. The rats’urine were collected within 12 hours prodose as well as within 0～6, 7～12, 13～24 and 25～48 hours after drug administration. The 1H nuclear magnetic resonance (1H NMR) spectroscopy of the rats’urine was measured.The integrated metabonomics study was applied to investigate the biochemical composition of urine obtained from two kinds of Lianbizhi Injections treated SD rats. It was found that the rat urinary metabonomic profile of dosed groups firstly deviated from the control group and then regressed the control level after dosing, reflecting the impacted information of animals by Lianbizhi Injections. The position of each sample in the principle components (PC) profile depending on its metabolic response and the changed of metabonomics profile of dosed groups were related to the toxicity of Lianbizhi Injections in 0 ~ 12 h phase urine, which the more toxic dose point was, the more distant its metabolic spectrum deviated from the control group. The levels of trimethylamine(TMA)(d2.90)and dimethylglycine(DMG)(d2.94)were increased and the levels of citric acid(d2.54,d2.66) andα-ketoglutarate (d2.46,d3.02)were reduced in 0 ~ 12 h phase urine of administrated groups compared with the control group. The mechanism may relate to the influence of the osmoregulation in renal medulla and the interference with the activity of mitochondrial enzymes due to high concerntration of lianbizhi injections, which result in reduction of energy metabolism in mitochondria, change of the mitochondrial function and destruction of membrane integrity, leading to renal cell injury, eventually affecting the concentration and dilution of the original urine.
2. Studies of the mechanism of the HK-2 cytotoxicity of Andrographolidi natrii bisulfis as follows: The morphological changes of HK-2 cells were examined with contrast microscopy and growth inhibitory rate of HK-2 cells were detected by MTT colorimetric assay. Cell-cycle arrest, apoptosis rate MMP and ROS were analyzed by flow cytometry, and the levels of apoptosis-related protein (cytochrome c (CytC), pro-Caspase-3, Bcl-2, Bax) were measured by Western blot. The studies indicated that the proliferation of HK-2 cells were markedly inhibited in dose-dependent manners and the IC50 values of andrographolidi natrii bisulfis for 24 and 48 h were (19.12±3.55), (11.56±3.881) mg/ml respectively. The toxic manifestations included: the inhibition of the cells proliferation,cells rounded and contraction, adhesion worse, some fall off, LDH leakage increasement, and cell apoptosis followed by the decreasment of mitoehondria membrane potential (MMP). Futhermore,andrographolidi natrii bisulfis can block the cell cyele, and the percentage of cells in G2/M phase was gradually inereased. ROS and MDA level increased and GSH, SOD and ATPase activity decreased in HK-2 cells. Western blotting analysis of apopotic related proteins revealed that the release of cytochrme C increased, the expression level of pro-caspase 3, Bcl-2and bax inceased signifieantly in dose- and time-dependence manner after treatment with andrographolidi natrii bisulfis, However, the ratio of Bcl-2 and Bax remained unchanged, indicating in a relatively balanced state of the two kinds proteins on the cells. These results implied that andrographolidi natrii bisulfis may lead to cell cycle arrest and apoptosis or necrosis though changing the redox state, depleting of intracellular GSH and SOD in, leading to a large accumulation of intracellular ROS,which induced lipid peroxidation, decreasing MMP, damaging the permeability of cell membrane, then apoptosis-inducing factor (AIF) releasing from mitochondria, including the CytC release, Caspase-3 zymogen activation, Bcl gene family members involved and so on.
3. To compare the differences of drug combination concerning andrographolidi natrii bisulfis and kanamycin sulfate on HK-2 cytotoxicity. The results showed that there were lots of differences in HK-2 cell morphology, cell cycle, MMP, and ROS respectively treated with andrographolidi natrii bisulfis and kanamycin sulfate, suggesting that the two drugs act on HK-2 cells in different pathways to produce toxic effects, respectively arresting the cells at different phases. Their combined effect can exacerbate the damage of renal cells.
In conclusion, based on the adverse reaction characteristics reported, we re-evaluated the safety of two kinds of Lianbizhi Injections. By general toxicity test, we found that adverse reactions of renal injury and allergenic reaction caused by Lianbizhi Injections can be reproduced on animal models. Preparation quality of Lianbizhi Injections plays an important role in these adverse reactions. Therefore, through re-evaluation of Lianbizhi Injections the pharmaceutical enterprises should improve the TCMI quality and control the content of related substances in order to reduce the risks of Lianbizhi Injection triggered adverse reactions. In clinical application we should pay attention to the dosage and the indication, monitoring the levels of urine KET, ERY, BUN, Crea, ALP, LDH and NAG so as to reduce the incidence of acute renal injury caused by Lianbizhi Injections.
The results of studies in vivo and in vitro confirmed that Lianbizhi Injections had potential nephrotoxicity in high concentration. The changes of Urinary trimethylamine, dimethyl glycine, citric acid andα– ketoglutarate in urine can be used as indicators to monitor the renal injury induced by Lianbizhi Injections. The mechanism of renal injury induced by Lianbizhi Injections may be related to affecting the osmolarity in kidney medulla and interferencing the activity of related mitochondria enzymes in renal cell, involving in affecting mitochondria energy metabolism, changing the redox state, resulting in excessive ROS, destructing the cell membrane permeability, declining MMP, mitochondrial dysfunction, leading to CytC releasing from mitochondria to trigger apoptosis or necrosis.
The toxic effects will occur when Lianbizhi Injections was used in large dose and unreasonable combination therapy. Therefore, the dosage should not exceed 10mg/kg a time, and functions of kidney should be monitored during administration. Patients with kidney disease need to adjust the dosage, and combination with kanamycinor or other aminoglycoside antibiotics must be forbidden in clinical usage. The related substances in the preparation may have great influence on the safety of Lianbizhi Injections. Therefore, quality criteria should be established to identify and control the related toxic components, as well as strengthen the monitoring of adverse reactions. With the development of metabonomics and cell biotechnology, these novel technologies might be increasingly applied in the toxicological effects of other Chinese herbal medicine in the future, which will contribute to the modernization of traditional Chinese Medicine.
莲必治注射液是我国70年代研制出的一种中药单体注射剂(成分为亚硫酸氢钠穿心莲内酯),主要用于细菌性痢疾、肺炎、急性扁桃体炎、腮腺炎、喉炎及上呼吸道感染等疾病。2008年,莲必治注射液的市场销售量占有率4.43%,医院销售额为8700多万元。随着临床用药量增加,其有关不良反应的报道也在日益增多,其中以急性肾损伤和过敏反应尤为引起人们的关注,国内已开始研究莲必治注射液引起的肾毒性,但现有研究未在动物中复制出临床上毒性特征,与临床出现不良反应的相关性不紧密,所以莲必治注射液引起的不良反应还有待进一步定论。藉此,本课题依据SFDA通知的指导精神,参考莲必治注射液临床报道的不良反应情况对其进行实验验证,找出发生原因,客观地对莲必治注射液进行安全性再评价研究。根据发生的不良反应类型进行实验设计,通过对两种纯度不同的莲必治注射液制剂(莲必治注射液A中亚硫酸氢钠穿心莲内酯含量235.5mg/ml,相关物质含量1.3%;莲必治注射液B中亚硫酸氢钠穿心莲内酯含量117.4mg/ml,相关物质含量50.9%)急性毒性试验(LD50)、单次静注毒性试验、30天重复给药试验和过敏试验等一般毒性试验,首次复制出临床不良反应症状特征的动物模型,通过对不同时间段尿液中敏感指标的测定,比较这两种莲必治注射液毒性大小,观察和测定药物对机体的损害和影响,探究其引起不良反应物质基础,为莲必治注射液的临床应用提供安全性再评价资料;其次利用现代技术代谢组学、体外细胞毒性试验及与卡那霉素联合用药试验,探究莲必治注射液引发急性肾损伤的可能机制,为莲必治注射液安全用药及中药安全性评价提供依据。一般毒性试验研究如下:
1.用上下法急性毒性试验检测得到莲必治注射液A对SD大鼠的LD50为2101mg/kg(2000～2350mg/kg),莲必治注射液B的LD50为612.4mg/kg(500～750mg/kg),按毒性分级均为低毒。动物染毒后立即出现中毒症状,死亡一般发生在染毒后0～30min之间,与临床报道的不良反应发病迅速短相符;莲必治注射液B的毒性明显大于莲必治注射液A,两个受试物的毒性大小差异可能与药物中相关物质含量有关。
2.一次给药毒性试验研究:SPF级雄性SD大鼠随机分成7组,每组10只。其中3组分别给予莲必治注射液A 400、800和1600mg/kg,3组分别给予莲必治注射液B 50、100和200mg/kg;另1组为空白对照组,给予生理盐水。每组按10ml/kg给药体积尾静脉单次注射。给药后观察大鼠活动和中毒表现。连续收集给药后0～6、7～12、13～18和19～24h各组大鼠尿液,进行尿常规,尿素氮(BUN)、尿肌酐(Crea)、尿酶测定。结果发现莲必治注射液在高浓度时能引起SD大鼠尿中KET、ERY、BUN、Crea、ALP、LDH和NAG增加,推测莲必治注射液高浓度时可能影响了肾小球滤过率,肾小管受损导致重吸收障碍。尿酶多在给药后0～12h升高显著,随着给药后时间的延长,尿酶变化恢复正常。这表明莲必治注射液引起尿酶改变是一过性的,与文献报道莲必治引起急性肾功能损害发病时间短相符。在观察尿酶增加与亚硫酸氢钠穿心莲内酯和相关物质含量关系时,发现NAG和β2-MG水平升高与相关物质呈剂量效应关系,表明莲必治注射液中所含的相关物质可加剧其对肾脏的损伤作用。
3. 30天重复试验研究:两种莲必治注射液高、中、低3个剂量组(莲必治注射液A,450,150,50mg/kg;B,150,50,25mg/kg)和生理盐水对照组,尾静脉注射每天给药1次,连续给药30天。观察包括SD大鼠一般行为、体重和摄食量、尿常规、血液学、血液生化学以及肾脏组织病理学等指标。试验结果:给药期间莲必治注射液A高剂量(450mg/kg)有一只动物死亡,莲必治注射液B高剂量(150mg/kg)组有4只只动物死亡。莲必治注射液A高剂量(450mg/kg)组和莲必治注射液B高剂量(150mg/kg)组能够引起动物摄食量减少和体重降低,尿中红细胞(ERY)增加,BUN、Crea和β2-MG水平显著增高,推测莲必治注射液可能影响了肾小球滤过率和肾小管功能,毒性作用与其纯度和相关物质可能相关。
4.按照《中药、天然药物免疫毒性(过敏性、光变态反应)研究的技术指导原则》的试脸方法对两种不同纯度的莲必治注射液进行被动皮肤过敏试验和全身主动过敏试验研究。过敏试验研究结果表明,在被动皮肤过敏试验中莲必治A和B的低剂量组中50%的动物出现阳性反应,莲必治注射液A和B的高剂量组中SD大鼠均出现阳性反应;而在全身主动过敏试验中,莲必治A和B的低剂量组均未出现阳性反应症状,莲必治A高剂量组只有33.3%动物出现阳性反应,而莲必治B高剂量组77.8%动物出现阳性反应。说明莲必治注射液在亚硫酸氢钠穿心莲内酯高剂量(大鼠200mg/kg,相当于成人临床常用剂量的等效剂量10mg/kg/次的20倍)均能引起过敏反应,从两种注射液过敏反应的差异推测相关物质可以增加莲必治注射液的过敏反应几率。
莲必治注射液引起急性肾损伤机制研究如下:
1.代谢组学技术:25只雄性SD大鼠,随机分为莲必治注射液A低剂量组(400mg/kg)、高剂量组(1600mg/kg);莲必治注射液B低剂量组(100mg/kg)、高剂量组(400mg/kg)和空白对照组(生理盐水),每组5只。各组动物均按10ml/kg单次尾静脉给药。收集各组大鼠给药前12h及给药后0～6、7～12、13～24和25～48h各时间段尿液,测定其核磁共振氢谱(~1H NMR),利用代谢组学技术研究动物尿样的~1H NMR谱变化情况,发现大鼠尿样的内源性代谢物图谱出现先偏离、再回归对照组的变化轨迹,反映出动物机体受到莲必治注射液的影响。每一个样本在主成分(PC)图上的位置由它的代谢反应决定,在给药后0～12h大鼠尿液1HNMR代谢谱变化与莲必治毒性作用强度密切相关,对于毒性越明显的剂量点,代谢谱偏离对照组也越远。给药组尿样中三甲胺(TMA)(δ2.70)和二甲基甘氨酸(DMG)(δ2.94)不同程度升高,柠檬酸(δ2.54,δ2.66)和α-酮戊二酸(δ2.46,δ3.02)降低。由此推测莲必治注射液在高浓度时能引起肾脏损伤作用,其机制可能是高浓度的莲必治影响肾髓质渗透压和干扰肾细胞中线粒体相关酶的活性,影响线粒体能量代谢,改变线粒体功能,破坏膜的完整性,最终引发肾细胞损伤,影响肾脏对原尿的浓缩和稀释作用。
2.亚硫酸氢钠穿心莲内酯对HK-2细胞毒性机制研究:通过倒置显微镜观察莲必治注射液A的原料药亚硫酸氢钠穿心莲内酯对HK-2细胞形态改变,应用噻唑蓝(MTT)比色法和流式细胞术法检测其对HK-2细胞的抑制率,细胞周期改变、凋亡率、线粒体膜电位(MMP)和活性氧(ROS),并用Western blot技术检测凋亡的相关蛋白(细胞色素c(CytC)、pro-Caspase-3、Bcl-2、Bax),探讨亚硫酸氢钠穿心莲内酯对HK-2细胞毒性机制。其研究表明,亚硫酸氢钠穿心莲内酯呈剂量依赖性抑制HK-2细胞增殖,作用24h和48h后IC50值分别为(19.12±3.55)和(11.56±3.88)mg/ml。细胞形态变化:细胞收缩变圆,黏附性变差,部分脱落;LDH漏出率显著增加,线粒体膜电位(MMP)下降,细胞出现明显凋亡,阻滞HK-2细胞周期于G2/M期,细胞内的ROS和上清液中的MDA水平明显增加,GSH、SOD和总ATP酶活性降低;Westernblot印迹分析凋亡相关蛋白显示,亚硫酸氢钠穿心莲内酯使HK-2细胞内细胞色素c(CytC)、pro-Caspase-3、Bcl-2和Bax蛋白水平均升高,并且呈现明显的剂量和时间效应关系,但Bcl-2和Bax比值不变,说明这两类蛋白的对细胞的作用处于相对平衡的状态。推测亚硫酸氢钠穿心莲酯对HK-2细胞的损伤作用可能改变HK-2细胞内氧化还原状态,耗竭细胞内GSH、SOD,导致细胞内ROS大量堆积,引发脂质过氧化作用,线粒体MMP下降,破坏细胞膜的通透性,使细胞损伤,凋亡诱导因子(AIF)从线粒体释放,包括CytC的释放、Caspase-3酶原激活、Bcl基因家族成员的介入等,最终导致细胞周期阻滞和细胞凋亡或坏死。
3.以HK-2细胞为模型,比较亚硫酸氢钠穿心莲内酯和卡那霉素对HK-2细胞毒性作用的差异。研究结果表明,亚硫酸氢钠穿心莲内酯和硫酸卡那霉素对HK-2细胞的形态、细胞周期、MMP和ROS的影响不同,推测亚硫酸氢钠穿心莲内酯和硫酸卡那霉素以不同的途径对HK-2细胞产生毒性作用,分别阻滞细胞周期于不同时期。它们联合作用,加重对肾脏损害作用。
总之,我们根据临床不良反应报道的特征,从一般毒性试验对两种莲必治注射液进行安全性再评价,首次在动物模型上复制出莲必治注射引起肾损伤和过敏反应等不良反应症状特征,发现引起这些不良反应与莲必治注射液的制剂质量有很大关系,因此通过对莲必治注射液的安全性再评价,需要制药企业提高中药注射剂的质量,控制制剂中相关物质的含量,减少莲必治注射液引发不良反应的风险。在临床应用中注意剂量和适应症,监测患者尿中KET、ERY、BUN、Crea、ALP、LDH和NAG水平,减少莲必治注射液引起急性肾损伤的发生率。
体内和体外机制研究证实,莲必治在高浓度时对肾脏有潜在的毒性作用,尿中三甲胺、二甲基甘氨酸、柠檬酸和α-酮戊二酸的变化情况可作为监测莲必治注射液肾损伤的指标。莲必治引起肾损伤的机制可能是通过影响肾髓质渗透压,干扰肾细胞线粒体中相关酶的活性,干扰线粒体能量代谢,改变细胞内氧化还原状态,产生过多的ROS,破坏细胞膜的通透性,线粒体MMP下降,线粒体功能紊乱,导致CytC等凋亡因子从线粒体释放,进而引发细胞凋亡或坏死。
莲必治注射液在大剂量使用和不合理的联合用药用会出现毒性反应,因此在临床上用药不要超过10mg/kg/次,用药期间要注意监测其肾功能,肾病患者应用时需要调整剂量。临床上禁止与卡那霉素等氨基糖苷类抗生素合用。制剂中相关物质对莲必治注射液的安全性影响很大,因此,应该建立质量标准,明确相关物质中的毒性成分,控制毒性成份的含量,加强不良反应监测。随着代谢组学技术和细胞生物技术的发展,这些新技术将会越来越多的用于中草药毒性毒理的研究,对于实现中药现代化具有重要的意义。
Traditional Chinese Medicine Injections (TCMIs) is an important landmark of modernization and progress of Traditional Chinese Medicine (TCM), and have played an important role in medical practice. However, with the widespread clinical applications, the safety issues of TCMI increasingly became the main obstacle for the development of TCMI. In order to develop these featured TCM formulations, the State Food and Drug Administration (SFDA) required in 2009 the reassessment of all existing TCMIs on aspects such as safety, effectiveness and drug combination, to control the safety risks of TCMI and ensure the safety, stability and effectiveness.
Lianbizhi Injection was developed as a single component of TCM injections in 1970s, with the main constituent being Andrographolidi Natrii Bisulfis. Lianbizhi Injections is mainly used for bacterial dysentery, pneumonia, acute tonsillitis, mumps, laryngitis and upper respiratory tract infections. The market share of Lianbizhi Injections within all TCMIs was 4.43% and the hospital sales had reached more than 8700 million in 2008.With the increased clinical applications, the relevant reports of adverse reactions were increased, in which acute renal injury and allergic reactions were particular cause for concerns. Nephrotoxicity caused by Lianbizhi injections has already been studied in China, but the current study did not replicate its clinical toxicity features, with little correlation with clinical adverse reactions. Therefore, it needs to further investigate the adverse reactions caused by Lianbizhi injections. Therefore, we carried out safety re-evaluation of the Lianbizhi Injection in this program, based on the guiding spirit of the SFDA note for the industry, and on the adverse reactions of Lianbizhi Injections reported in clinicals. In this study, firstly, the general toxicity tests such as acute toxicity test, single dose kidney toxicity test, 30-day repeated dose toxicity test and allergy test had been conducted to explore the toxicity of two kinds of formulations of Lianbizhi Injections (the concentrations of Andrographolidi Natrii Bisulfis for Lianbizhi Injections A and B were 235.5 and 117.4mg/ml, respectively, with relevant impurities contents being 1.3% and 50.9%, respectively), which first replicated the clinical adverse reactions in animal models, to compare the toxicity of these two kinds of formulations by detecting the sensitive indices in urine at different time point, to observe the biological damage to animals, to explored the potential toxicity biomarkers and the material fundation of the clinical adverse reactions, and to provide toxicological informations for its clinical applications. Secondly, the nephrotoxicity mechanisms are explored, by using modern metabonomic technologies, in vitro cytotoxicity test and the combination effect study with of kanamycin, we aim to provide some evidences on safe administration of Lianbizhi injections as well as the safe evaluation of TCM.
Results of the general toxicity test are as follows:
1. By the up and down acute toxicity test, the LD50 of Lianbizhi Injections A and B in male SD rats were determined to be 2101mg/kg (95% Confidential Interval, CI, 2000 to 2350 mg/kg) and 612.4 mg/kg (CI, 500 to 750mg/kg), respectively, with both formulations being low toxic. Toxic signs appeared immediately after dosing, with animal death usually evidented within 30 minutes after dosing, similar to the clinical adverse reaction symptoms induced by Lianbizhi Injections. The toxicity differences of these two kinds of formulations may be related with the concentrations of relevant impurities in the formulations.
2. Studies of the single-dose kidney toxicity test are as follows: SPF male SD rats were assigned into seven groups at random: the first three groups were intravenously (i.v.) injected with 400, 800, 1600mg/kg of Lianbizhi Injections A, the second three groups were i.v. injected with 50, 100, 200mg/kg of Lianbizhi Injections B and the control animals were injected with saline (10 rats in each group). The dosing volume is 10ml/kg via caudal vein. Activities and intoxication signs of animals were observed after dosing, and urine were collected in the intervals of 0-6h, 7-12h, 13-18h and 19-24 h after dosing, for urine analysis, and the urea nitrogen (BUN), urine creatinine (Crea) and urine enzymes measurement. Then blood clinical chemistry and histopathology examination were also performed. The results showed that the levels of KET, ERY, BUN, Crea, ALP, LDH, and NAG in urine were increased in the high dose Lianbizhi Injections treated rats, suggested that Lianbizhi Injections may affect the glomerular filtration rate and cause the renal tubular reabsorption dysfunction in at high dose level. Urine enzyme level increased significantly in 0-12h after dosing, while urine enzyme changes had no significant difference compared with the control group with the extension of time after administration, which showed that a transient change of urine enzymes caused by the Lianbizhi Injections was close to the clinical reported fact that Lianbizhi Injections caused acute renal injury in a short time after administration. Meanwhile inspecting the relationship between the changes of urine enzymes and the concentration of andrographolide sodium bisulfite and related substances, the increase in levels of NAG andβ2-MG was associated with the amount of related substances, indicating the amount of related substance in injection potential intensify the nephrotoxic effects of Lianbizhi Injections.
3. The methods of the chronic toxicity test for thirty days: the male SD rats were administered intravenously with 50, 150, and 450mg/kg of Lianbizhi Injections A, 25, 50, and 150mg/kg of Lianbizhi Injections B, and the physiological saline for 30 days, respectively. The clinical signs, body weight gain and the food consumption, examine the haematology (ERY), blood chemistry, the change of routine urine and urine enzymess were measured and necropsy and histopathology were recorded. One male in high dose (450mg/kg) Lianbizhi A group and four males in high dose (150mg/kg) Lianbizhi B group died during the administration. Clinical signs of toxicity in SD rats of the high dose Lianbizhi A and B group included: reduced food intake, slow body weight gain, significant increases the levels of urine ERY, BUN, Crea, andβ2-MG, et al. From these results, the Lianbizhi Injections were estimated to influence the glomerular filtration rate and tubular reabsorption and its potential nephnotoxic effects on rats is related to its purity and dosage.
4. In accordance with the traditional Chinese medicine, natural medicine, immunity toxicity (allergic, photoallergic) of the technical guidelines, the passive cutaneous anaphylaxis (PCA) and active systemic anaphylaxis (ASA) tests were carried out on two different purity of lianbizhi injections. The results of the allergy testing showed that positive symptoms in SD rat of the high dose Lianbizhi A and B group all occurred, and 50 percent animals in the low dose Lianbizhi A and B group showed the positive symptoms in PCA toxicity study. While in ASA toxicity study, 33.3 percent animals appeared weak positive in high dose Lianbizhi A group and 77.8 percent animals appeared positive in in high dose Lianbizhi B group and there are no anminal in the low dose Lianbizhi A and B group occurring the positive symptoms. It is demonstrated that Lianbizhi Injections in high concentrations of Andrographolidi Natrii Bisulfis (200mg·Kg-1 in SD rats, which is approximately equal to 20 times of adult clinical dosage) can cause allergic reactions. From the allergic differences of two kinds of formulations, the probability of allergic reaction would be increased by the concentration of non- Andrographolidi Natrii Bisulfis.
Main studies on the mechanisms of acute kidney injury induced by Lianbizhi Injections are as follow:
1. Technique on Metabonomics:Twenty five male SD rats were randomly divided into the five groups: the low-dose (400 mg/kg) and the high-dose(1600 mg/kg)Lianbizhi A groups, the low-dose (100 mg/kg) and the high-dose(400 mg/kg)Lianbizhi B groups, and the blank control group (normal saline). Each group comprised 5 rats. All rats received respectively a single intravenous injection of Lianbizhi 10 ml/kg via caudal vein. The rats’urine were collected within 12 hours prodose as well as within 0～6, 7～12, 13～24 and 25～48 hours after drug administration. The 1H nuclear magnetic resonance (1H NMR) spectroscopy of the rats’urine was measured.The integrated metabonomics study was applied to investigate the biochemical composition of urine obtained from two kinds of Lianbizhi Injections treated SD rats. It was found that the rat urinary metabonomic profile of dosed groups firstly deviated from the control group and then regressed the control level after dosing, reflecting the impacted information of animals by Lianbizhi Injections. The position of each sample in the principle components (PC) profile depending on its metabolic response and the changed of metabonomics profile of dosed groups were related to the toxicity of Lianbizhi Injections in 0 ~ 12 h phase urine, which the more toxic dose point was, the more distant its metabolic spectrum deviated from the control group. The levels of trimethylamine(TMA)(d2.90)and dimethylglycine(DMG)(d2.94)were increased and the levels of citric acid(d2.54,d2.66) andα-ketoglutarate (d2.46,d3.02)were reduced in 0 ~ 12 h phase urine of administrated groups compared with the control group. The mechanism may relate to the influence of the osmoregulation in renal medulla and the interference with the activity of mitochondrial enzymes due to high concerntration of lianbizhi injections, which result in reduction of energy metabolism in mitochondria, change of the mitochondrial function and destruction of membrane integrity, leading to renal cell injury, eventually affecting the concentration and dilution of the original urine.
2. Studies of the mechanism of the HK-2 cytotoxicity of Andrographolidi natrii bisulfis as follows: The morphological changes of HK-2 cells were examined with contrast microscopy and growth inhibitory rate of HK-2 cells were detected by MTT colorimetric assay. Cell-cycle arrest, apoptosis rate MMP and ROS were analyzed by flow cytometry, and the levels of apoptosis-related protein (cytochrome c (CytC), pro-Caspase-3, Bcl-2, Bax) were measured by Western blot. The studies indicated that the proliferation of HK-2 cells were markedly inhibited in dose-dependent manners and the IC50 values of andrographolidi natrii bisulfis for 24 and 48 h were (19.12±3.55), (11.56±3.881) mg/ml respectively. The toxic manifestations included: the inhibition of the cells proliferation,cells rounded and contraction, adhesion worse, some fall off, LDH leakage increasement, and cell apoptosis followed by the decreasment of mitoehondria membrane potential (MMP). Futhermore,andrographolidi natrii bisulfis can block the cell cyele, and the percentage of cells in G2/M phase was gradually inereased. ROS and MDA level increased and GSH, SOD and ATPase activity decreased in HK-2 cells. Western blotting analysis of apopotic related proteins revealed that the release of cytochrme C increased, the expression level of pro-caspase 3, Bcl-2and bax inceased signifieantly in dose- and time-dependence manner after treatment with andrographolidi natrii bisulfis, However, the ratio of Bcl-2 and Bax remained unchanged, indicating in a relatively balanced state of the two kinds proteins on the cells. These results implied that andrographolidi natrii bisulfis may lead to cell cycle arrest and apoptosis or necrosis though changing the redox state, depleting of intracellular GSH and SOD in, leading to a large accumulation of intracellular ROS,which induced lipid peroxidation, decreasing MMP, damaging the permeability of cell membrane, then apoptosis-inducing factor (AIF) releasing from mitochondria, including the CytC release, Caspase-3 zymogen activation, Bcl gene family members involved and so on.
3. To compare the differences of drug combination concerning andrographolidi natrii bisulfis and kanamycin sulfate on HK-2 cytotoxicity. The results showed that there were lots of differences in HK-2 cell morphology, cell cycle, MMP, and ROS respectively treated with andrographolidi natrii bisulfis and kanamycin sulfate, suggesting that the two drugs act on HK-2 cells in different pathways to produce toxic effects, respectively arresting the cells at different phases. Their combined effect can exacerbate the damage of renal cells.
In conclusion, based on the adverse reaction characteristics reported, we re-evaluated the safety of two kinds of Lianbizhi Injections. By general toxicity test, we found that adverse reactions of renal injury and allergenic reaction caused by Lianbizhi Injections can be reproduced on animal models. Preparation quality of Lianbizhi Injections plays an important role in these adverse reactions. Therefore, through re-evaluation of Lianbizhi Injections the pharmaceutical enterprises should improve the TCMI quality and control the content of related substances in order to reduce the risks of Lianbizhi Injection triggered adverse reactions. In clinical application we should pay attention to the dosage and the indication, monitoring the levels of urine KET, ERY, BUN, Crea, ALP, LDH and NAG so as to reduce the incidence of acute renal injury caused by Lianbizhi Injections.
The results of studies in vivo and in vitro confirmed that Lianbizhi Injections had potential nephrotoxicity in high concentration. The changes of Urinary trimethylamine, dimethyl glycine, citric acid andα– ketoglutarate in urine can be used as indicators to monitor the renal injury induced by Lianbizhi Injections. The mechanism of renal injury induced by Lianbizhi Injections may be related to affecting the osmolarity in kidney medulla and interferencing the activity of related mitochondria enzymes in renal cell, involving in affecting mitochondria energy metabolism, changing the redox state, resulting in excessive ROS, destructing the cell membrane permeability, declining MMP, mitochondrial dysfunction, leading to CytC releasing from mitochondria to trigger apoptosis or necrosis.
The toxic effects will occur when Lianbizhi Injections was used in large dose and unreasonable combination therapy. Therefore, the dosage should not exceed 10mg/kg a time, and functions of kidney should be monitored during administration. Patients with kidney disease need to adjust the dosage, and combination with kanamycinor or other aminoglycoside antibiotics must be forbidden in clinical usage. The related substances in the preparation may have great influence on the safety of Lianbizhi Injections. Therefore, quality criteria should be established to identify and control the related toxic components, as well as strengthen the monitoring of adverse reactions. With the development of metabonomics and cell biotechnology, these novel technologies might be increasingly applied in the toxicological effects of other Chinese herbal medicine in the future, which will contribute to the modernization of traditional Chinese Medicine.
引文
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