Study On The Safety Re-evaluation Of The Lianbizhi Injectas

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.