Security Research And Toxicity Mechanism Of TR

Objective: To study the toxicity, the toxicity severity, the main target organ and the reversibility of damage in SD rats and Beagle dogs with successive TR injection, so as to find the NOAEL dose as the references for human use in clinical design and the main observation indicators. Methods: According to the weight, 80 healthy SD rats were randomly divided into 0, 25, 50, 100 mg/kg dose groups (m:f=1:1), with 20 SD rats in each group, to take intraperitoneal injection. According to the weight, 26 healthy Beagle dogs were divided into 0, 5, 15, 45 mg/kg dose groups(m:f=1:1), with 6 animals in 0, 5, 15 mg/kg groups and 8 animals in 45 mg/kg group, to take intravenous injection. The drug was administrated every day except Sunday for excessive 90 days and 30 days of recovery period. One hour after administration, the symptoms and five general indicators, including general food intake, body weight, respiration, rectal temperature, pupil were monitored in the experimental animals. At 90th day (withdrawal period) and 120th day (recovery period) after administration, half rates of each group were selected to collect blood samples to measure the hematological indexes of red blood cell, hemoglobin, hematocrit, mean cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet, white blood corpuscle, neutrophils, lymphocytes, monocytes, eosinophils, basophils, granulocytes, large unstained cells, reticulocyte, prothrombin time, activated partial thromboplastin time, thrombin time and plasma fibrinogen and blood biochemical indexes of serum alanine transarninase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, total bilirubin, blood urea nitrogen, creatinine, total protein, albumin, glucose, total cholesterol, triglyceride , creatine phosphate kinase, Ca, P, K, Na and Cl. Before administration and at 45th ,90th and 120th days after administration, measurements were conducted on the following items: the ECG indicators of heart rate, P wave voltage, PR interval, R wave voltage, QRS time, ST segment, T wave voltage and the QT interval ; the hematological indexes of red blood cell, hemoglobin, hematocrit, mean cell volume, mean corpuscular hemoglobin volume, mean corpuscular hemoglobin concentration, platelet, white blood corpuscle, WBC, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, reticulocyte, prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen; the blood biochemical indexes of serum alanine transarninase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, total bilirubin, blood urea nitrogen, creatinine, total protein, albumin, glucose, total cholesterol, triglyceride , creatine phosphate kinase, serum Ca, P, K, Na and Cl; urine indictors of urine glucose, protein, bilirubin, urobilinogen, Ph, specific gravity, erythrocyte, ketones, nitrite and leukocytes.At 90th day and 120th day after administration, half of the experimental animals undergone necropsy to make the histopathological examination and bone marrow cell count and classification. Results: 1. Rat toxicity test of consecutive 90-day TR injection( 25 ~ 100 mg/kg, ip) (1) At a dose of 100mg/kg, the body weight of female rats were lightly affected. (2) No significant effect on rat hematological indices. (3) At a dose of 100 mg/kg, the Cr values of d90 in female rats were significantly higher. (4) There was no significant bone marrow toxicity. (5) No significant effect were observed on the absolute weight and relative weight of the main organs including heart, liver, kidney, lung, brain of rats. (6) No local stimulation was observed in the administration sites. 2. Beagle dogs 90-day toxicity test of TR Injection (5 ~ 45 mg/kg,iv): (1) At a dose of 45 mg/kg, the animals were observed with limbs stiff, standing instability, side lying, salivation, vomiting (stomach contents), defecation, howling (some animals), opisthotonus (n=2) and gradually became quiet, which were recovered within 1 h , indicating that there was toxic and side effects in the digestive system, nervous system and endocrine system, but the effects were reversible. The compound had no significant effect on body weight, rectal temperature, respiration and pupil of Beagle dogs. (2) There was no effect on the ECG. (3) There was no effect on hematological indices. (4) There was no effect on blood biochemical parameters. (5) There was no effect on the urine. (6) There was no significant bone marrow toxicity. (7) Pathological examination suggested no morphological evidence of injury on Beagle dogs and no blood vessels and muscle irritation. Conclusions: (1) In consecutive 90-day of rat toxicity test of TR injection (25 ~ 100 mg/kg, ip), the safe dose and toxic dose are 50 mg/kg and 100 mg/kg respectively. The target organs are digestive system and urinary system (kidney), but the toxic effects are all functional and reversible. (2) In Beagle dogs 90 days of TR Injection (5 ~ 45 mg/kg, iv), the toxic dose and severe toxic dose are 15 mg/kg and 45 mg/kg respectively. The target organs are digestive system nervous system, nervous system and endocrine system, but the toxic effects of the role are both functional and reversible. Objective: To study the whole genome changes of Beagle dog's brain and find the possible mechanism of neurotoxicity based on TR injection 90 days long-term toxicity tests of Beagle dogs. Methods: Eight brain tissues of Beagle dogs (3 from 0 mg/kg dose group, 4 from 45 mg/kg dose group, 1 from 45 mg/kg dose recovery group) were extracted to collect the RNA. After labeling and amplification, the RNA was hybridized with gene chips. The chips were scanned and differentially expressed genes were selected. The results of gene microarray were verified by Real-time PCR and Western Blot. Results: (1) The signal of hybridized chip was clear with low background and high signal to noise ratio. (2) The differentially expressed genes were screened with significant up-expression (signal ratio≧ 2) or significant down-expression (signal ratio≦ 0.5) with T-tests. (3) The results of Gene Ontology showed that there were 110 different genes, which were further divided into 16 categories, including positive regulation of cell communication, regulation of system process, regulation of multicellular organismal process, regulation of DNA metabolic process, positive regulation of signal transduction, positive regulation of protein amino acid phosphorylation, negative regulation of biological process and so on. (4) The possible signaling pathways of drug neurotoxicity included cell adhesion molecules, ECM-receptor interaction, focal adhesion, T cell receptor signaling pathway, leukocyte transendothelial migration, ribosome and glycine/serine/threonine metabolism. (5) Real-time PCR has the same result with GENE CHIP, indicating that the chip change in the gene can be verified from RT-PCR. (6) LAMC2 protein level form TR treatment group was significantly higher than the control group and NRXN1 protein level form TR treatment group was significantly lower than the control group. Conclusions: (1) The results of gene microarray and cluster analysis are valid. (2) The central nervous system toxicity of TR may be related to the following effects: by regulating genes of cell adhesion molecules pathway such as NXRN1, the neuronin was down-expressed to further affect the synaptic transmission between neurons and cell signal transduction; by regulating genes of ECM pathway such as LAMC2, the laminin was up-expressed to affect the structure and the function of central nervous system; by regulating genes of focal adhesion pathway such as ITGA6, the integrin was down-expressed to affect biological behavior between cells; by regulating genes of T cell receptor pathway ,the structure and function of T cell were interfered; by regulating genes of leukocyte transendothelial migration pathway, the inflammatory response of target organs were realized; by regulating genes of glycine/serine/threonine metabolism pathway, the synthesis of phosphatidylserine was affected to cause the central nervous system injury. Therefore, the neurotoxicity of TR is caused through multiple signaling pathways.