| 1. Establishment of a biological background database of rhesus monkey (M. m. Lasiotis) A rhesus monkey (M. m. lasiotis) population that contains 2000 monkeys was established under the support of National Science & Technology Infrastructure program "National Source Base of Experimental Rhesus Monkey Subspecies M. m. Lasiotis". And the research on the biological background data of these monkeys has been conducted. (1) Results showed that the body weight and ength of monkeys under age of 3.5 were significantly less than those of other domes(?)c rhesus monkeys. However, as their body weight was similar to that of Cynomolg(?)s monkeys, M. m. lasiotis may have the feature of relatively small. (2) The reference range for blood biochemistry and hemagglutination was established based on over 400 monkeys aged 2.5 to 5. The influences of different conditions of artificial feeding and different levels of domestication (e.g. introduction of wild species, adaptive breeding for 30 days, standardized breeding for one year, standardized breeding for two years, and completely domesticated) on blood biochemistry were analyzed. The results showed that standardized artificial breeding and domestication had dramatic influences on the experimental data, and therefore feeding and domestication hould be carefully planned and implemented during experiment. During the artificial domestication, the fluctuation ranges of various indicators including ALT, AST, ALP GGT, BUN, GLU, and CK gradually declined and these values were in the reference range of human beings. After the monkeys were successfully domesticated, the values of serum biochemistry and hemagglutination were most similiar with the reference ranges of human beings. These results provided important basic data for the use of M. m. lasiotis in the safety evaluation of new drugs and in the research of human diseases.2. Assessment of long-term toxicity of metacavir on rhesus monkey (M. m. lasiotis) after three months of intravenous administration. Metacavir is a novel deoxyguanosine analog for the treatment of HBV. As a part of the safety assessment of metacavir, a 3-month repeated dose toxicity study was performed on rhesus monkey (M m. lasiotis).24 monkeys were randomly divided into metacavir 120 mg/kg group, metacavir 40 mg/kg group, metacavir 13 mg/kg group, and control group. Drugs (or placebo) were intravenously administered for three months. The appearance, ECG, and indicators including urine, hematology, blood biochemistry, T cell subsets and CD4+/CD8+ ratio, serum IgG titer, serum IFN-r, and bone marrow were observed or measured. The results showed that the effects of long-term administration of metacavir on the body weight, temperature, fundus, and ECG were not biologically significant. Apparent gastrointestinal toxicities occurred in the metacavir 120 mg/kg group and the animals experienced nausea, vomiting, and decreased food consumption in the early stage of dosing; however, all these adverse effects gradually abated. Hematological injuries showed as anemia in this group, including decrease of hemoglobin level, compensatory increase of bone marrow erythrocytes, and increase of reticulocytes. Hepatic functions were also damaged at certain levels, which affected the production of proteins (e(?)pecially albumin), resulting in the decrease of total proteins and albumin. The above changes were reversible after drug withdrawal but it is important to monitor the changes of hematological and blood biochemical indexes in clinical trials, especially the changes of hemoglobin and albumin. Metacavir 120 mg/kg can reversibly increase the number of T lymphocyte subset CD3+, and the ratio of CD3+/CD4+, CD3+/CD8+ of monkeys. Furthermore, the administration of metacavir did not cause remarkable change of CD4+/CD8+ ratio.No metacavir-related immune toxicity was observed. Therefore, the main toxic target organs of metacavir are gastrointestinal tract, liver, and blood. The no-observable-adverse-effect-level (NOAEL) of metacavir in rhesus monkeys is considered to be 13 mg/kg.3. Research on the mitochondrial toxicity of metacavir on HepG2 cells to validate the sensitivity of experimental monkey models Objective:To evaluate the potential mitochondrial toxicity of drugs using human hepatoma HepG2 cells, and to provide evidence for clinical dose design and main indicato(?)s in clinical trials. Method:HepG2 cells were treated with drugs including 250μM metacavir,50μM metacavir, 10μM metacavir,50μM AZT (6 times of Cmax), and 50μM ADV (745 times of Cmax) for 9 days. Lactate content in culture solution, and activities of mitochondrial respiratory chain complexes were measured. The morphology and structure of mitochondria were observed under transmission electron microscope. The mitochondrial DNA (cytochrome b gene) and nuclear DNA (Actin gene) were quantified by qPCR. Cytochrome b/actin ratio was used to assess the effects on mitochondrial mtDNA content and the level of injury. Results:After the HepG2 cells were treated with 50μM AZT for 9 days, the cell inhibition rate (?)as about 55%. The lactate content in the culture solution remarkably increased. The mitochondrial structure was severely damaged and became swollen; some of the cristae disappeared, resulting in vacuolization.The mitochondrial respiratory chain proteins were remarkably decreased about 60%, and the content of mtDNA significantly decreased over 70%. After the HepG2 cells were treated with 50μM ADV for 9 days, the cell inhibition rate was about 30%. The lactate content in the culture solution remarkably increased. The mitochondria showed relatively complete structure. The activities of mitochondrial respiratory chain complexes increased and the content of mtDNA decreased slightly. After the HepG2 cells were treated with 250uM PNA for 9 days, the cell inhibition rate was about 48%. The lactate content in the culture solution remarkably increased. The mitochondrial structure was complete and the density of cristae became relatively low. The activities of mitochondria respiratory chain complexes and the content of mtDNA somehow declined. After tho HepG2 cells were treated with 50μM metacavir or 10μM metacavir for 9 days, tht cell inhibition rate was about 20% and 10%, respectively. The lactate content in the (ulture solution was not increased. The mitochondria showed relatively complete structure and no obvious damage was observed. The treatment had few effects on the activities of mitochondrial respiratory chain complexes and the content of mtDNA. Conclusion: 50μM AZT can damage the mitochondria of HepG2.250μM PNA and 50μM ADV can cause certain mitochondrial injuries. The potential mitochondrial toxicity of 50μM and 10μM PNA to the liver tissues are relatively weak.4. Comparative study on the clinical features of type 2 diabetic rhesus monkey (M. m. lasiotis) and human being. Objective:To establish classification and diagnosis criterias for type 2 diabetic rhesus monkey. Method:60 male monkeys (PPG<5mmol/L) were enrolled in the study. During the two-year experiment,50 monkeys aged 7-20 years were continuously fed with hign-fat monkey diet (containing 8% fat) and 10 monkeys aged 4-10 years were fed with standard monkey diet (containing .3%fat). The body weight, body mass index (BMI), fasting blood glucose, fasting plasma insulin level, and HbA1c level were measured. Intravenous glucose tolerance test (IVGTT) was performed to diagnose IGT monkeys and the insulin secretion sensitivity. WHO 2006 diagnostic criteria for diabetes were adopted to analyze the data in this study. Results:Of 50 monkeys fed with high-fat monkey diet,8 developed overt diabetes,26 showed a tendency to diabetes (at the stages of IFG or FPG), and blood glucose of 16 monkeys were normal. All IGT and at the early stage of T2DM monkeys showed obesity and compensatory increase of EPI. These monkeys’rate of glucose clearance (KGluc5-20) after meals re(?)larkably decreased, secretion of first-phase insulin decreased, and secretion of second-phase insulin was retarded, compared with the healthy ones. HbA1c level increa(?)ed along with the glucose level. The clinical features of monkeys at the advanced stage of chronic hyperglycemia were emaciation and weakness, polyuria, and polydipsia, increased FPG; HbA1c≥8.9%; remarkably declined FPI; insulin secretion increasing within half an hour after meal but declining immediately. An HbAlc level of 4.5-5% was a high risk factor of the development of T2MD. Conclusion:The disease course, clinical manifestations, and risk factors of T2DM rhesus monkey (M. m lasiotis) were similar with those of human being. Therefore, rhesus monkey (M. m lasiotis) can be a good animal model for research on the new therapies for type 2 di betes. |
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