Toxicology Of The Recombinant Fusion Protein RMBP-NAP

The recombinant Helicobacter pylori neutrophil activating protein(Hp-NAP) fused with the maltose binding protein of Escherichia coli(rMBP-NAP),as a novel immunomodulatory drug candidate which can be used for immunotherapy of cancer and other diseases related to abnormal immune regulation. This study aimed to investigate the toxicologic profiles of r MBP-NAP in animal models, containing acute toxicity, long-term toxicity, micronucleus test and mutagenicity test.(1)Preparation of rMBP-NAPProliferation of E. coli TB1 with exogenous gene pMAL-c2x-napA and pMAL-c2 x was proceeded at 37 °C for overnight. The positive clones of TB1(pMAL-c2x-napA) were cultured at 37 °C with IPTG for 3 h. The harvested cells were sonicated, followed by centrifugation at 4°C to obtain the supernatant for SDS-PAGE. Then, the protein rMBP NAP were collected after affinity chromatography purification and dialysis with PBS. The expression level was evaluated by the percentage of rMBP-NAP accounted for total protein by the gel image analysis system together with the program Image J2 X. The protein amount was determined by BCA kit following the manufacturer’s instructions.(2)acute toxicity of rMBP-NAPAccording to the results of pre-test, we used the method of maximal torlerance dose(MTD) to evaluate the acute toxicity of rMBP-NAP in mice. Kunming mice of each sex were randomly placed into 2 groups of 30 animals, 20 of the treatment group and 10 of the negative control group with PBS. Mice in the experimental group were administered a maximal concentration of rMBP- NAP(125mg/kg,25-fold of the proposed clinical dose) by iv single injection in the tail. After injection, the average food consumption, body weight, and lethality were observed everydays. In acute toxicity tests in mice, the maximum tolerated dose was 125mg/kg and no deaths were recorded during the treatment days.(3)long-term toxicity of rMBP-NAPAccording to the results of pre-test, the doses of 21 and 42mg/kg/day rMBP- NAP for 90 days was chosen. Healthy male and female rats were randomly assigned to two experimental groups and a control group. Each group included 10 rats of each sex. PBS was given to the animals in the vehicle control group. After 90 days, some rats were killed and the blood samples were taken for haematological examination and biochemisty determinations. The body weight and their relative organ weight(percentage of body weight) was also calculated. The other rats were also killed after the 30 day’s recovery and same parameters were checked. In long-term toxicological studies, there were statistically significant differences in the body weights between the control and treatment males. The administration of 21 mg/kg rMBP- NAP resulted in a significant decline, while 42 mg/kg rMBP- NAP also caused a significant reduction in the final body weight of the animals when compared with the control. There were no treatment-related gross findings at the time of necropsy, with the exception of the enlargement of the liver and spleen exposed to all the rats treated with rMBP- NAP. Some biochemisty changes were observed in the treatment groups. The total protein exhibited a drug dose dependent elevation and albumin showed a significant decrease, when compared with the saline treated group. The estimation of creatinine and cholesterol revealed significant differences in male animals. No significant differences were noted with regards to hematology between the control and treatment groups.After 30 days recovery phase,all the rats have been able to return to the normal.(4)micronucleus test of rMBP-NAPKunming mice of each sex were randomly placed into 5 groups of 50 animals, including negative control(PBS), positive control(cyclophosphamide) and three treatment groups of rMBP-NAP(5mg/kg, 25mg/kg, 50mg/kg i.v). After five days for consecutive injection, all the mice were killed and the bone marrow were taken. The results showed that no significantly increased proportion of micronucleated polychromatic erythrocyte(MnPCE) was observed when rMBP-NAP were administered by intravenous injection at all doses.(5)Bacterial reverse mutation assay of rMBP-NAPThe study designed four S.typhimurium(TA97,TA98,TA100 and TA102) to test the mutagenicity of rMBP-NAP in two conditions, including added S9 to the medium or not. The dose levels of rMBP-NAP were 50μg/plate, 200μg/plate, 1000μg/plate and 4000μg/plate respectively. The results showed that rMBP-NAP caused genetic mutations about TA98, TA100 and TA102 in the range of 2000-4000μg/plate, and in the range of 1000-4000μg/plate about TA97.In conclusion, these studies used acute toxicity, long-termtoxicity, micronucleus test and bacterial reverse mutation assay to test the safety of rMBP-NAP. The results showed that the target organs were liver and kidney and rMBP-NAP was mutagenic when treated in an overdose. No significantly increased proportion of micronucleated polychromatic erythrocyte was observed,too. If further drug development of the rMBP-NAP fusion protein is successful, our study strongly support for the candidature of rMBP-NAP for preclinical trials.