Non-clinical Safety Evaluation Of Liver-targeting Interferon(IFN-pfCSP)

Background and objective:Hepatitis b virus is a kind of epidemic virus,which do great harm to human health.The infection of HBV may cause chronic hepatitis B,hepatocirrhosis and even liver cancer.So the hepatitis b virus infection has become a global health problem.Currectly,FDA recommended drugs for hepatitis b virus(HBV)infection include biological preparation interferon,pegylated interferons and nucleoside analogue lamivudine,telbivudine,adefovir dipivoxil,entecavir and tenofovir.However,they has strong side effects,resulting in poor compliance of patients prone to drug resistance in clinical use.The complete response rate of interferon for HBV was 30%-40%,use of it can cause various adverse reactions such as leukopenia and weight loss.So now the urgent need to develop more effective and safer drugs.Malaria parasites circumsporozoite protein(pfCSP)is on the surface of the plasmodium falciparum sporozoite,containing about 400 amino acids,it consists of three structure,including conservative area I in N terminal end,the C terminal conservative II area and the central repeat area.Through intravenous injection,the recombinant pfCSP can be combined into surface of hepatocytes within a few minutes,show that CSP has a highly efficient and specific liver targeting property.Targeting peptide pfCSP I-plus and IFNa2b are fusioned through genetic engineering technology in our previous work and so we obtained liver targeting interferon IFN-pfCSP,study found that IFN-pfCSP inhibit the action of the HBV increased significantly and the effect of its specific activity is as high as 108,in rats,the action time was prolonged,AUC were increased and Its bioavailability was improved show in our Pharmacokinetic studies.Besides,Targeted evaluation show IFN-pfCSP has good liver-targeting property.On the base of the first stage of research,we evaluate the safety of IFN-pfCSP through long-term toxicity,immune toxicity,local irritation,genetic toxicity and teratogenicity.It well Predict possible toxic effects or adverse reactions in clinical trials and provide experimental basis for clinical trials of liver targeting interferon.methods and contents:1.Long-term toxicity experiment of IFN-pfCSPSD rats were divided into five groups randomly.Normal saline group,IFN-pfCSP high-dose group(6.3×107IU/kg),middle-dose group(3.15×107 IU/kg),low-dose group(1.5×107 IU/kg),IFNa2b group respectively.The dose of IFN-pfCSP administration groups in accordance with clinical equivalent dosage,generally 3 months of long term experiment,rats were administrated with 50-100 times,25 to 50 times,10 to 20 times of clinical dosage,the dosage of IFN-pfCSP high,middle and low dose group was 100,50,25 times of the maximum clinical equivalent dosage,respectively.All the rats were administrated subcutaneously once a day for 13 weeks.In the experimental period of time we observed the appearance,activity,body weight,death of each rat and so on.The blood samples were collected on the 2th,3th,5th 7th,9th,11th,and 13th week during the administration time,the anti-IFN-pfCSP binding antibodies in sera were detected by indirect ELISA and the anti-IFN-pfCSP neutralizing antibodies were detected by cytopathic inhibition method.at the end of experiment,the urine was examined.In addition,we executed 10 female and 10 male rats in each group to detect hemology index,blood biochemics index and blood coagulation index,recorded the weight of main organs and calculated viscera coefficient,at last,each tissue was determined bu pathologic observation.After 30 days observation,the rest rats of each groups were sacrificed and the above examinations were repeated.2.Immune toxicity experiment of IFN-pfCSP2.1 Delayed-type hypersensitivity experiment40 Balb/c mice were divided into five groups randomly.IFN-pfCSP low-dose group(1.87×107 IU/kg),middle-dose group(5.6×117 IU/kg),high-dose group(1.68×108 IU/kg)(corresponding to 24,72 and 216 times of human clinical dosage respectively),the negative control group received same quantity of normal saline,positive control groups were administered cyclophosphamide(150 mg/kg·bw).The mice of IFN-pfCSP treatment groups and negative control group were administrated subcutaneously once a day for 14 days,while the mice of positive control group were administrated subcutaneously only once on the 9th day.The mouse model of delayed type hypersensitivity was established by sensitizing the skin of mice in the groups except the negative control group with 50 ?l,1%2,4-dinitrofluorobenzene(2,4-DNFB)on the 9th day of treatment,after 5 days,all animals were again challenged with 10 ?l,1%2,4-dinitrochlorobenzenein smearing on the right ear(effector phase).The left ear was smeared with the same volume of solvent serve as control.After 24 hour,all the mice were sacrificed and the weights of ear,thymus and spleen were analyzed.2.2 Haemolytic plaque test40 male Balb/c mice were randomly divided into 5 groups according to the weight,each of 8 animals,the dosage of IFN-pfCSP treatment groups were the same as delayed-type hypersensitivity experiment groups.The mice of IFN-pfCSP treatment groups and negative control group were administrated subcutaneously once a day for 14 days,while the mice of positive control group were administrated subcutaneously four times.3 days before the last administration all mice were immunized by i.p.injection of 0.2 mL sheep red blood cells,after 4 days,all the mice were sacrificed and spleen single-cell suspensions were prepared at a cell density of ×106 cells/mL in Hanks.0.7%agar powder was dissolved and 0.5 mL of it was added to a test tube at 48?,0.1 mL SRBC,0.05 mL of 1%dextran,0.1 mL spleen cells were preheated to 40 degrees and then were added to the test tube in order,it was poured into the 40?underlying agar surface after mixed quickly and fully,and then incubated for 1 h at 37? after 15 minutes,1.5 mL of 1:30 dilution of guinea pig complement was added to it and incubated again for 30 minutes at 37?,incubated for 4 hours at room temperature and then overnight at 4 0C,the number of plaques in each sample was then counted.3.Local irritation experiment of IFN-pfCSPMethod of self-contrast left and right side in rabbit was adopted to study the local irritation experiment of IFN-pfCSP,4 rabbits,weighting about 2 kg,before the experient,animal hairs on the back of left and right sides was removed with an electeic razor.In advance,use the 3*3 cm square of the mark and administration of drugs in this area.Due to the local irritation experiment should be given in accordance with the principles of clinical use,the dose was 0.136 mg/mL,0.5 mL/kg,subcutaneous injection of IFN-pfCSP on the left side of rabbit according to the prediction of clinical dosage and subcutaneous injection of normal saline on the left side.All the rabbits were administrated once a day for 3 days.during the administration period,the animals and injection sites were observed by visual inspection,stimulation phenomenon such as redness,congestion in the skin at the site of the injection were recorded.After the last administration,two rabbits were sacrificed and skin tissues from the injection sites were taken for pathological examination.The other two animals were continue observed for 2 weeks,and the recovery of the injection sites were recorded every two days.After the observation,the animals were sacrificed and the same pathological examination was performed.4.Teratogenicity test of IFN-pfCSPAdult SD rats were kept in the same cage and the ratio of female and male was 2:1,morning the finding of sperm in the female vaginal smear identified the copulation of the rats and the day was regarded as the zero-day of fertilization.The detection of pregnant rats were randomly divided into five groups,IFN-pfCSP high-dose group,middle-dose group,low-dose group,the dosage of IFN-pfCSP high-dose group was the same as maximum ineffective dosage in the long-term toxicity experiment,the dosage of IFN-pfCSP dose groups were 100,50,25 times of the maximum clinical equivalent dosage,respectively.Other two groups were the negative control group and Positive control groups.Rats in the three test groups and negative control group was administrated in the gestation of 6 to 15 days,the positive control group was injected subcutaneously with cyclophosphamide(12 mg/kg)on gestational day 12.Record the weight of pregnant rats during pregnancy and on the 20th day,the pregnant rats were sacrificed and uterus weight,sex,wet weight,dry weight,appearance,body length,tail length,embryo rate and teratogenic rate of fetus were examined.5.Genetic toxicity experiment of IFN-pfCSP5.1 Bone marrow micronucleus test30 male kunning mice,were randomly divided into five groups:IFN-pfCSP high-dose group,middle-dose group,low-dose group,the maximum dose as high dose group base on national standards of the People's Republic of China GB15193.5,the mice were given 0.4 mL IFN-pfCSP per 20 g body-weight,the dosage of IFN-pfCSP dose groups were 1128.2?112.82?11.282 times of the maximum clinical equivalent dosage,respectively.The other groups were the negative control group and positive control groups(Cyclophosphamide,40 mg/kg·bw).all the mice were administrated twice with a 24 h interval,at 6 hour after second subcutaneous injection,mice were sacrificed and femur marrow was smeared on slides,fixed with methanol.stained with Giemsa dye,red blood cells(RBC)and polychromatic erythrocytes(PCE)were examined under microscope,number of micronucleus normochromic erythrocytes(NCE)in 200 NCE,and nmber of the micronucleus PCE in total 1000 PCE per mouse were counted.5.2 Mice sperm abnormality test30 male kunming mice were divided into five groups:IFN-pfCSP high-dose group,middle-dose group,low-dose group,base on national standards of the People's Republic of China GB15193.7,and the dosage of IFN-pfCSP treatment groups were the same as mice sperm abnormality test groups,the other groups were the negative control group and positive control groups(clophosphamide,40 mg/kg·bw).mice were injected subcutaneously once a day for 5 days,thirty-five days after the first administration,all the mice were sacrificed,weight gain and testicular weight were recorded,the epididymis was isolated,placed into a culture dish containing normal saline and cut into pieces,the treated suspension was smeared onto the slide,air-dried,fixed with methanol,the slide was stained with 2%eosin,a total of 1000 structurally completed sperms were scored for each animal under microscope at high magnification to evaluate the incidence of sperm deformity,sperms appeared as normal,folded tail,fat head,double heads,double tails banana-shaped,no hook and amorphous.5.3 Ames assayFour characterized histidine-dependent stains of Salmonella typhimurium(TA97,TA98,TA100,and TA102)were utilized for bacterial reverse mutation assay.First to identify the biological characteristics of the four strains,including histidine require-ment,R factor and pAQl plasmid,uvrB mutation,deep rough type and spontaneous revertant colonies.After strains identification qualified,the plate incorporation method was used for assay,experiment is divided into negative control group,five different concentrations of IFN-pfCSP groups(44.00 ?g/plate,8.80 ?g/plate,1.76 ?g/plate,0.35 ?g/plate,0.07 ?g/plate),positive control group.The positive control agents included 2-AF,4-NQO,NaN3 and MMC.2.0 mL top agar containing 0.5 mmol/L histidine and 0.5 mmol/L biotin solution was added to a sterile tube(45?),0.1 mL bacteria culture solution,0.1 mL test substance,0.5 mL S9 mix or sodium phosphate buffer(when S9 mixture was not addedd)were added and mixed,then poured onto minimal glucose agar plates.After top agar hardened,the plates were incubated for 48 h at 37?,the revertant colonies were counted manually.Results:1.Long-term toxicity experiment results show that no animal death,animal taking food,drinking water,moving were normal in the term of taking drugs and following observation for 4 weeks.Body mass in IFN-pfCSP experiment groups was no significant difference compared with solvent control group.Bisides,IFN-pfCSP had no obvious harmful influence on the index of hematology,urine and blood coagulation function.Blood biochemical tests results showed that administration of IFN-pfCSP may cause liver dysfunction in the middle of administration,administration of IFN?2b may also cause liver dysfunction in the middle of administration and their damage degree have no great difference,however at the end of administration,ALT,AST and ALP in each group were turned to normal,only ALP of male rats in high dose group increased as compared with the solvent control group.In the convalescence of the test,the inspections results found that the indexes of liver function were no significant difference compared with solvent control group,indicating the damage of liver function can be restored.The statistical results show that the organic coefficient of male rats,thymus increased in the IFN-pfCSP low dose group,with statistical significance as compared with the control group(P<0.05)in the end of the test,while the organ coeffocent of other IFN-pfCSP administrated group was no significant difference compared with solvent control group,the thymus index increased only observed in male low dose group,it did not show a dose-response and thymus in histological examination also didn't find abnormal,so IFN-pfCSP will have little impact on the viscera coefficient,besides the histologic examination did not see obvious pathological changes area.The detection results of binding antibodies show that the binding antibodies were induced after 2 weeks of administration and showed a certain dose-response relationship,the level of binding antibody tilers continue to increased after 8 weeks of administration and reached the maximum value at 11 weeks which was about more than 10 times compared with IFNa2b treated group.Abnormal toxicity related to administration with IFN-pfCSP was not found in the long-term toxicity experiment,so elevated binding antibody levels of little significance and no special significance to clinical guideline.2.Delayed-type hypersensitivity results show that when the concentration of IFN-pfCSP was 1.87×107 IU/kg(clinical dosage of quasi 24 times),5.6×107 IU/kg(72 times of clinical dosage of quasi)or 1.68×108 IU/kg(216 times of clinical dosage of quasi),weight gain in mice was not affected,the ear swelling degree,spleen index and thymus index compared with solvent control group had no significant difference.Hemolytic plaque test results show that there were no significant difference in the number of hemolytic plaque between the three administered groups and the vehicle group.3.The rabbits in skin irritation experiment showed good mental state and normal activities,macroscopic observation and histopathological examination did not find erythema,purple scab,swelling,congestion,ulcers and other skin irritation.4.In the traditional teratogenic test,the number of fetal rats,wet weight,body length,tail length,dry weight in all IFN-pfCSP treated groups shows no significant difference compared with those in the solvent control group,in each IFN-pfCSP treated groups,fetuses appearance had no abnormal changes,skeletal and visceral also found no abnormalities.5.The negative results were observed in the genetic toxicity test of micronucleus test of bone marrow cells and sperm shape abnormality test in mice when the dosage of IFN-pfCSP was 1128.2 times higher than clinical dosage.Otherwise,the Ames test showed that IFN-pfCSP treatment did not significantly increase the revertant colonies in plates of four strains TA97,TA98,TA100 and TA102 with or without S9 activation when the concentration in the 0.07 ?g/plate-44.00 ?g/plate range,respectively.Conclusion:Long-term toxicity tests show that liver function of IFN-pfCSP treated groups may have a transient damage,but liver function of IFNa2b control group also have a transient injury,other physiological and biochemical indices no significant toxicity changes,suggesting that repeated use of IFN-pfCSP is safe;Besides IFN-pfCSP had no significant effect on cellular immunity and humoral immunity when the dosage of IFN-pfCSP was 216 times higher than its largest clinical dosage;skin irritation experiment showed that the subcutaneous injection of IFN-pfCSP to normal skin without no irritation;In the Traditional teratogenic test,IFN-pfCSP had on developmental toxicity on fetuses and had not teratogenic effects on mouse embryos;Three genetic toxicity experiment results are negativethe,so the non-clinical safety evaluation showed that IFN-pfCSP had no significant side effects on the body.