| Objective: The aim of study was to observe the hemolysis,allergy reaction,toxic reaction,and the target organs of fibrin sealant,which provided references for clinical dosage and main measurements in human.Methods: 1.Defibrinated rabbit blood was washed by the normal saline repeatedly to gain the precipitated erythrocytes.Dilute proper precipitated erythrocytes with normal saline to 2% erythrocyte suspension.Nine test tubes were prepared for the test: NO.1-5th tubes were fibrin sealant of different concentrations,the 6th tube was positive one with bovine albumin,the 7th tube was vehicle one with distilled water,the 8th tube and the 9th tube were componentⅠand componentⅡ,respectively.Agitated the 9 tubes lightly and observed the hemolysis outcome in 37 oC in 3 hr.2.The single dose toxicity study for mice: Forty ICR mice were randomly divided into 2 dosage groups(0,2280 mg/kg)by body weight after quarantine.All animals were intraperitoneally injected with vehicle or fibrin sealant(50 ml/kg of body weight)2 times a day and followed by a 21-day recovery period.The body weights were measured on d7,d14 and d21.A half of the survival mice were dissected and macroscopic observed on d14 and d21.3.The single dose toxicity study for rats: Forty SD rats were randomly divided into 2 dosage groups(0,342 mg/kg)by body weight after quarantine.All animals were intraperitoneally injected with vehicle or fibrin sealant(15 ml/kg of body weight)2 times a day and followed by a 21-day recovery period.The body weights were measured on d7,d14 and d21.A half of the survival rats were dissected and macroscopic observed on d14 and d21.4.The single dose liver treatment toxicity study for rats: Forty SD rats were randomly divided into 2 dosage groups(0 and 342 mg/kg)by body weight after quarantine.All animals were laparotomized and sprinkled livers with vehicle or fibrin sealant(15 ml/kg of body weight)2 times a day and followed by a 21-day recovery period.The body weights were measured on d7,d14 and d21.A half of the survival rats were dissected and macroscopic observed on d14 and d21.5.The repetitive dose toxicity study for rats: Eighty SD rats were randomly divided into 4 dosage groups(0,86,171,and 342 mg/kg)by body weight after quarantine.All animals were intraperitoneally injected with vehicle or fibrin sealant daily for 14 days and followed by a 28-day recovery period.The clinical signs,hematological and biochemical indices were measured on d14,and d42.A half of the survival rats were dissected at d14 and d42,and performed with the examination of histopathology.6.The repetitive dose toxicity study for dogs: Twenty four dogs were randomly divided into 4 dosage groups(0,8.6,17.1,and 34.2 mg/kg)by body weight after quarantine.All animals were intraperitoneally injected with vehicle or fibrin sealant(1.5ml/kg of body weight)daily for 14 days and followed by a 28-day recovery period.The clinical signs,ECGⅡ,hematological,biochemical and urine indices were measured on d0,d7,d14,and d42.The average of the results,which were tested two times before the administration,was used as that of d0.A half of the dogs were dissected on d14 and d42,and performed with the examination of histopathology.7.Rat passive cutaneous anaphylaxis: Male and female rats were randomly divided into 4 groups(the low and the high group of fibrin sealant,the positive and the vehicle group)by body weight.The 4 rats with half males and half females were in each group of test B(the test for preparation of antibodies).The 6 rats with half males and half females were in each group of test A(the test for sensitization and motivation).In test B,the low and high groups of fibrin sealant(34,and 171 mg/kg)were intraperitoneally injected with fibrin sealant(7.5ml/kg of body weight)every other day.The vehicle group was dosed with normal saline by 7.5ml/kg and the positive group was dosed by bovine albumin by 5 mg/alone.All of them were administrated 3 times totally.Fourteen days after the last administration,we collected their blood to get antiserum.In test A,rats of the four groups were intradermal injected with their own antiserum(1:2,1:4,1:8,and 1:32)in 0.1ml to precede the passive sensitization.Forty eight hours after intraderma injection,rats of the positive group were intravenous injected with 0.5 ml bovine albumin and 0.5 ml 1 % Evans blue to provocation.Rats of the other 3 groups were intraperitoneally injected with 7.5 ml/kg provocative antigens and intravenous injected with 0.5 ml 1 % Evans blue to provocation.Thirty minutes after provocation,we executed all the rats from 4 groups by anesthesia,cut their skin of back,and assay the locus ceruleus.8.Guinea pigs passive cutaneous anaphylaxis: 24 guinea pigs were randomly divided into 4 dosage groups(the low and the high group of fibrin sealant,the positive and the vehicle group)by body weight after quarantine.Rats of each group were 6 with half male and half female.The low and high groups(34,and 171 mg/kg)were intraperitoneally injected with fibrin sealant.The vehicle group was dosed by normal saline with 5 ml/kg body weight and the positive group was dosed by bovine albumin with 5 mg/alone.All of them were administrated five times in 10 days.Fourteen days after the last administration,we treated the guinea pigs with 3 times of the allergize dosage to provocation.Results: 1.No erythroprecipitin or hemolysis were observed in 3 hr after administration.2.No absolutely adverse reaction was observed in ICR mice after administration on the 1st day.One mouse of dosage group was died on two days after administration,with no obviously abnormality after dissection.Survival mice dissected on d14 and d21 were found decreased residual drugs in abdomen by the time.And we didn’t find other abnormalities in the mice.The weights of survival mice increased on d7,d14,and d21.3.The weights of rats increased on d7,d14,and d21.The weights of male dosage rats had a decrease tendency compared with the vehicle group,and have the statistical significance vs.the vehicle group on d7.Residual drugs were found in abdomen on d14.However,no residual drugs were in abdomen on d21.4.The weights of rats increased on d7,d14,d21.The weights of male dosage rats had a decrease tendency compared with the vehicle group,and have the statistical significance vs.the vehicle group on d7 and d14.Rats dissected on d14 were found residual drugs on the surface of the liver.Only one rat was found residual drugs on liver after dissection on d21.5.Increase of white blood cell count and decrease of fibrinogen in d14 were found in 171.0 mg/kg and 342.0 mg/kg dosage groups.Furthermore,the tendency of increased weights of spleen was found in 171.0 mg/kg and 342.0 mg/kg dosage groups.Pathological exams of peritoneal cavity found that there were granulation tissues containing the FS in some of the rats in 342.0 mg/kg group.All of these changes reversed after the recovery period.6.(1)Part of dogs from low and high dosage groups were partly eating,vomiting and loosing stools after administration.It gradually recovered 3~5 d after first administration.(2)The indices of eosinophil of the high dosage group had a rise tendency on d14,and recovered on d42.(3)The index of alanine aminotransferase of one dog from the high dosage group raise on d7 and d14.The indices of glucose of the high dosage group were lower on d14 than d0.The indices of lactate dehydrogenase were higher on d7 and d14 than d0,and basically recovered on d42.7.No locus ceruleus were found in the vehicle group or the high dosage group.Locus ceruleus with diameter exceed 5 mm was found in the positive group.Locus ceruleus with diameter exceed 5 mm was found in one rat of the low dosage group.8.No allergy symptom was found in the vehicle group.Obviously allergy symptoms were found in the positive group.The low and high dosage groups were found weakly positive allergy symptoms.Conclusion: Fibrin sealant didn’t provoke the hemolysis in vivo;The lethal median dose of fibrin sealant administrated on ICR mice was exceed 2280 mg/kg;The maximum tolerated dose of fibrin sealant administrated on SD rats was 684 mg/kg;The maximum tolerated dose of fibrin sealant sprinkled on SD rats’ livers after celiotomize was 342 mg/kg;The safety dose in repeated test of SD rats is considered to be 86 mg/kg,and the toxicity dose is 171.0 mg/kg.The target toxicity system of fibrin sealant in SD rats is hematological systemand the injection site;The safety dose in repeated test of Beagle dogs is considered to be 8.6 mg/kg,the toxicity dose is 17.1 mg/kg,and found no severe toxicity dose on 34.2 mg/kg.The target toxicity system is digestive system(liver and gastrointestinal tract),and the toxic effect of fibrin sealant is reversible;SD rats intraperitoneally injected by fibrin sealant at 34mg/kg caused the passive cutaneous anaphylaxis;Guinea pigs administrated by fibrin sealant at 34~171 mg/kg caused weakly positive active systemic anaphylaxis.We should focus on the influences of fibrin sealant on digestive system(liver and gastrointestinal tract)and hematological system(blood cell differential and blood clotting indices)in clinical trials.As the fibrin sealant is made from pig,which is heterogenetic to people,attentions should be paid on the influences of immune system in clinical trials as well.Withdrawal the fibrin sealant or treatment the symptoms should be done once the adverse reaction happened. |
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