Exploration And Application Research On Key Techniques Of Juvenile Animal Toxicity Study For Pediatric Drugs

Safety must be considered in paediatric medicine.The safety data of adult animals or the clinical data of adults directly used in pediatric drugs are risky,so it is necessary to use juvenile animals to investigate the safety and effectiveness of pediatric drugs.This study established and optimized the juvenile animal toxicology evaluation technology system,and successfully applied it to the safety evaluation research of new pediatric Chinese medicine drugs.1.The technical system of toxicology test in juvenile animals(rats)was established and optimized,main key technologies were improved.a)Comparison of different rearing methods for post-weaning:to observe the effects of the two rearing methods on the general clinical symptoms,body weight and growth and development of juvenile SD rats.The results showed that the two rearing methods had no significant effect on the growth and development of young rats.b)Study on the earliest start age and maximum administration volume of different administration routes: research on juvenile rats from 3 days to 20 days of birth.Oral administration,juvenile rats can be given pure water at the volume of 1.0m L/100 g and2.0m L/100 g at the earliest 3 days after birth,and 3.0m L/100 g at the volume of 5 days after birth.Intravenous administration can be administered as early as PND20 with a maximum dose of 3.0m L/100 g.Intramuscular injection can be administered at the earliest in PND3-PND5 at the maximum dose(unilateral)0.5m L/100 g.For subcutaneous injection,juvenile rats can be given at a volume of 0.3m L/100g(injected at 4 sites,1.2ml/100g)in PND2,and1.0m L/100g(injected at 4 sites,4ml/100g)in PND5.c)Establishment and optimization of multiple jugular vein blood sampling techniques inconscious juvenile rats: after optimizing blood collection tools and blood collection methods,six jugular vein blood samples were taken from juvenile rats in PND45,and the success rate was over 90%.d)Preliminarily explore the blood and biochemical reference values of juvenile SD rats of different ages.e)Research on optimization and standardization of observation indicators: In this study,the growth and development indicators of juvenile SD rats(weight,top hip length,femur length),bone development indicators(bone density,osteocalcin,femur length),behavioral indicators(modified Irwin’s behavior evaluation,Observation indicators and technical methods such as water maze and open field experiment have been screened,optimized,and standardized,making these indicators easier to operate in a unified manner and more in line with GLP specifications.2.Study on the application of juvenile animal toxicology test technology system in the safety evaluation of new pediatric drugs.In this study,a comprehensive young animal toxicology safety evaluation study was carried out on a new pediatric traditional Chinese medicine-compound Taraxacum mongolicum extract(CTME),based on the preliminarily established technical system for juvenile animal safety evaluation test.In the experiment,CTME was administered orally to juvenile SD rats(PND27～PND54,covering early childhood to adolescence)for 4 consecutive weeks.After drug-withdrawal and during the recovery period,through the study of physical growth,behavior(learning and memory),bone development,immune development,sex hormone secretion and other inspection indicators,to study the possible adverse reactions of the drug to juvenile rats,doseeffect relationship and time-effect relationship,reversibility.The experimental result shows in the high dose group,the male juvenile rats slowly grew,reduced food intake and the juvenile rats of both genders demonstrated salivation,some changes in hematology(decreased red blood cell count and hemoglobin levels,increased numbers and percentage of reticulocytes),biochemistry(decreased total carbohydrates,triglycerides and glucose levels),urine(increased ketones,protein,leukocytes,specific gravity,p H and microalbumin),immunology(increased spleen weight and coefficient,and increased Ig G in male rats),mild extramedullary hematopoiesis in spleen and liver,and mild proliferation of erythroid hematopoietic cells in bone marrow.The influence could be reversed after 4 weeks of withdrawal.There were no obvious toxic effects on sexual development,skeletal development,growth hormone,behavior,memory,ophthalmology,sex hormones,or bone marrow cell morphology.No significant toxic effects were observed in the middle or low dose groups.Under these test conditions,the no-observed adverse effect level(NOAEL)of CTME in juvenile rats(PND27～PND54)was 3.8g/kg.However,a dose of 12 g/kg resulted in reversible changes in some indexes,with red blood cells being the main toxicity target.The research on the key technology system of juvenile animal toxicology test established in this study not only covers the toxicological indicators of conventional repeated dosing,but also involves specific key evaluation indicators related to the growth and development of juvenile animals,which can well predict the safety of the test drug in the pediatric population.The 4-week toxicity test of CTME in juvenile rats well verified that the key technical system is feasible.This research is based on the ICH S11 guidelines and the non-clinical safety evaluation guidelines for pediatric drugs of National Medical Products Administration in this GLP laboratory to carry out practical exploration of the optimization of the toxicity test system of pediatric drugs in juvenile animals.This study provides a scientific reference for further improving the application of juvenile animal toxicity testing in the field of non-clinical safety evaluation of pediatric drugs and the implementation of ICH S11 guidelines in China.