| The children are within a specific stage of growth and development; the physiological functions of most organs are still immature. Major functional (dynamic) and kinetic differences between neonates/infants and adults occur on body compositions, the drug disposal systems for absorption, distribution, protein binding, metabolism and elimination, and the susceptibility to the adverse effects of the drugs. However,because of the limitations of traditional evaluation paradigm for paediatric drugs, most medicinal products currently used in paediatric patients have not been properly developed for the use in this age group. Within more than a decade, we have witnessed a paradigm shift for the development of the paediatric drugs. Along with the issue of international and national regulational rules on paediatric clinical trials, the qualities and the number for the paediatric clinical trials have been substantially enhanced. Meanwhile, the pharmaceutical regulative authorities have requested more juvenile toxicological data submissions, which have provide more and more theoretical and practical significances for the clinical risk assessment of the paediatric drugs. Currently, the test guidelines for the juvenile animal toxicological studies in developed countries have been in ripen, however, the CROs in home are still lacking the experiences on the conduct of nonclinical juvenile safety studies, and the State Food and Drug Administration also has not formulated the test guideline on the Nonclinical Juvenile Safety Studies. It is an emergent issue to proveide some nonclinical juvenile safety database for such studies.Yunpi Zhixie Granule Prescription (YZGP) is a new drug currently developed by Beijing Shouyi Technology Limited; its main ingredients included muscatel, hoelen, caryophyllus, and Halloysitum Rubrum. The treatment indication for this drug is child diarrhea, and this drug has been applicated for many years in Beijing Children Hospital. The exports in Beijing Shouyi Technology Limited had taken extensively pharmacological studies on it,however, the toxicology studies on this drug is still lacking. To evaluate the safety of YZGP in pediatric patients and to provide safety data for the furture clinical trial, we have conducted a single dosing comparative toxicity study between juvenile and adult rats on YZGP and a repeated dosing toxicity study on juvenile rats. The purpose of these studies is to evaluate the potential effects on the growth and development of the juvenile animals, the general toxicity characteristics, to determine the NOAEL of toxicity effects, and to provide the nonclinical experimental data in general toxicity evaluation of this product.The toxicity of oral YZGP was determined in newborn rats, and compared with that in young rats. In newborn rats, males and females were given YZGP at 0, 12g/kg on postnatal days (PND) 14. The animals were observed for appearance or behaviors on skin, mucosa, hair and fur, eyes, respiration, autonomic activity, and central neural system activity for 2 weeks after dosing. The result showed that upon oral administration 12 g/kg of YZGP to juvenile and adult rats, there are not any specific changes on clinical manifestations and gross examinations. However, the effects on the body weight of animals are obviously severe in juvenile animals than in adult animals; and the male juvenile rats are more sensitive.The oral administration of YZGP was designed to encompass the major part of postnatal development in the rat and to evaluate potential chronic effects. The Sprague Dawley rats were crossfostered on postpartum day 9 in order to minimize possible maternal effects and assigned to dose groups. YZGP was administered orally by gavage as suspensions in sterized water, at doses of 1.33, 4, and 12g/kg/day on postnatal days 10 to 102. Control animals received an equivalent dosing volume (10 mL/kg) of sterized water. The clinical signs for dams on lactation, littering and pup feeding, and those for pups on appearance, behaviors were recorded daily. Body weight was determined daily on post-partum day 10 through 25, then twice weekly beginning on post-partum day 25 and on the day of sacrifice. Food consumption was determined twice weekly after weaning (post-partum days 28), until initiation of terminal sacrifice or, if selected for fertility assessment, until cohousing. The developmental, sex matural and reflex milestones were recordes at appropriate timepoints and inclded upper incisor eruption, eye openning, ear separation, testes descent, balanopreputial separation, vaginal patency, air righting reflex, auditory startle reflex, and negative geotaxis. Part of the study animals were taken open field motor activity testing at PND 50. The male and female animals assigned to fertility evaluations were cohoused at PND 100. The animals were sacrificed at mid-term (PND 53), dosing termination (PND103) and ending of recovery period (PND131) for gross and histological pathology examination, the blood were collected for clinical hematology and biochemistry evaluations, and the femural bones were collected for bone density determunations.The results showed that the main toxicity were manifested in the high dosage group animals which include slow body weight gain, yellow loose stools and local alopecia , during 2 weeks of preweaning dosing period. Meanwhile, the food intake of dams and juvenile pups in the last measuring timepoints of preweaning dosing period was decreased, and the eye opening day for the YZGP treated pups was delayed. There are no obvious effects on open field motor activity testing,the other milestones of growth and reflecx, fertility indices, bone density and clinical pathology parameters. After 13 weeks of YZGP treatment, the male rats in the 12 and 4g/kg dosing groups have developedα2μ- globulin nephropathy, and the male rats in 1.33g/kg dosing group have only manifested some preliminary changes ofα2μ- globulin nephropathy, such as slight renal tubule atrophy. Four weeks after dosing termination, theα2μ- globulin nephropathy in 12 and 4g/kg dosing groups have not obviously recovered, but the renal tubule atrophy in the males in 1.33g/kg group have completely recovered. On the other hand, YZGP has not exerted any obvious toxic damages to famle rats under all dosage levels, and any other pathological lesions except theα2μ- globulin nephropathy have been observed. Based on aforementional experimental result, after the Sprague Dawley rats was administration YZGP for 13 weeks, the Lowest Observed Adverse Effect Level (LOAEL) for general toxicity is 1.33g/kg. However, if theα2μ- globulin nephropathy of male rats (which are not relevant to human) is not considered, the NOAEL of general toxicity will be 4g/kg.The juvenile studies should been rationally designed and strictly conducted on a''case-by-case''basis, and being relevant to the pharmacological, pharmacokinetic characteristics of the test article, based on the requirements of corresponding test guidelines. During the conduct process of the juvenile studies, some technological difficults should be handled properly, i.e. animal dosing, animal assignment, and the statistical analyses of the data. Referred to the test guidelines on the nonclinical studies of pediatric drugs for US FDA and the European Medicine Evaluation Agency, and using YZGP as a test substance, and juvenile rata as experimental model we have gotten some technique expeiences and basic database for the juvenile animal studies (the hematology, clinical chemistry, and pathology of the juvenile rats), and have provided some suggestions on the Chinese test guideline on the Nonclinical Juvenile Studies of Pediatric Drugs. In the study, we have found, for the first time, that Traditional Chinese Medicine (TCM) formulation could induceα2μ- globulin nephropathy in male rats, which should be differiated and analyzed during the conduct of the preclinical safety evaluation of TCM formulations. By the conduct of the juvenile rat toxicological test of YZGP, our center has acquired the relevant experiences, equipments and conditions for the performance of the juvenile toxicological tests. |
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