| Objective:ZBH201001 was designed and synthesized based on the chemical structure of alamifovir by the researchers in Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences. In this study, both single and repeat dose toxicity testing were carried out in Beagle dogs to investigate the toxic effects and acute toxicity of ZBH201001, the reversibility of damage and the toxic target organs, then to confirm non-toxic reaction dose (NOAEL) of this drug. This paper is aimed at providing a reference and data for clinical trial dose and safe range, as well as potential guidance for its clinical application and new drug registration.Methods:(1) The acute oral toxicity test in Beagle dogs:The approximate lethal dose method was adopted in our study. The toxic indices of ZBH201001in Beagle dogs were investigated following a single gavage administration at different doses:118.0,264.0,594.0,890.0,1334.0 and 2000.0mg/kg body weight, respectively. Then we observed the general clinical observations, bodyweight, food intake, body temperature, ECG, hematology, blood biochemistry, and urine chemistry.3 weeks after administration, all the animals were anesthetized following gross anatomy and histopathological examination.(2) Repeated dose 30-day oral toxicity study in Beagle dogs:three dose groups were designed:6.0,12.0,24.0 mg/kg body weight and corn oil solvent control group, and administered dogs once daily for 9 consecutive days, then adjusted the doses down to 3.0,6.0 and 9.0 mg/kg till the end of the test. After the end of administration, there was 4 weeks for recovery. During the administration and recovery period, general clinical observations, body weight, food intake, body temperature, ECG, hematology, blood biochemistry and urine chemistry were documented. And at the end of the administration or recovery period, gross anatomy and histopathological examination were carried out according to the plan.Results:(1) Beagle Dog’s single oral gavage:in the group of 594.0 mg/kg and above, vomiting and occasional loose stools were observed in all the animals after administration and/or the next day. Then we observed the animal feeding conditions were poor, the weight continued to decrease. After 3weeks, the animal feeding condition gradually improved and weight also increased, weight increased. In the 890.0 mg/kg dose group, a female animal was accidental dead at 12 days after administration. So a supplemental male Beagle Dog was administered at the same dose of ZBH201001, then the animal survived. At the group of 118 mg/kg and 264.0 mg/kg, we observed the weight of animals was decreased slightly and the grazing was reduced after administration. Except the hematological, blood biochemistry, and urine chemical indicator were changed at individual time points in higher dose group, the body temperature and electrocardiogram have no significant toxicological relevant and other changes. Histopathological examination:The accidental death animal autopsy showed no obvious abnormality except for the weight loss in the group of 890.0 mg/kg. Microscopic examination showed severe pulmonary edema, hemorrhage, alveolar epithelium, gastrointestinal system lesions appear; the survived 6 animals were sacrificed and visceral surface inspection showed no abnormalities. The animal in group of 1334.0 mg/kg showed body recombination liver focal liver cell degeneration, necrosis scattered, colonic bleeding, epithelial cell necrosis, inflammatory cell infiltration; The animal in group of 2000.0mg/kg showed body recombinant animal gastric mucosal necrosis, edema. Other animal organs showed no abnormalities.(2) One month Beagle dogs repeated dose toxicity tests:after administrated the animal with 6.0,12.0 and 24.0 mg/kg ZBH201001 for 9 days, male and female animals were exhibited gastrointestinal toxicity, including loose stools, poor grazing conditions, weight loss and other symptoms, and showed obvious dose-and time-response effects. So, we adjusted the dose to 9.0,6.0 and 3.0 mg/kg. The animals in 9.0 mg/kg showed significant food-intake reduction and body weight decrease, and gradually sparse and/or bloody stool, with weight loss, reduced activity and other clinical signs; the body weight of animals in 3.0 mg/kg and 6.0 mg/kg were lower than the control at the same time point, but it remained at a stable level, not further reduced. Both before and after the dose adjustment, the food consumption and body weight changes were showed similar trends in all groups. The ECG, body temperature, and ophthalmologic examination were showed that no toxicological relevant changes in low, medium and high dose group. The blood tests showed that the activated partial thromboplastin time (Activated Partial Thromboplastin Time, APTT) was decreased both in the middle and end of the administion in high dose group animals. This change was recovery to baseline level in the recovery period, suggesting that the drug may affect the endogenous coagulation system. No obviously changes were observed in urine analysis. Histopathology, we observed significant toxic effects in gastrointestinal system in medium and high doses. We also observed an obvious toxic effects on thymus, testis, prostate, pancreatic atrophy, bone marrow, erythropoiesis inhibition of fatty liver cell swelling, degeneration. Above-mentioned organizations/organ lesions were recovery in recovery period, which indicating the toxic effects were recoverability. At the end of administration, all the animals were observed pulmonary edema, alveolar epithelium, inflammatory cell infiltration and other diseases, these phenomena were not found in all of the animals after the recovery period. Considering oral administration with suspension can induce occasional cough reaction ocationally. Also, the incidence and severity of lung lesions did not have dose-response effects. So, we considered the observed lung disease may due to the operation procedures, such as administration.Conclusion:The conclusions of our study are showed as follow, (1) The MTD of the acute oral toxicity test in Beagle dogs is more than 2000 mg/kg, the minimum dose causing toxic response is 118 mg/kg, and the gastrointestinal tract may be the toxic target organ/tissue of ZBH201001; (2) In Repeated dose 30-day oral toxicity study, medium dose and/or high dose of ZBH201001 caused certain toxic effects in esophagus, liver, thymus, testis, prostate, pancreas, bone marrow, kidneys, gastrointestinal system, and may affect endogenous coagulation system, but these toxicities could be reversed after stopping administration. Therefore, the NOAEL of ZBH201001 in Beagle dogs is 3.0 mg /kg body weight (about 18 times of the clinical dose for human, which is supposed as 10 mg/day). |
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