Pharmacokinetics and Safety Evaluation of anemoside B4 in Healthy Beagle Dogs

JiJinzhao Ji^1,2Yuqiao Ma^2,3Shaobing Wan^2,3Xiaoqing Ding^2,3Jingyu Wang^2,3Yongcheng Zhong^2,3Yangyang Song⁴junqing Zhao⁵Zhetong Su⁵Kun Jia^2,3Shoujun Li^2,3*

• ¹College of Veterinary Medicine, South China Agricultural University,, Guangzhou, China
• ²Guangdong Technological Engineering Research Center for Pet, Guangzhou, China
• ³College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
• ⁴Guangxi Innovate Pharmaceutical Co., Ltd,, Guangxi, China
• ⁵Guangxi Innovate Pharmaceutical Co., Ltd, Guangxi, China

Anemoside B4 (AB4), a pentacyclic triterpenoid saponin isolated from the traditional Chinese medicinal herb Pulsatilla chinensis, has demonstrated potent anti-inflammatory and immunomodulatory effects in both preclinical studies and clinical trials. However, data on the pharmacokinetics and safety of AB4 in dogs remain lacking. A comprehensive understanding of its pharmacokinetic profile, bioavailability, and safety margin in dogs is essential to support its future study.This study included three parts: (1) A randomized parallel-controlled study was conducted in 40 Beagle dogs, which received single subcutaneous injections of AB4 at low (10 mg/kg), medium (20 mg/kg), or high (40 mg/kg) doses, or intravenous injection bolus at 20 mg/kg. Plasma concentrations were analyzed using a validated HPLC-MS/MS method to establish concentrationtime profiles and determine pharmacokinetic parameters, including absolute bioavailability, dose proportionality, and sex-based differences. (2) In a multiple-dose study, 10 Beagle dogs were administered 20 mg/kg AB4 subcutaneously once daily for 7 consecutive days to assess drug accumulation and degree of fluctuation. (3) In a target animal safety study, 32 healthy Beagle dogs were randomly assigned to receive 20, 60, or 100 mg/kg of AB4, or saline, by daily subcutaneous injection for 7 days. Clinical signs, hematology, serum biochemistry, gross necropsy, and histopathology were evaluated.In the safety study, no mortality or near-death events occurred in dogs receiving 20-100 mg/kg AB4. Dogs exhibited normal clinical signs throughout the study period, with no observable toxic reactions or adverse effects. No significant effects were observed on liver and kidney function, hematopoiesis, coagulation, or related physiological parameters. Histopathological analysis revealed no consistent or target-organ specific lesions.These findings provide fundamental pharmacokinetic and safety data to support the rational clinical use of AB4 in veterinary medicine.