Anemoside B4 (AB4, purity: 96.4%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). The investigational drug, AB4 for injection (purity: 94.2%) was provided by Guangxi Innovate Pharmaceutical Co., Ltd. (Guangxi, China). Normal saline for injection was obtained from HFQ Co., Ltd. (Jiangsu, China). All reagents and solvents used were of analytical or HPLC grade.

The HPLC-MS/MS analysis was performed using a SHIMADZU LCMS-8045 system (Shimadzu Corporation, Kyoto, Japan) equipped with a ZORBAX Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm; Agilent Technologies, USA). The mobile phase consisted of solvent A (ultrapure water) and solvent B (acetonitrile containing 0.1% formic acid). The gradient elution conditions are summarized in Table 1.

The method was evaluated in terms of specificity, linearity, accuracy, precision, recovery, matrix effect, and stability, in accordance with standard bioanalytical guidelines (ICH M10, 2022).

Healthy Beagle dogs (9–11 months old) purchased from Zhenhe Laboratory Animal Co., Ltd. (Fuzhou, China) were used in this study. All procedures were approved by the Animal Clinical Research Ethics Committee of the Veterinary Drug Evaluation Center, South China Agricultural University (Approval No. 2023E007). Before the study, dogs underwent a 7-day acclimation. During the study, dogs were fed a standardized diet with daily intake calculated based on resting energy requirement (RER) and maintenance energy requirement (MER) to ensure nutritional balance and appropriate caloric intake. Water was freely available.

In the single-dose pharmacokinetic (PK) study, 40 Beagle dogs (equal numbers of males and females) were randomly assigned into four parallel groups: low-dose (10 mg/kg), medium-dose (20 mg/kg), high-dose (40 mg/kg), and intravenous (20 mg/kg). AB4 was administered as a single subcutaneous injection in the dorsal neck or intravenously into the cephalic vein; In the multiple-dose PK study, 10 dogs (five males and five females) received 20 mg/kg AB4 once daily (8:00 a.m.) by subcutaneous injection for 7 consecutive days.

In the safety study, 40 dogs (equal numbers of males and females) were randomized into four groups: low-dose (20 mg/kg), medium-dose (60 mg/kg), high-dose (100 mg/kg), and a saline control group (0 mg/kg). AB4 was administered subcutaneously once daily (8:00 a.m.) for 7 days at 24-h intervals.

All AB4 solutions were prepared at a concentration of 48 mg/mL, and the administered volume was adjusted according to body weight and dosing requirements. Detailed dosing information is provided in the Supplementary materials 7–9.

In the single-dose pharmacokinetic (PK) study, a blank blood sample was collected before dosing. After administration, 2 mL blood samples were collected from the cephalic vein at 5, 10, 15, 30, and 45 min, and at 1, 2, 3, 3.5, 4, 6, 9, 12, 16, 24, 36, and 48 h.

In the multiple-dose PK study, a pre-dose blood sample was collected before the first administration, followed by sampling at the same time points as the single-dose study. Additional trough samples were collected within 5 min before each daily administration. On days 2 to 6, samples were collected at 3, 3.25, 3.5, 3.75, and 4 h. After the final dose, samples were taken at the same time points as the single-dose group, with additional collections at 60 and 72 h.

Plasma was separated by centrifugation at 4000 rpm for 10 min. Subsequently, 300 μL of plasma was mixed with 900 μL of methanol, vortexed for 3 min, and centrifuged at 14,000 rpm for 10 min at 4°C. The resulting supernatant was filtered through a 0.22 μm PTFE membrane and subjected to LC–MS/MS analysis.

In the safety study, blood was collected from the cephalic vein for hematology, serum biochemistry. According to the AVMA Guidelines for the Euthanasia of Animals (2020 Edition), dogs were rapidly administered propofol intravenously at a dose of 10 mg/kg. After confirming deep anesthesia, bilateral axillary vessels were incised for exsanguination, ensuring a painless procedure. Following euthanasia, major organs were collected, weighed for organ coefficient calculation, then fixed in 10% neutral-buffered formalin, and prepared for histopathological examination using H&E staining.

Plasma concentration–time data were analyzed using non-compartmental analysis (NCA) in Phoenix WinNonlin software (Certara, USA) (22). Pharmacokinetic Parameters were compared between male and female dogs within each dose group to assess sex-based differences. Bioavailability was calculated based on AUC comparison between IV bolus and SC groups. Dose linearity for key PK parameters (C_max, AUC_0–t, and AUC_0-∞) was assessed using the power model approach.

Safety assessments were conducted on Days 0 (D0), 4 (D4), 7 (D7), and 14 (D14). Blood samples were collected for laboratory testing, including hematology and serum biochemistry. Local tolerance at the injection site was also monitored. Body weight was recorded on D0 and D14 (after fasting). On D14, gross anatomical examinations and organ weight measurements were performed in the control and high-dose groups. Throughout the study, dogs were monitored for clinical signs and adverse events (AEs).

Statistical analyses were conducted using SPSS software (version 25.0; IBM, Armonk, NY, USA), with a two-sided significance level of α = 0.05.

In the pharmacokinetic study, continuous variables were tested for normality and homogeneity of variance. Normally distributed data with equal variances were analyzed using independent samples t-tests; those with unequal variances were analyzed using Welch’s ANOVA. Non-normally distributed data were assessed using the Mann–Whitney U test.

In the safety study, normally distributed data are presented as mean ± SD and analyzed using one-way ANOVA, followed by Dunnett’s test or Tamhane’s T2 test depending on variance equality. Non-normally distributed data are expressed as median (P25, P75) and analyzed using the Wilcoxon rank-sum test. Categorical data are shown as frequency and percentage (n, %) and compared using the chi-square or Fisher’s exact test. Paired data were evaluated using the paired t-test or Wilcoxon signed-rank test.

The HPLC–MS/MS method established for AB4 quantification in Beagle dog plasma demonstrated acceptable selectivity, sensitivity, accuracy, and reproducibility. No significant endogenous interference or system carryover was observed. The method showed good linearity over 0.2–20 μg/mL (R² > 0.99). Precision and accuracy were within ±15% (±20% for Lower Limit of Quantification), and recovery exceeded 80%. Dilution integrity and stability under various conditions (freeze–thaw, short−/long-term storage, post-preparative) were confirmed. Detailed validation data are provided in the Supplementary materials 1–6, 36–37.

At baseline, no statistically significant differences in body weight were observed among groups (F = 0.397, p = 0.756). Pharmacokinetic parameters of AB4 were analyzed using a non-compartmental model. Following single-dose administration, AB4 exhibited a short elimination half-life (t₁/₂ ≈ 3–4 h), a high clearance rate, and a moderate apparent volume of distribution (V_d/F < 300 mL/kg), indicating rapid elimination and limited tissue distribution. Moreover, AB4 showed high absolute bioavailability.

Sex-based comparisons revealed statistically significant differences in terminal elimination rate constant(λ_z) and t₁/₂ in the low-dose subcutaneous group: λ_z was 0.23 ± 0.02 h⁻¹ in females and 0.20 ± 0.02 h⁻¹ in males (t = −2.361, p = 0.046), while t₁/₂ was 3.08 ± 0.22 h vs. 3.48 ± 0.22 h, respectively (t = 2.374, p = 0.045). In the intravenous group, only steady-state volume of distribution (V_ss) showed a significant difference (164 ± 9 mL/kg in females vs. 207 ± 30 mL/kg in males; F = 8.935, p = 0.033). No other pharmacokinetic parameters showed statistically significant sex differences (p > 0.05).

Detailed pharmacokinetic parameters are summarized in Table 2, and mean plasma concentration-time profiles are illustrated in Figure 1.

Dose proportionality of AB4 was assessed in Beagle dogs following single subcutaneous administration at 10, 20, and 40 mg/kg. After normalization by body weight, the systemic exposure parameters(C_max, AUC_0–t, and AUC_0–∞)increased approximately in proportion to the dose (1:2:4), indicating a dose-proportional relationship.

A power model analysis was performed to further evaluate linear pharmacokinetics. The estimated exponents (β) for C_max, AUC_0–t, and AUC_0–∞ were 0.9417, 1.032, and 1.027, respectively, all of which are close to 1. These results indicated that AB4 exhibits linear pharmacokinetic behavior over 10-40 mg/kg dose range in dogs.

Dose proportionality and fitted curves of systemic exposure are presented in Table 3. Linear regression plots based on the power function model for key pharmacokinetic parameters are shown in Figure 2.

Following repeated subcutaneous administration of AB4 at 20 mg/kg once daily for 7 days, the mean steady-state trough concentration (C_ss-min) was 1.35 ± 0.51 μg/mL, and the mean steady-state average concentration (C_ss-avg) was 19.61 ± 5.27 μg/mL. The degree of fluctuation (DF) was 2.52 ± 0.42.

The accumulation ratios estimated by three different methods were as follows: R (based on the terminal elimination rate constant) was 1.03 ± 0.01, R_AUC was 1.22 ± 0.52, and R_Cmax was 1.16 ± 0.18. These results indicate that AB4 exhibits no significant accumulation in dogs under the tested dosing regimen. The concentration-time curve of AB4 in dogs after multiple dosing is presented in Figure 3, and pharmacokinetic parameters following repeated subcutaneous administration (n = 10) are summarized in Table 4.

At baseline, body weight in the 5 × dose group was significantly lower than the saline control group (p = 0.008), while no significant differences were observed in other treatment groups (p > 0.05). During the entire observation period (Days 0–14), Dogs in treatment and control groups maintained stable vital signs. Body temperature, respiratory rate, and heart rate remained within normal physiological ranges, with no signs of fever, hypothermia, tachypnea, or arrhythmia. Food and water intake were normal, with no cases of anorexia or polydipsia. Fecal and urinary characteristics appeared normal. Dogs were alert and active, with bright eyes and responsive behavior. Mucous membranes were moist and pink, and the skin on the lips and nose remained normal. No injection site reactions (e.g., erythema, edema, pain, or pruritus) were observed, apart from mild bleeding due to needle puncture. No significant differences in body weight gain were observed between groups (p > 0.05), indicating no treatment-related effects. No adverse events or mortality occurred during the study. Injection site manifestations are provided in the Supplementary material 38.

Hematological results showed that during the administration period, red blood cell parameters, platelet counts, and white blood cell counts in dogs remained within normal ranges, with no statistically significant differences compared to the saline control group. On Day 14 (seven days after drug withdrawal), statistically significant differences in neutrophil (NEUT) and total white blood cell (WBC) counts were observed between the treatment groups and the control group.

Serum biochemistry results indicated that serum markers of liver function (AST, ALT, ALP, GGT, and TBIL) remained within reference ranges, with no statistically significant differences from the control group on any test day. Renal function parameters (CREA and UREA) showed minor statistical differences at isolated time points, however, all values remained within physiological ranges and did not persist throughout the study. Indicators of protein metabolism (TP, ALB, GLOB) and lipid/glucose metabolism (TG, GLU, TC) were also within reference ranges. Although some intergroup differences were observed at certain time points, no trends related to drug intervention were identified, and the variations were primarily attributed to individual differences among animals. No significant effects were observed on muscle function (CK) or on calcium and phosphorus levels.

These findings indicated that AB4 administered at doses of 20–100 mg/kg did not induce clinically significant effects on hematological or serum biochemical profiles in dogs. All data are detailed in Supplementary materials 12–35.

Gross necropsy findings in the 5 × dose group revealed no visible abnormalities in the heart, liver, spleen, lungs, kidneys, thymus, pancreas, gastrointestinal tract, and lymph nodes. All examined organs exhibited normal color, intact serosal surfaces, and well-defined margins, with no signs of hemorrhage, ulceration, necrosis, or other gross lesions. No apparent differences were noted compared to the saline control group.

Organ coefficient analysis showed no statistically significant differences (p > 0.05) in the weights of major organs (heart, liver, spleen, lungs, and kidneys) between the 5 × dose group and the saline control group. These results indicated that AB4 administration did not cause hypertrophy, atrophy, or abnormal enlargement of the major organs. Detailed results are presented in Table 5.

Histopathological changes observed in the 5 × dose group, diffuse hepatic vacuolar degeneration, lymphoid follicle hyperplasia in the renal cortex (1/8, 12.5%), splenic nodule edema with loosely arranged lymphocytes, intestinal villus edema, lymph node edema (1/8, 12.5%), and congestion of hepatic vessels and sinusoids (1/8, 12.5%) were observed. In the saline control group, focal pulmonary interstitial hyperplasia (1/8, 12.5%), decreased lymphocytes in the splenic red pulp, proteinaceous exudate in renal tubules accompanied by epithelial necrosis, and pancreatic edema (1/8, 12.5%) were noted. These histopathological findings did not exhibit consistency or directional trends and were determined to be incidental or attributable to individual variation. There was no statistically significant difference in the incidence of lesions between the two groups (p > 0.05). Detailed results are presented in Table 6. Representative histopathological sections are shown in Figures 4, 5, and additional sections are provided in the Supplementary material 39.