For WDP preparation, Korean angelica extraction was obtained by filtering grinded Korean angelica root 1 kg that was mixed at 80 rpm with 70% ethanol 5 L, during 6 hours at 60°C. Then Angelica extraction 5 kg (extract + extract solvent) was condensed to 370 g Angelica concentration that included decursin and decursinol angelate. The Angelica extract and an emulsifier were quantified to the reaction beaker. To make water-soluble, WFI (Water for Injection-Fine FA, Korea) was added to the beaker and stir by high speed stirrer. Water solution was adjusted to pH 7.3 by NaOH solution. A filter (1 μm, satorius) equipment was set up between preparation tank and filling tank and emulsified solution was transferred to filling tank by N2 gas. After fractioning in the sterilized vial bottle, the bottle was kept refrigerated at 4°C after autoclaving 30 minutes at 121°C in the low temperature sterilizer (high pressure sterilizer, fine FA, KOREA).
In the study, Sprague-Dawley rats (ORIENTBIO INC., Korea) were used. When taking-in, visual inspection and weight measurement of the rats by electronic scale (CP3202S, Sartorius, Germany) were performed. during 7-day adaptation period, general symptoms were examined every day. To fetch animals, rats were transferred from quarantine to the animal room after observing general symptoms for 3 days. At the end of the adaptation day, weights were measured, and general symptoms were checked to confirm normality. At the time of IV injection, weight range of 6-week rats were 191.2 ~ 212.9 g males and 148.9 ~ 167.2 g females, 20 rats each. The raise condition were as follows: temperature 21.0 ~ 22.9°C, ventilation frequency 10 ~ 15 time/hour, relative humidity 43.2 ~ 59.6%, lighting time 12 hour/day (7:00–19:00), and illumination intensity 150 ~ 300 Lux. Rats were allowed to free intake of Telkad Certified Irradiated Global 18% Protein Rodent Diet 2918C (Harlan Laboratories, INC., USA) solid feed from the feeding system.
The study was approved by the approval of the Institutional Animal Care and Use Committee according to Laboratory Animal Act in Biotoxtech Co. (Approval No: 130366). The study was followed by Good Laboratory Practice regulation (Ministry of Food and Drug Safety notification; MFDSn) and performed by Standard for Toxicity Study of Pharmaceuticals (MFDSn).
Groups were separated at the end of the adaptation day. At the group separation day, 20 males and 20 females rats that approximate mean weight, were selected. Selected animals were randomized into 4 groups to have equal mean weight, 5 rats per each group (Table 1). During observation period, individuals were marked at the tail of the rats with blue oil-based pen, and color individual identification cards were attached at the breeding cage.
As clinical application route will be intravenous, WDP was injected intravenously. Injection doses were 0.5 mL/animal for control and high-dose group, 0.125 mL/animal for low-dose group, and 0.25 mL/animal for middle-dose group (Table 1). The solutions were injected once at a speed of approximately 2 mL/min at the tail vein with a disposable syringe (1 mL, 26G). The scheduled WDP clinical dose is 1.0 mL/time.
The pilot test of the study (Biotoxtech Study No.: B13480P) that single dose IV injection to the male and female rats of 0.5 mL/animal, did not reported death case. Based on the pilot test results and discussion with the sponsor, the administration dose of the study was set to 0.5 mL/animal high-dose, and applied at the ratio of 2 for middle-dose and low-dose, 0.25 and 0.125 mL/animal, respectively. The same dose of high-dose group of normal saline was administrated to the control group.
At the injection day (day 0), general conditions (toxicity symptom types, onset time, recovery time, etc.) and death were observed 30 minutes, 1 hour, 2hour, 4 hour and 6 hour after the injection. General symptoms were observed daily during injection day 1 to day 14. Weights were measured at injection day (before the injection), 3, 7, and 14 days after the injection.
All animals were fasted more than 18 hours before the autopsy for hematological test. At the autopsy day (15 days after the administration), animals were anesthetized with isoflurane and blood was gathered from the abdominal aorta. Blood 1 mL was collected in the EDTA tube to analyze by the blood corpuscle analyzer (ADVIA 120, SIEMENS, Germany). Erythrocyte count (RBC), hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), platelet (PLT), leucocyte count (WBC), neutrophils (NEU), eosinophils (EOS), basophils (BASO), reticulocytes (Reti), lymphocytes (LYM), monocytes (MONO) were measured.
For blood coagulation test, blood plasma was taken by following methods: collected blood 2 mL were kept in the 3.2% sodium citrate tube, followed by 3,000 rpm centrifuge for 10 minutes. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were checked by the coagulation time analyzer (Coapresta 2000, SEKISUI, Japan).
For blood biochemical examination, the remained blood collected from the abdominal aorta was centrifuged by 3,000 rpm for 10 minutes to get blood serum. Blood biochemical analyzer (7180, HITACHI, Japan) were applied to analyze alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), g amma glutamyl transpeptidase (γ-GTP), total bilirubin (T-Bili), total protein (TP), albumin (Alb), A/G ratio, total cholesterol (T-Chol), triglycerides (TG), blood urea nitrogen (BUN), creatinine (Cr), glucose (Glu), and calcium (Ca), phosphorus (P) were measured. In addition, electrolyte analyzer (AVL9181, Roche, Germany) was used to measure chloride (Cl), sodium (Na), and potassium (K).
For autopsy, detail macroscopic inspection was conducted for organs and tissues of the entire bodies of all animals. All organs and tissues were removed and fixed in the 10% neutral buffered formalin from the autopsied animals for histopathological examination. Fixed organs and tissues were turned, dehydrated, and praffin embedded to make tissue sections. The tissue sections were cut into thin slices and stained with hematoxylin & eosin (H&E). The grey matters of the bone tissues were removed by Calci-Clear-RapidTM solution (National diagnostics, U.S.A.). The remained organs and tissues were kept in the 10% neutral buffered formalin. A local tolerance test was done at the injection site of all animals.
For statistical analyses, weight, hematological and blood biochemical examination results were tested by SAS (version 9.3, SAS Institute Inc., U.S.A.). Bartlett test was used for equal variance assumption (significance level at 0.05). When equal variance assumption was satisfied, one-way analysis of variance (ANOVA) was applied at the significance level 0.05. For post-hoc analysis, Dunnett’s t-test was used (significance level at 0.05 or 0.01). When equal variance assumption was not satisfied, Kruskal-Wallis test was used (significance level 0.05) for testing the differences between groups.