Subtilisin QK was provided by Wuhan Zhenfu Pharmaceutical Co., Ltd. (Wuhan, China). Subtilisin QK was derived from B. subtilis QK02 liquid fermentation. The fermentation broth was made into a powder by centrifugation, microfiltration, ultrafiltration concentration, and spray drying. Subtilisin QK activity was measured using a fibrin degradation assay developed by Japan Bio Science Laboratory Co., Ltd. (Walnut Creek, CA, USA). A spray-dried powder was tested for microbial limit, pH, biological activity, moisture, MW, protein content, and percentage. Analytical data from three batches of subtilisin QK are presented in Supplementary Table 1.
Healthy female and male SD rats, aged 6–8 weeks, were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd. (Shanghai, China), and all animals received 7 days of adaptive feeding. All SD rats were housed in a specific pathogen-free room (License No. SYXK (Su) 2016–0043). The room was maintained at 20.0–23.7 °C and a relative humidity of 49.4–55.1%, with a 12 h light/12 h dark cycle and minimum air change ≥15 times/h.
In a previous study of nattokinase, the acute toxicity results showed that nattokinase did not cause apparent toxicity at a dose of 2000 mg/kg (49,400 FU/kg) . Subtilisin QK and nattokinase are highly homologous, and belong to the subtilisin family. Therefore, we considered that subtilisin QK has low toxicity, and conducted a limit test. For some low-toxic subjects, the maximum dose method can be used (a single dose < 5000 mg/kg; National Food and Drug Administration, China, May 13, 2014). Since the maximum solubility of subtilisin QK is about 2500 FU/mL, and the maximum feeding volume is 40 mL/kg in a single day. The acute toxicity of subtilisin QK was determined using the maximum dose method in SD rats at a concentration of 10,0000 FU/kg (~ 4000 mg/kg). The results of our study on mouse tail vein thrombolysis showed that the lowest effective dose was 1250 FU/kg (the equivalent dose in rats is ~ 500 FU/kg). Therefore, in a subchronic toxicity test, the low, medium, and high doses were 2500, 7500, and 25,000 FU/kg, respectively, which were approximately 5, 15, and 50 times the equivalent dose in rats. In the pharmacology safety test, the low, medium, and high doses were 500, 1500, and 5000 FU/kg, respectively, which were approximately 1, 3, and 10 times the equivalent dose in rats.
Acute toxicity study
Forty SD rats were randomly allocated to two groups (10 per sex). The study protocol was conducted in compliance with Chinese technical guidelines for single-drug toxicity studies and Chinese general principles for technical evaluation of non-clinical safety of therapeutic biologic products (National Food and Drug Administration, China, May 13, 2014). The treatment group was administered a single dose of 100,000 FU/kg subtilisin QK dissolved in saline, in a volume of 40 mL/kg. The control group was administered an equal volume of saline. Clinical signs of toxicity were closely monitored for 1, 3, and 6 h after administration, and all rats were monitored twice daily for 14 days. Body weights and food consumption of all animals were recorded before administration and on days 2, 7, and 14. On day 15, all animals were euthanized by carbon dioxide inhalation and were subjected to macroscopic examination. According to AVMA guidelines, carbon dioxide exposure is less likely to cause pain by a gradual-fill method. Therefore, euthanasia of rodents by inhaling carbon dioxide in their cages can help reduce stress and pain.
Subchronic toxicity study
In total, 120 SD rats were randomly allocated to four groups (15 per sex). According to OECD 407-Repeated Dose 28-day Oral Toxicity Study in Rodents regulations, at least 10 animals (5 females and 5 males) should be used at each dose level. For the additional satellite group, 10 animals (5 per sex) were observed for reversibility, persistence, or delayed occurrence of toxic effects for at least 14 days post-treatment. To obtain sufficient oral toxicity and delayed toxicity data on rodents for 28 days prior to human use, we used 30 animals in each group (15 per sex). The study protocol was conducted in compliance with the Chinese Technical Guidelines for Repeated-Dose Toxicity Studies (National Food and Drug Administration, China, May 13, 2014). The dosing groups were administered single doses of 2500, 7500, and 25,000 FU/kg subtilisin QK dissolved in saline for 28 days, in a volume of 10 mL/kg. On day 29, the remaining animals (5 rats of each sex in each group) as the satellite group, were continuously monitored for a 28-day recovery period. Clinical signs of toxicity were monitored twice daily during the dosing period and during the recovery period. Food consumption of each animal was measured on days 7, 14, 21, 28, 35, 42, 49, and 56. Body weights of each animal were recorded on days 0, 7, 14, 21, 28, 35, 42, 49, and 56. Urinalysis, biochemical, hematological and coagulation parameters were conducted on days 28 and 56. Ophthalmological examination was conducted on days 0, 28 and 56, and pathological examination on days 28 and 56. Note: Day 0 represents before the dosing day, and day 1 represents the first day of dosing.
Ophthalmoscopy and urinalysis
Each animal underwent an eye examination with a slit lamp microscope and binocular indirect ophthalmoscope including eyelid, conjunctiva, cornea, sclera, iris, pupil, lens, vitreous, and fundus. Animals were placed in metabolic cages to collect fresh urine on days 28 and 56. After collection, urine samples were placed in a sample transport box and detected within 2 h. Urine samples of all animals were used for urinalysis with a urine chemistry analyzer (AUTION MAX AX-4280; ARKRAY Inc., Kyoto, Japan) including pH, color, turbidity, nitrite, glucose, specific gravity (SG), occult blood, protein, bilirubin (BIL), urobilinogen, and ketone.
Macroscopic findings and organ weights
All animals were anesthetized by intramuscular injection of Zoletil (75 mg/kg) at the end of the experiment. Blood was collected from the abdominal aorta after anesthesia, and then the animals were killed. During dissection, the general condition, body surface, thoracic cavity, abdominal cavity, pelvic cavity, and intracranial tissues/organs of the animal were examined, and all abnormal changes were recorded.
Organ weights were recorded (including brain, thymus, kidney, adrenals, thyroid, liver, spleen, testis, epididymis, uterus, and ovaries), and the relative organ weights were calculated by the following formula: relative organ weight = absolute organ weight (g)/body weight (g) × 100%.
Hematological, biochemical, and coagulation parameters
The blood samples collected in EDTA-K2-coated tubes were analyzed for hematology using an auto-hematology analyzer (XT-2000iV; Sysmex Co., Kobe, Japan) including white blood cell (WBC), WBC classification count (N, L, M, E, B) and classification percentage (N%, L%, M%, E%, B%), red blood cell, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet, and reticulocyte count. The blood samples collected by inert separating gel coagulant tube were analyzed for blood biochemistry parameters including K+, Na+, Cl−, total BIL (TBIL), total protein, albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transferase, urea, creatinine, glucose, triglyceride, and total cholesterol. Blood biochemistry was determined using an automatic biochemistry analyzer (c8000; Abbott Laboratories, Chicago, IL, USA). Na+, K+, and Cl− were determined using an electrolyte analysis instrument (XI-931 T; Shenzhen City Kate Bio-Medical Electronics Co., Ltd., Shenzhen, China). The blood samples were collected in sodium citrate-coated tubes. Plasma was separated for coagulation detection using an automated coagulation analyzer (STAGO STA-R Evolution; Diagnostica Stago, Asnières-sur-Seine, France) including thrombin time (TT), prothrombin time, (PT) fibrinogen, and activated partial thromboplastin time (APTT).
Routine histopathological examinations were performed on related organs in the high-dose group (25,000 FU/kg) and control group (0 FU/kg). The brain, spinal cord, pituitary, thyroid, parathyroid glands, heart, aorta, trachea, lungs, main bronchi, salivary glands, esophagus, pancreas, stomach, small/large intestines, liver, gallbladder, kidneys, urinary bladder, testes, epididymis, uterus, oviduct, ovaries, cervix prostate, vaginal, sciatic nerve, skeletal muscle, adrenals, spleen, thymus, mesenteric lymph node, submandibular lymph node, bone marrow, skin, mammary gland, eye, and optic nerve were removed from each animal. The testis, epididymis, eyeball, and optic nerve were fixed in modified Davidson fixation solution, and the remaining tissues/organs were fixed in 10% neutral buffer formalin fixation solution. Then the sections were routinely processed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined under a microscope.
Safety pharmacology study
Forty SD rats were randomly allocated to four groups (5 per sex). The dosing groups were administered single doses of 500, 1500, and 5000 FU/kg subtilisin QK dissolved in saline, in a volume of 10 mL/kg. The control group was administered an equal volume of saline.
Functional observation battery (FOB) was recorded at 1 h prior to dosing and at 0.5, 1, 2, 4, 6, 8, and 24 h after dosing including in-cage observation (animal sleep, exercise, vertical hair, attacking caged animals, abnormal vocalization and grooming behavior), open field observation (position, autonomic activity, ataxia, hypotonia, convulsions, convulsions, stereotypes, vertical tail, vertical hair, erect, urination, respiration and panic reaction), and hand-held observation (location passive response, catalepsy, visual location, corneal response, auricular reflex, skin color, cyanosis, blinking, eyeball protrusion, pupillary light reflection, tear secretion, saliva secretion, tail-tail reaction, righting reflex, positive reflex, aggression/stress, abnormal vocalization, viscosity of bowel movements, and death and body temperature). Functional tests were recorded at 1 h prior to dosing and at 0.5, 1, 2, 4, 6, 8, and 24 h after dosing including spontaneous activity total distance, number of activities, and holding power. In the functional test, the universal spontaneous activity video analysis system (JLBehv-LAG-4; Shanghai Jinliang Software Technology Co. Ltd., Shanghai, China) was used to detect the spontaneous activity of the rats, and the number of spontaneous activities of the animals within 3 min was recorded at each time point. Rats were examined for limb grip using a grip tester (YLS-13A; Jinan Yiyan Technology Development Co. Ltd., Jinan, China). At least one adaptation training was performed on the autonomous animals before the test.
Levene’s test was used to test the homogeneity of variance. If the variance was uniform (p > 0.01), the analysis of variance results were directly used to determine the statistical significance of the overall difference. If the variance was not uniform (p ≤ 0.01), the statistical significance of the overall difference was judged using results from Welch’s t-test. If the analysis of variance or Welch’s t-test results were statistically significant, multiple comparisons of differences between groups were performed using the Bonferroni test to further determine which differences were statistically significant. P < 0.05 was considered statistically significant.