2.1 Reagents
Recombinant human FGF-21 for injection (batch number: 20150101) was produced and provided by Wenzhou Medical University Biological and Natural Medicine Development Center Co., Ltd. The human FGF-21 Immunoassay Kit was purchased from IMD Company (Product No.: 31180, Lot number: HFT012, Shenzhen, China).
2.2 Methods
2.2.1 Method validation: accuracy and precision
Linearity: A series of FGF-21 standard solutions was prepared with final concentrations of 4000, 2000, 1000, 500, 250, 125, and 62.5 pg/mL. The absorbance (A) was plotted against the drug concentration (C), and a sigmoid curve was drawn based on a four-parameter log logistic (4-PL) model. The experiment was repeated 10 times to obtain the standard curve of the FGF-21 ELISA kit.
Precision and accuracy: Five QC samples (4000, 3200, 800, 160, and 62.5 pg/mL) were used to analyze the precision and accuracy. The intra-batch precision and accuracy were determined by measuring each quality control sample five times on the same enzyme-labeled plate. Two wells were included for each QC sample, and the average value was taken. The inter-batch precision and accuracy were investigated by measuring OD values of each quality control sample on different enzyme-labeled plates of six independent analysis batches. The ratio of the standard deviation to the mean value is the precision, expressed as RSD%. The ratio of the average value of the measured results to the theoretical value is the accuracy, expressed as RE%.
2.3 Animals, dosage, and injection of FGF-21
Twenty cynomolgus monkeys (SPF grade, 3–5 years old, 10 female and 10 male, males weighing 2.85–3.90 and females 2.75–3.50 kg) were purchased from Suzhou Xishan Zhongke Laboratory Animal Co., Ltd. (Suzhou, China). Monkeys were randomly divided into two groups (n = 10 animals per group). Low- and high-dose FGF-21 were subcutaneously injected for 86 days. After the last injection (d86), the animals were allowed to recover for 29 days. The animals were fed normally during the drug administration period.
2.4 Determination of serum FGF-21 levels
The serum concentration of FGF-21 was measured using the ELISA method. The OD value was measured in a 96-well plate using a microplate reader at a wavelength of 450 nm.
2.5 Clinical observation and histopathology
Cynomolgus monkeys were weighed using an electronic scale (OCS-W-100kg) three times per day on d0, d24, d57, and d87 after injecting FGF-21. Cynomolgus monkeys were also weighed at the end of the recovery period (rd29). After the administration period (d87, n = 6) and after the recovery period (rd29, n = 4), animals were dissected.
The brain, cerebellum, brain stem, pituitary gland, eyeball, heart, aorta, lung (main bronchus), trachea, salivary glands, liver, pancreas, gallbladder, kidney, spleen, thymus, spinal cord (neck, chest, waist), sciatic nerve, mammary gland, uterus, ovary, fallopian tube, vagina, cervix, prostate gland, testis, epididymis, seminal vesicle, adrenal gland, thyroid gland, parathyroid gland and distal lymph node, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, bladder, skin, skeletal muscle, the administration site, lymph nodes related to administration, bone marrow (sternum), and femur were resected. Finally, the adipose tissue and connective tissue were removed and blood and tissue fluid were absorbed with filter paper. HE staining was performed to analyze organ histopathology. The heart, liver, spleen, kidney, brain, adrenal gland, thymus, testis, epididymis, uterus, ovary, and thyroid (including parathyroid gland) were weighed. For each organ, the organ coefficient was calculated using the following formula:
2.6 Toxicokinetic analysis
Serum was collected and FGF-21 concentrations were determined on d1, d37, and d87 at 0, 0.5, 1.5, 3, 5, 8, 12, and 24 h after injecting FGF-21. The plasma FGF-21 concentration–time relationship was analyzed using Drug and Statistics (DAS) 3.2.8 software (Beijing Bozhiyin Technology Co., Ltd.). The noncompartmental model was used to calculate the pharmacokinetic parameters, including the area under the drug–time curve (AUC(0-t)), t1/2z, Tmax, Cmax, and CLz/F. Except for individual data, all toxicokinetic parameters in each test group are expressed as mean ± standard deviation ( ). Mapping and statistical analysis results were obtained by GraphPad Prism 8.