动物造模 z-bio 2022-01-31 498

　　Animals: Approximately 14 months old at the start of dosing, weighing 7.8 to 10.1 kg. Vehicle control group (0 mg/kg/day, 1 female and 1 male), low-dose group (2 mg/kg/day, 1 female and 1 male) and medium-dose group (10 mg/kg/day, 1 female and 1 male), High-dose group (50mg/kg/day, 2 females and 2 males). The dose groups were staggered on the first day of the dose, starting with the lowest dose. The animals in the control group were given 10 mg/kg/day at the same time. All dogs were given a dose of 5 ml/kg at approximately 8:00 a day. Clinical symptoms were recorded daily, and all animals were assessed twice daily (morning and afternoon) for mortality, abnormalities, and signs of pain or distress. The body weights of all surviving dogs were recorded on days 1, 4, 8, 11, and 15. Determine quantitative food consumption from 1 to 4, 4 to 8, 8 to 11 and 11 to 15 days (survival period). The 2 surviving high-dose animals underwent detailed observation, body weight and food consumption 3 days before unplanned euthanasia. On days 1, 4 and 13, the ECG was measured using the vest telemetry system. Blood samples were collected from the jugular vein for toxicokinetic analysis at about 0.5, 1, 3, 7 and 24 hours after administration on 1, 4, and 13 days. All animals have pre-collected blood samples for hematology, clinical chemistry and coagulation evaluation. In addition to the high-dose male animals euthanized on the second day of the study, blood samples were also collected from the early euthanized animals. All animals were weighed immediately before necropsy. All animals (including those that were euthanized early) were autopsied, and tissues were collected in 10% neutral buffered formalin (adrenal gland, aorta, brain, cecum, cervix, colon, duodenum, epididymis, esophagus, Femur with bone marrow [distal articular surface], gallbladder, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph node, breast, mesenteric, skeletal muscle, ovary, pancreas, pituitary, prostate, rectum, salivary gland, Sciatic nerve, skin/subcutaneous, spinal cord [neck, chest, waist], spleen, sternum and bone marrow, stomach, thymus, thyroid/parathyroid gland, tongue, trachea, ureter, bladder, uterus and vagina) or modified Davidson fixative fixation ( Eyes, optic nerve, testes). Use the anatomical landmarks on the ventral side of the brain to sample three coronal (transverse) levels: level 1 samples between the optic chiasm and the papillary body, level 2 samples at the end of the pontine, and level 3 samples through the pontine/medulla to get the right fit The representation. From each level of the cerebellum, the left hemisphere is sampled. Due to the large size of the dog’s brain, the cerebral hemispheres from grades 1 and 2 are subdivided into dorsal and ventral parts, and each animal has a total of 5 brain parts. In addition, formalin fixation, brain tissue and liver tissue were embedded in paraffin and cut into 5μm, and then stained by special or immunohistochemistry (IHC). All slides used for special stains were baked at 60°C for 1 hour, demyelinated, and hydrated before staining. Perl's Prussian blue staining method detects iron. The slides are immersed in potassium ferrocyanide/hydrochloric acid solution for 30 minutes, rinsed in deionized (DI) water, and stained with nuclear fast red. The severity and extent of the changes in the respective organs are as follows: 1=minimal, 2=slight, 3=moderate, 4=significant, 5=severe.

　　Naphthoquine plasma concentration analysis: All samples are kept below -60℃ before analysis. Plasma samples were processed for protein precipitation with acetonitrile/methanol mixture. The samples were centrifuged and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with positive pressure chemical ionization as the interface. The calibration curve is valid from 0.500 to 10,000 ng/ml, using 0.0400 ml of plasma. At all concentration levels, the calibration standards are within ±30%. . The plasma Cmax (highest concentration) of each group of male and female dogs is reported separately or as the average of 2 animals in the high-dose group. Similarly, the time to observe the Cmax value is reported separately as the Tmax value or as the average of the high-dose group. The trapezoidal rule was used to calculate the area under the plasma curve of each animal at 0-24 hours (AUC0-24hr) after the dose, and reported separately or as the average of 2 males and females in the high-dose group.

　　Result: Clinical observation: According to clinical observation, animals with a dose of 10 or 50 mg/kg/day before the end of the experiment were euthanized. After the second dose of 50 mg/kg/day, 1 male and 1 female developed convulsions, combined with ataxia, lying down, excessive saliva, and mucous stools, which led to the early termination of the experiment that day. The remaining 2 animals given 50 mg/kg/d were euthanized on the 3rd day because of the unfavorable clinical observations related to NP observed at this dose level and the presence of a significant increase in body temperature that may be secondary to convulsions. The dosage was considered inappropriate. Both dogs taking 10 mg/kg daily lost weight significantly on the 11th day, and the food consumption during the 8th to 11th day was significantly reduced compared with the early intermittent period and the vehicle control group during the same period. The clinical observation of vomiting and fever began on the 10th day, and the animal was not taken on the 11th day and was euthanized. Both groups of animals survived to the 15th day planned euthanasia at 2 mg/kg/day. The only clinical observations noted with this dose were reduced food consumption and slight weight loss (0.5 kg) in females from day 11 to day 15. When the control animals were administered at the same dose as the 10 mg/kg/day dose animals, they were also euthanized on the 11th day. There are no NP-related changes in ECG parameters (PR interval, QRS interval, QT interval, corrected QT interval, or heart rate), and no abnormal rhythm or qualitative time or dose-dependent changes caused by NP during the qualitative ECG evaluation .

　　Clinicopathology: In 50 mg/kg/day female animals euthanized on day 2, only hematology or coagulation changes related to NP were observed. These findings include a mild increase in red blood cell mass (hematocrit relative to baseline +9%) and absolute neutrophil count (relative to baseline +52%) and a mild decrease in absolute lymphocyte and eosinophil count (relative to baseline) Baseline, respectively -34% and -71%). The increase in the mass of red blood cells is consistent with the contraction of the spleen, and the changes in white blood cells are consistent with the stress response to the release of endogenous catecholamines and glucocorticoids. These effects may be secondary to the convulsions observed before euthanasia. The only clinical chemistry findings related to NP in this study were found in 50 mg/kg/day female animals euthanized on the second day, and it is likely to be secondary to convulsions. These findings include a moderate reduction in glucose concentration (-67% relative to baseline), a moderate increase in aspartate aminotransferase and creatine kinase activity (5.0 and 8.4 times the baseline, respectively), and a moderate reduction in inorganic phosphorus and Potassium concentration (-28% and -31%, respectively). The decrease in blood glucose concentration and the increase in muscle enzyme activity are consistent with glucose consumption and muscle damage associated with twitch activity. The specific mechanism of inorganic phosphorus and potassium concentration changes is not obvious, but there is no similar change in other NP dose animals.

　　Histopathology: The female dose exceeds 2 mg/kg/day, and the male dose exceeds 10 mg/kg/day. The main microscopic changes related to NP include slight to obvious vascular central inflammation in the central nervous system and adjacent meninges, occasionally Appear. Central nervous system inflammation is characterized by lymphocytes and macrophages containing scarce neutrophils, filling and expanding the Virchow-Robin space, and often obscuring the blood vessel wall, multifocal, usually surrounding blood vessels, areas with increased cytoplasm . Among the 4 females taking NP, 2 had similar vascular center inflammatory changes and/or gliosis focal areas in the white matter and gray matter of the spinal cord. In the meninges of 3 female animals, white and similar focal, sparsely distributed vascular central inflammation changes were observed. Although the microscopic changes were always centered on the vascular system, no morphological evidence of direct vascular injury, such as fibrinoid necrosis or pus fragments, was observed. Compared with the animals euthanized on days 11-15, the microscopic findings in the brain of 2 male animals at 50 mg/kg/day are usually less severe and more acute (in inflammatory infiltrates). The increase in the proportion of neutrophils) may be due to the short treatment time of these animals (2-3 days). One female took 50 mg/kg daily and was euthanized on the third day of the study, showing significant inflammation with a distribution similar to that of the euthanized later. Compared with the control group, the affected animals showed no difference in the nature, intensity, or pattern of glial fibrillary acidic protein (GFAP) markers (astroglia). NP-related vascular center microscopic changes are also reflected in the liver, and are characterized by minimal to slight vascular inflammation and perivascular inflammation, mainly concentrated in female doses ≥ 2 mg/kg/d and male doses ≥ 10 mg/kg/d Central lobular vein. In addition, 1 case of females with 50 mg/kg per day had mild single cell necrosis of hepatocytes with parenchymal inflammation, and iron pigmentation was found in female Kupffer cells with ≥10 mg/kg per day. In various other tissues and organs, a small amount of NP-related microscopic changes were observed, and these changes tended to be in the high-dose group. These changes include vacuolated alveolar macrophage infiltration, vacuolation of adrenal cortex, vacuolation of pancreatic acinar cells, occasional single-cell necrosis, neutrophil infiltration of the gastrointestinal mucosa, and single-cell necrosis of the cecum and colon. (Mucous epithelial cells), bone marrow has low hematopoietic function, and lymphocyte depletion/necrosis in gastrointestinal-related lymphoid tissues, spleen, retropharyngeal lymph nodes, and mesenteric lymph nodes.

　　Toxicokinetic evaluation: No measurable test article concentration was observed in any samples of vehicle control animals. Plasma NP exposure is generally between 2-10mg/kg/d and is proportional to the increase in dose, and there is accumulation.

　　Conclusion: Beagle dogs were given NP by oral gavage for 2 weeks at the doses of 10 and 50 mg/kg/day, respectively, which cannot be tolerated. 50 mg/kg/day has obvious neurological clinical symptoms. Lead to early euthanasia. In addition, female doses ≥2mg/kg/d and male doses ≥10mg/kg/d can cause central nervous system and hepatic vascular central inflammation.