动物造模 z-bio 2022-04-13 574

　　Introduction: Cardiac troponins (cTnT and cTnI) serve as clinical blood biomarkers for myocardial injury such as myocardial infarction because of their high diagnostic sensitivity and tissue specificity. Since their structure and function are highly conserved across species, cardiac troponins have also been used as translational biomarkers in animal studies. , despite the availability of actin in cardiac injury, its high diagnostic sensitivity remains a challenge, as increased diagnostic sensitivity inevitably leads to decreased diagnostic specificity (ie, increased number of false positives). When they are used in toxicology studies, it is often difficult to distinguish treatment-related changes from procedural changes. Therefore, it is very important to obtain data on troponin levels in the blood of intact animals. Schultze et al. reported blood cTnI measurements in SD rats and cynomolgus monkeys. The experiment included careful measurements at multiple time points after gavage administration. While these studies provide much-needed data for future cTnI studies, serial blood sampling was performed using automated cannulation, which differs from the standard procedure for most toxicity studies. Our aim was to obtain background data similar to typical drug toxicology studies performed in animals. We measured the concentrations of cTnI and cTnT in the blood of SD rats, beagle dogs and cynomolgus monkeys, and after a single oral administration of 0.5 W/V% methylcellulose solution vehicle, blood was repeatedly collected until 24 h in a restrained state.

　　Materials and methods: Rats: 7-week-old female and male SD rats, the animals were acclimated for 2 weeks. Five male and female animals were orally administered 0.5 W/V% methylcellulose solution (5 mL/kg) using flexible gastric tubes and syringes. About 0.25 ml/animal blood was collected from the tail vein at 0.5, 1, 2, 4, 8 h after dosing for awake and restrained animals. Additionally, approximately 2 ml/animal blood was collected through the abdominal aorta under isoflurane anesthesia after 24 hours of treatment. The blood sample in the sodium heparin tube was immediately placed on ice, centrifuged at 10,000 rpm for 2 minutes at 4°C, and the plasma was collected and stored at -80°C until analysis.

　　Dogs: Male and female Beagles, 10 to 13 months old. Animals were exposed to light for 12 hours from 7:00 am to 7:00 pm under the conditions of a temperature of 23±3°C and a relative humidity of 50±20%. A single dose of 0.5 W/V% methylcellulose solution (5 mL/kg) was administered orally to 30 male and 30 female animals using a disposable catheter and syringe. Approximately 7.8 ml (only on D6) or 2.3 ml/animal per time point. Collected blood samples were left at room temperature for 20-60 minutes, centrifuged (room temperature, 3000 rpm, 10 minutes) to obtain serum, and measured on the same day or stored at -70 °C until measurement.

　　Cynomolgus monkeys: 3-7 years old, female and male cynomolgus monkeys, the animals are housed at a temperature of 26±3°C, a relative humidity of 50±20%, and 12 hours of light per day from 7:00 am to 7:00 pm. Animals were provided approximately 108 g/animal/day of HF primate J12G pellets and had free access to drinking water. Animals were acclimated under test conditions for 2 weeks, during which time they were treated with drinking water (10 ml/animal) in the same manner as methylcellulose solution. 10 male and 10 female animals were administered a 0.5 W/V% methylcellulose solution (5 mL/kg) using a disposable catheter and syringe. 4.5 ml (-D13) blood was drawn from the femoral vein 13 days before administration and before administration and at 0.5, 1, 2, 4, 8 and 24 h after administration for monkeys under non-anesthesia and restraint.

　　Clinical test method: cTnI and cTnT levels were determined in rats, dogs and cynomolgus monkeys. CK and LDH levels were also measured in dogs and monkeys to monitor the effects of vigorous exercise. All test methods were validated for assay accuracy, assay precision, and freezing stability.

　　RESULTS: Rats: Plasma cTnI levels were below the lower limit of quantification at nearly all time points except for one male and two females at 2 hours post-dose and 1 male at 4 hours post-dose. The detection level was 0.015-0.028 ng/ml. Plasma cTnT levels were below the lower limit of quantification (BLUQ) at all time points.

　　Dogs: Except for 2 male dogs, serum cTnI levels were detected in other animals. Blood cTnT levels, one male and five female dogs showed detectable but low levels throughout the examination period. Other animals showed below the lower limit of quantification at all points. No animal exhibited abnormal LDH values throughout the examination period. One male and two female dogs had higher CK values at 8 hours post-dose than at pre-dose. No corresponding changes in higher CK values were found in cTnI or cTnT in these animals.

　　Cynomolgus monkeys: Three males and one female showed low levels of cTnI during examination. Only two males showed low but detectable cTnT values. Although CK or LDH levels were higher, there was no significant correlation in cTnI or cTnT levels.

　　Conclusion: For cTnT, its value is mostly BLQ, which is more frequent than cTnI. This may be due to differences in the measurement systems used. For rats, all cTnT measurements are BLOQ, therefore, we do not need to account for individual variation or manipulation effects.