大鼠胎盘转移,乳腺排泄 z-bo 2020-11-17 379

　　Background: Hypertension is one of the most common medical problems during pregnancy, affecting 6-8% of pregnant women. Although many pregnant women with high blood pressure have healthy babies without serious problems, high blood pressure may increase maternal and perinatal risks. For example, the mother suffers from pre-eclampsia, which may affect the placenta and the mother's kidneys, liver, and brain, and cause fetal complications such as low weight and premature birth. In the most severe cases, preeclampsia can progress to convulsions and life-threatening. However, drug treatment for pregnant women may cause various developmental toxicity. Some drugs that lower blood pressure are considered safe during pregnancy. However, some of the most effective antihypertensive drugs, such as angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs), are usually avoided during pregnancy. For example, Losartan has been designated by the FDA as pregnancy category D, which means that based on adverse reaction data, there is strong evidence that it has an effect on the development of human fetuses. The fetal toxicity associated with ACE inhibitors and ARBs is related to the reduction of angiotensin II and the dysfunction of the renin-angiotensin system during fetal development. ACE inhibitors can cause fetal toxicity including severe renal insufficiency, neonatal anuria, skull ossification defects, fetal growth retardation, stillbirth, and abnormal features. Intrauterine exposure to drugs is evidence of fetal or neonatal toxicity, renal insufficiency and the use of ARBs have been shown to reduce fetal weight, cause renal damage, and even cause fetal death. In addition to the toxic effects of the drug itself, the distribution characteristics of the drug are directly related to the safety of the fetus and newborn. The large distribution of the drug in the fetus and breast milk is one of the important factors leading to the toxicity of the fetus and the newborn, and the less distribution will lead to the reduction of toxicity. Placental transfer and milk excretion are characteristics of ARBs. The distribution of FMS in fetus or breast milk is unclear, and it is the key to evaluate its safety in pregnancy and lactation. In this study, the placental metastasis and milk secretion in pregnant and lactating rats were detected. After intravenous infusion, the pharmacokinetics of FMS in placenta, amniotic fluid, fetus and milk were evaluated.

　　Method: Animal test: Female SD rats (8-10 weeks old, weight 250-330 g), 12-hour light/dark cycle animal facility, temperature 23±3°C, relative humidity 55±15%, change every hour Gas 10-20 times. There are 3 animals in each cage.

　　Mother-to-child transfer of FMS: At 16-17 days of gestation (GD), the size of the fetus is sufficient for analysis. Female rats are anesthetized by intraperitoneal injection of Shutai 50 (20 mg/kg), and polyethylene tubing is implanted into the internal jugular vein and Femoral artery. One day after recovery, FMS was dissolved in physiological saline and injected through the jugular vein at doses of 2.70 and 5.50 mg/kg at rates of 0.17 and 0.34 mg/h/kg, respectively, to reach target concentrations of 100 and 200 ng/ml. The initial intravenous bolus loading dose and continuous intravenous infusion rate were determined by the target steady-state concentration (CSS) multiplied by the steady-state volume of distribution and FMS clearance rate. Administration without anesthesia. Blood was taken from the femoral artery before and 4, 8, 24, 28, and 32 hours after the operation. At each sampling time, collect about 0.3 ml of blood and mix with the same volume of heparinized saline (50 IU/ml). Centrifuge at 13000 g for 5 min and store at -20°C until plasma sample analysis. 32 hours after the start of intravenous infusion of drugs, the animals were anesthetized by intravenous injection of Shutai (2 mg/kg), and the animals were killed by cervical dislocation. Three samples of each tissue, such as placenta, amniotic fluid, and fetus. The placenta and fetus are homogenized with a homogenizer after adding saline. The samples are stored at -20°C until analysis. The average concentration of 24-32hde FMS represents the steady-state plasma concentration. The tissue-plasma partition coefficient (KP) is calculated by dividing the steady-state plasma FMS concentration by the 32-hour average tissue FMS concentration.

　　FMS in milk secretion: During mid-lactation, on the 12th-13th day of lactation (LD), female rats were injected with Serate 50 (20 mg/kg) intraperitoneally under anesthesia, the internal jugular vein and femoral artery were implanted with poly Vinyl pipe. One day after recovery, FMS was dissolved in physiological saline and injected through the jugular vein at doses of 2.70 and 5.50 mg/kg at rates of 0.17 and 0.34 mg/h/kg, respectively, to reach target concentrations of 100 and 200 ng/ml. The dose is given in a non-fasting state. Blood samples were collected before administration and 4, 8, 24, 28, and 32 hours later. After 32 hours of continuous intravenous infusion, Shutai 50, 2 mg/kg was injected intravenously to obtain milk under mild anesthesia. 5 IU of oxytocin was injected subcutaneously 30 minutes before breast milk sampling to facilitate breast milk collection. Pull out the nipple gently by hand to stimulate milk discharge, and collect the milk in a polypropylene tube. The samples are stored at -20°C until analysis. The average concentration of 24-32hde FMS represents the steady-state plasma concentration. The KP of milk is calculated as the 32-hour milk concentration exceeding the plasma FMS concentration.

　　Liquid chromatography-mass spectrometry combined to determine the concentration of FMS: The concentration of FMS in biological samples was determined by the previously reported LC-MS/MS method. 200μL of acetonitrile and 50μL of internal standard solution were added to 50μl of the thawed biological sample and vortexed for 1 minute. The mixed sample was centrifuged at 15000G for 10 min at 4°C. The supernatant was transferred to a polypropylene tube and diluted with the same volume of distilled water. Inject 10uL volume of liquid into the LC-MS/MS.

　　Result: Liquid chromatography-mass spectrometry combined to determine FMS: the lower limit of detection in plasma, placenta, amniotic fluid, fetus and milk matrix is 0.5 ng/ml. The accuracy rate is 94.2-117.9% in plasma, 89.2-111% in placenta, 87.7-116.9% in amniotic fluid, 89-110.7% in fetus, and 88.8-109.5% in milk. The accuracy of plasma, placenta, amniotic fluid, fetus, and milk samples were 8, 12.3, 3.8, 10.4, and 8.5%, respectively.

　　FMS placental transfer: When the steady-state concentration of FMS (CSS) in the plasma of pregnant rats is 100 and 200 ng/ml, the average plasma and tissue FMS concentration changes with time. The prediction interval of CSS is 10 and 90%, mainly because FMS is cleared. The 10 and 90% CSS prediction intervals are 81.4 and 138.6 ng/ml, respectively, and the target is 100 ng/ml (intravenous infusion, rate = 0.17 mg/h/kg) and 162.8 and 277.3 ng/ml, the target is 200 ng/ml (intravenous infusion, rate = 0.34 mg/h/kg). After administration of FMS, the plasma FMS concentration increased rapidly, reaching 114.1±22.0 and 213±89.4 ng/ml, which were close to the expected target CSS=100 and 200 ng/ml within 24 hours, respectively. There was no significant difference in FMS concentration after 24, 28 and 32 hours, indicating that the FMS concentration has reached a steady state. FMS concentration in placenta, amniotic fluid, fetus and plasma 32 hours after administration. The target dose group of CSS=200 ng/ml showed that the concentration of placental FMS was 112.2±51.2 ng/g and the concentration of placental FMS of CSS=100 ng/ml group 7 was 4.9±24.5 ng/g. The FMS mass concentrations of the amniotic fluid in the CSS=200 ng/ml and CSS=100 ng/ml dose groups were 3.3±3 and 2.3±1.3 ng/ml, respectively, and the fetal FMS mass concentrations were 37.4±23.6 vs. 19.2±4.8 Nanograms/gram. There was no statistically significant difference in tissue concentration between the CSS=200 ng/ml and CSS=100 ng/ml groups. Table 2 summarizes the average tissue-to-plasma partition coefficient (KP). The partition coefficient of placenta and plasma (KP, placenta) is 44.6-59%, and the KP values of amniotic fluid and fetus are low, 1.3-1.7% and 14.9-17%, respectively. The KP values of all tissues in the high-dose and low-dose groups are comparable, indicating that as the plasma concentration increases, the concentration in the tested tissue increases proportionally, while the placental metastasis of FMS is not affected by the dose.

　　FMS in milk secretion: The target steady-state concentration (CSS) of 100 and 200 ng/ml is 13-14 days after delivery. With the use of FMS, the average FMS concentration in plasma and milk changes with time. The Css prediction interval is 10-90%, and the plasma FMS concentration increases rapidly, close to the target CSS of 100 and 200 ng/ml, and maintained throughout the study period. There was no statistically significant difference in plasma FMS concentration at each sampling time. 32 hours after administration, the plasma concentrations in the 100 and 200 ng/ml groups were 126.4±49.3 and 198.8±40.8 ng/ml, respectively. The concentration of FMS in plasma and milk increased significantly in a dose-dependent manner. The CSS=200 ng/ml group had significantly higher plasma and breast FMS concentrations than the CSS=100 ng/ml group. The calculated plasma and plasma partition coefficient (KP) is 10.4 to 15.2%. The comparison of the milk and plasma partition coefficients between dose groups (100 vs. 200 ng/ml) showed that as the plasma concentration increased, the concentration in the milk increased proportionally, while the amount of milk secretion was not affected by the dose.

　　Conclusion: This is the first report on exposure of fetuses and newborns to FMS. Our data indicate that FMS transfer to the fetus and breast milk is relatively lower than other ARBs. Further research is needed to evaluate the clinical impact of FMS transfer to the fetus and breast milk, and reveal the potential mechanism of FMS in the fetus or breast milk.