NEW USE OF CARBAMATE Beta PHENYLETHANOLAMINE ANALOGUES FOR ENHANCING INTRACELLULAR CLEARANCE OF LDL CHOLESTEROL AND FOR COMBINING THERAPY WITH STATINS TO ENHANCE THE EFFICACY AND REDUCE ADVERSE EFFECTS
A technology of carbamate and phenylethanolamine, which is applied in the direction of drug combination, active ingredients of amines, active ingredients of esters, etc.
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Embodiment 1
[0070] R-Bambuterol increases the expression of LDL protein receptor and promotes the uptake of LDL-cholesterol
[0071] Detection method
[0072] Mouse liver AML12 cells were seeded in 24-well culture plates and cultured in DMEM / F12 medium containing 10% heat-inactivated fetal bovine serum. Incubate cells with fluorescently labeled LDL to study cellular uptake and metabolism of LDL-cholesterol. Cell-expressed LDL receptors were detected with an LDL-specific antibody.
[0073] Divide the cells into different groups, add R-bambuterol (R-BM), atorvastatin (Statin) or R-BM+Statin respectively, including the following groups: control group, LDL protein, LDL protein+Statin 10 μM, LDL protein + R-BM 10 μM or 20 μM, LDL protein + R-BM 10 μM + Statin 10 μM, LDL protein + R-BM 20 μM + Statin 20 μM.
[0074] After 24 hours of treatment, cells were washed and fixed with paraformaldehyde. Then the primary antibody against LDL protein receptor diluted 1:200 was added and incubated for ...
Embodiment 2
[0084] R-BM can improve the clearance or transport of LDL-C, and has a synergistic effect with statins.
[0085] Experimental content
[0086]HepG2 cells are human hepatocytes, which were seeded in 6-well plates and cultured in DMEM / F12 medium containing 10% heat-inactivated fetal bovine serum. Cells were exposed to 40 μg / mL LDL environment and incubated with different doses of R-BM. After incubation for 24 h, the total intracellular and extracellular cholesterol contents were detected using a total cholesterol assay kit (Total Cholesterol Assay Kit E105, Applygen Technologies Inc., Beijing, China). In addition, in the mouse liver cell (AML12) experiment, under the same cell culture conditions, in the environment of 40 μg / mL LDL, 10 μM statins, 10 μM R-BM or a combination of the two were given respectively, and the same method was used to detect intracellular LDL -C content.
[0087] Experimental results
[0088] 1) Increased clearance of intracellular LDL-C
[0089] In t...
Embodiment 3
[0098] R-BM can counteract statin toxicity, thus exhibiting a protective effect when combined with statin administration.
[0099] Experimental content
[0100] HepG2 cells or pulmonary artery smooth muscle cells (Pulmonary artery smooth muscle cell, PASMC) were seeded in 96-well plates. When the cell density reached about 50%, the medium was replaced with a serum-free medium, and atorvastatin or R-BM was given and incubated at room temperature for 24 hours. Afterwards, the serum-free medium containing 10% CCK-8 was replaced, cultured in the dark for 3 h, and the cell viability was detected by a multifunctional microplate reader (TriStar2S LB942). The drug-free group was used as the control group, and the cell viability of the R-BM, statin or both groups were normalized to the control group. In all groups except the control group, the concentration of R-BM was 20 μM.
[0101] Experimental results
[0102] (1) Experimental results of HepG cells
[0103] All concentrations ...
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