The Pharmacodynamics Evaluation Of Endothelin-receptor Antagonist In Pulmonary Hypertension And The Preliminary Research Of Its Mechanism In Myocardial Hypertrophy

Objective:The aim of this project is to evaluated the pharmacodynamics of neotype endothelin-receptor antagonist synthesized by Beijing institute of pharmacology and toxicology at the base of study of structure-function relationship of previous compounds in pulmonary hypertension, meanwhile to investigate its mechanism of action in myocardial hypertrophy, in order to provide evidence for discover new anti-pulmonary hypertension drugs.Methods:1. The toxicity of compounds was evaluated through acute toxicity test of mice and dermat-toxicity test of rats;2. The preliminary pharmacodynamics of compounds was evaluated by radio-ligand receptor binding assay and test of anti-vasoconstriction effects in vitro.3. The integrative pharmacodynamics of compounds was evaluated by detecting pulmonary artery pressure, right ventricular hypertrophy index, heart rate, hematology, the content of ET-1 or ATⅡin plasma and lung and morphology of pulmonary vessel in hypoxic- or MCT-induced rat pulmonary hypertension.4. The preliminary research of mechanism of action was investigated in primary culture of neonatal rat cardiomyocytes by detecting change of protein synthesis and expression of ANPmRNA in ET-1-induced myocardial hypertrophy through [3H]-Leu incorporation and RT-PCR.Result:1. The dermat-toxicity of new compounds in rats and the acute toxicity of YJ009 in mice were significantly lower than previous compounds, and the value of LD50 of YJ009 in mice was 756.8mg/kg.2. In radio-ligand receptor binding assay, YJ007, YJ008 and YJ009 exhibited high affinities for ETA receptor, with Ki values in inhibiting [125I]-ET-1 binding in the nM andμM range and with a ratio of 1/1000-1/100 for ETA and ETB receptor, indicating that compounds were selective ETA-receptor ligands. In rat thoracic aorta ring experiment in vitro, the compounds inhibited the effect of vasoconstriction induced by ET-1 in concentration dependent manner, with value of IC50 in 10-8M levels, and the activity was more potent compared with previous compounds.3.In primary screening of compounds, pretreatment with YJ008 orYJ009 (40mg/kg, administration twice a day, sc) can significantly decrease mean pulmonary artery pressure (MPAP), alleviate right ventricular hypertrophy and the remodeling of the pulmonary vessels (P<0.05), with the ratio of inhibition in MPAP was 39.7% or 43.1%, and 16.7% or 35.6% in right ventricular hypertrophy respectively in hypoxia-induced rat pulmonary hypertension models.4.The further pharmacodynamics research of YJ009. In hypoxia-induced rat pulmonary hypertension models, positive control Bosentan (120mg/kg/d, ig) can significantly decrease MPAP(P<0.05)and right ventricular hypertrophy index (RVHI), with the ratio of inhibition was 34.2% and 23.9% in MPAP and RVHI; pretreatment with YJ009 (5mg/kg, 10mg/kg, 20mg/kg, 40mg/kg, administration twice a day, sc) can decrease MPAP, RVHI and wall thicken of pulmonary vessels (WT) in dose-dependent manner. Especially, 20mg/kg and 40mg/kg groups had significant difference compared with control (P<0.05), with the ratio of inhibition was 41.1% and 64.1% in MPAP and 31.7% and 41.2% in RVHI, respectively. In monocrotaline(MCT)-induced rat pulmonary hypertension models, Bosentan (120mg/kg/d, ig) can partly decrease MPAP and RVHI; pretreatment with YJ009 (10mg/kg,20mg/kg,40mg/kg, administration twice a day,sc) can decrease MPAP, RVHI and WT in dose-dependent manner, and 40mg/kg group had significant difference compared with control (P<0.05), with the ratio of inhibition in MPAP and RVHI was 60.7% and 65.9%; all groups were no effect in heart rate and hematology. Remarkable, YJ009 (40mg/kg, administration twice a day, sc) was no effect by itself to MPAP, RVHI and WT in normal animals.5. In primary culture of neonatal rat cardiomyocytes, ET-1(1,10 and 100nM) can significantly augment the volume of myocardial cells and promote the [3H]-Leu incorporation and ANPmRNA expression in concentration-dependent manner, the [3H]-Leu incorporation was higher than control group by 50%, 73% and 82%, and ANPmRNA expression by 63%, 80% and 98%, respectively (P<0.05); YJ009, BQ123, GF063 and ETP508 (10μM) can significantly inhibit the [3H]-Leu incorporation and ANPmRNA expression induced by ET-1( P<0.05), but GF004 can not.Conclusion:1. The toxicity of new compounds was lowered and the activity was strengthened compared with previous compounds.Especially, YJ009 showed better pharmacological effect in pulmonary hypertension induced by hypoxia- or MCT.2. One of the mechanisms of compounds in improvement of myocardial hypertrophy may be the result of compensation of pulmonary artery pressure decrease indirectly and effect by blocking the ET-receptor in myocardial cells directly.