Studies On The Discovery And Evaluation Of New Drugs For The Treatment Of Vascular Diseases

The new drug research and development is closely related to the health of human beings. The model of drug discovery has transferred from random development to specific target-oriented research, which exhibits several characteristics such as high-performance, rapid and specificity. The pharmacological research is involved in the every step of drug discovery and development, including discovery and validation of drug target, establishment of drug screening model, obtaining of lead compound, preclinical research of drug candidate and clinical evaluation of drug. Therefore, development of new theory and technology of pharmacological research and development would advance the discovery of new drugs.Cerebrocardiovascular diseases are the top cause of mortality among all the human diseases worldwide at the present. More than a third of all deaths annually are caused by cerebrocardiovascular diseases. The pathological changes of vasculature, including vascular structure abnormality and vascular dysfunction, are the key etiology of most cerebrocardiovascular diseases. Those kinds of diseases are classified into vascular diseases. Various medications have been clinically available nowadays, most of which have been proven to be effective in treating these diseases. However, the patients have to take the medications through their lifetime because these drugs are targeting the symptom rather than the pathogeny. And some of the drugs have shown serious side effects, which may interfere with the compliance and curative effects. More importantly, the morbility of vascular diseases is increasing with more and more uncontrolled cases every year, despite the availability of numerous medications. Therefore, the discovery of novel drugs for preventing and treating vascular diseases, as well as the evaluation of the efficacy for these drugs, is the primary purpose and task for the healthcare workers and researchers all over the world.The aim of the present study was to find new drug candidates through evaluating the novel drug targets for vascular diseases and establishing related drug screening models, and to test the efficacy of bioactive compounds on the animal models of vascular diseases further in order to pave the way for their clinical trials.PartⅠDrug screening for urotensinⅡreceptor antagonistUrotensinⅡ(UⅡ) exhibits extensive bioacitivities, and it is known as the most potent vasoconstrictor so far. UrotensinⅡreceptor (UTR), expressed in many tissues and organs in human body, is a recently identified member of G-protein-coupled receptor family. It is reported that UII plays an important role in the etiology of many vascular diseases, which makes it the potential drug target for several cardiovascular diseases such as hypertension and atherosclerosis. Our study was to evaluate this drug target and screen UTR antagonist.Firstly, we found that the expression of cytoskeletal protein and vinculin in vascular smooth muscle cells (VSMC) was enhanced by UII treatment, indicated that UII could induce the transformation of VSMC phenotype by promoting the rearrangement of cytoskeleton of VSMC, which ultimately enhanced VSMC migration and influenced the structure and function of vascular wall. Furthermore, our study showed that the secretion of ICAM-1 and VCAM-1 and the expression of mRNA of iNOS and VEGF were increased in VSMC by UII stimulation; additionally, the release of NO by vascular endothelial cells was decreased by UⅡstimulation. Thus, the mechanism of enhanced VSMC proliferation and migration was attributed to the activated inflammatory reaction.Secondly, we established a drug screening model for this new drug target. We constructed a three dimensional structure of UTR by homology modeling using the stereochemical structure ofβ2 adrenergic receptor as a model at the first step. Then the receptor-based virtual screening was applied to screen more than ten thousand compounds in our sample library using docking of stimulated UTR model with these compounds, and the top 500 compounds which showed strongest ability to dock with UTR model were selected for further cell-based drug screening. Meanwhile, we established a stable HEK293 cell line expressing UTR, and performed the receptor antagonism screening for the 500 compounds on the UTR-HEK293 cells by homogeneous time resolved fluorescence resonance energy transfer technology. The results showed 12 compounds exerted UTR antagonism in the first round of screening. However, they failed to exhibit any activity in the second screening. In this part, we successfully established a cell-based high throughput drug screening model for UTR antagonist, which might be applied to other GPCR antagonist screening. This method shows a prosperous application in drug research in the future.Part II Establishment of in vivo vascular injury model and its application on evaluation of the efficacy of Rho-kinase inhibitorsFirst of all, we used the L-NAME-induced endothelial dysfunction model and vitamin D3/nicotine induced vascular calcification model to exam the extent of vascular injury and blood pressure imbalance. The results demonstrated that a sustained systemic hypertension was induced by chronic L-NAME treatment in rats, along with blunted vasoconstriction and vasodilation in thoracic aorta, whereas vascular calcification affected little on the blood pressure and vascular function of rats. Therefore, we chose the L-NAME-induced hypertensive rat model to evaluate the efficacy of Rho-kinase inhibitors.Rho-kinase is a serine/threonine protein kinase, which is the downstream effector of the small GTP binding protein Rho, which plays a key role in the development of various cerebrocardiovascular diseases such as hypertension, artherosclerosis, pulmonary hypertension, cerebral angiospasm, myocardial infarction and heart failure. Thus, currently Rho-kinase is a novel potential drug target for vascular diseases. In our pilot study, several compounds were identified as the Rho-kinase inhibitors through high throughput screening. We chose two of them, coptisine and DL0805, to evaluate the efficacy on L-NAME-induced hypertensive rat model, as well as the drug effects on the vascular structure and function, renal and cardiac structure and function in the rats.We found that:(1) Coptisine and DL0805 showed no antihypertensive effect on the L-NAME-induced hypertensive rat model, without any significant effect on the heart rate, either. (2) Coptisine and DL0805 decreased the reactivity of aorta to NE, KC1, CaCl2 and PDBu through blocking the voltage-dependent and receptor-dependent Ca2+channels, while they exerted little effects on the vasorelaxation. (3) HE staining of aorta showed that DL0805 and high-dose coptisine reduced the vascular wall diameter, which suggested the antihypertorphic effect of Rho-kinase on vasculature. (4) The results of plasma biochemical analysis showed that coptisine and DL0805 not only increased the NO, GSH levels and SOD activity, but also decreased the ROS and MDA contents, which indicated the Rho-kinase inhibitor could exhibit protective effect on target organs by its anti-oxidative action. (5) Coptisine and DL0805 significantly down-regulated the over-expression of RhoA/ROCK and reduced the expression level of p-eNOS, AMPK and Akt slightly in the vessels with regard to the Western blotting results.The results on renal and cardiac mitochondrial function showed that:(1) Coptisine and DL0805 elevated the RCR and ADP/O in the renal and cardiac mitochondria and enhanced the state III and IV respiratory rate when using glutamate-malate as the substrates. In addition, these drugs increased the ATP content, as well as the activities of SOD and SDH in the mitochondria of kidney and heart.The results on renal tissue study showed that:(1) Coptisine and DL0805 reduced the areas of renal glomerulus and improved the pathological changes of kidney, according to the histopathological results. (2) Coptisine and DL0805 reduced the over-expression of RhoA and reduced the expression of total ERK, while increased the expression of p-ERK in kidney, which suggested that these two drugs exhibited renoprotective potentials through influencing the pathway of RhoA and ERK.In all, the Rho-kinase inhibitors coptisine and DL0805 showed no antihypertensive effect on the L-NAME-induced hypertensive rat model, however, they regulated the vasoconstriction by influencing the Ca2+channels, reduced the overactivated oxdidative stress, reversed the vascular hypertrophy by down-regulation of RhoA/ROCK. Moreover, coptisine and DL0805 improved the renal and cardiac mitochondrial function in L-NAME treated rats, and reduced the structural and functional changes of kidney by affecting the pathways of RhoA/ROCK and ERK. These abovementioned results illustrated that inhibition of Rho-kinase activity leaded to the protection of targeted organs in hypertension such as vessel, kidney and heart.Part III Protective effect of pinocembrin on cerebrovascular diseases and its mechanismIschemic stroke (cerebral ischemia), caused by blockade of blood supply due to embolism of cerebral vessels, can lead to a series of physiopathological dysfunction, which is also one kind of vascular diseases. Most of the currently available medications for stroke are classified to neuroprotective drugs, except for thrombolytic drug. Pinocembrin has been studied on the effects of neuroprotection and vasodilation in our laboratory for more than a decade. In order to verify its efficacy on cerebral ischemia, we investigated the therapeutic time window of pinocembrin in the transient middle cerebral artery occlusion rat model (MCAO) and the protective effect of pinocembrin against ischemic injury on the global cerebral ischemia rat model induced by four vessels occlusion (4VO).Our results showed that the neurological defect and brain edema were ameliorated significantly by 1 mg/kg and 5 mg/kg pinocembrin therapy at 1,4,6 h after MCAO, along with reduced brain infarction in drug-treated groups, indicating the therapeutic time window for pinocembrin on MCAO is 6 h. In 4VO study, the results showed that pinocembrin alleviated the brain edema and neurobehavioral dysfunction in the rats suffering cerebral ischemia/reperfusion, and reversed the neural death evidenced by histopathological examination using Nissl staining for the pyramidal neurons in hippocampal CA1 region. Furthermore, pinocembrin dose-dependently reduced the MDA content and SOD activity, decreased MPO activity to lessen inflammatory reaction, and generally regulated the metabolic balance between excitatory amino acids and inhibitory amino acids in the central nervous system.These abovementioned results demonstrated that pinocembrin exhibits a significant anti-cerebral ischemia and neuroprotective effect against ischemic injury, which may pave the way for its clinical development for stroke therapy in the future.In summary, the present study, which includes three main different research contents, focused on the key steps of discovery for new drugs, such as validation of drug target, establishment of drug screening model and compound screening for new targets, evaluation of drug candidate for efficacy. This study represents the characteristic principle of drug discovery, which may supply with abundant experimental data to the research and development on novel drug of preventing and treating vascular diseases.