| Globally,cardiocerebravascular diseases is of highest incidence and mortality among all diseases. In all factors that induce cardio-cerebral vascular diseases, high blood pressure and its complications play a vital role. The last report shows that at present there are 300 million patients with hypertension in China with an incidence at about 25% and 10 million new cases each year. Since hypertension features high prevalence, high morbidity, high mortality, low awareness, low treatment rate and low control, therefore, hypertension and its complications threaten health of people all the time and bring heavy burden to people and society.It is of great significance that effectively controls blood pressure and its complications and decreases the morbidity and mortality of cardio-cerebral vascular diseases. At present, the commonly used medicines in clinical practice mainly include: Calcium antagonists, Beta Adrenoceptor blockers, Diuretics, Angiotensin Receptor Blockers(ARBs), Angiotensin-converting enzyme inhibitor. Among these, ARBs has a clear effect of lowering blood pressure, less side effects, and it can be well tolerated by patients. Moreover, it is the only drug that can lower blood pressure and at the same time protect heart, brain, kidney and other end-organs, which is important for the more comprehensive improvement in cardio-cerebral vascular diseases. In recent years, with gradually development of pharmacological study on ARBs, it is found that its protective effect to target-organ is not only related to its Angiotensin II Type I Receptor(AT1)but also to peroxisome proliferators-activated receptors γ. Other antihypertensive drugs don’t possess the feature that combines Angiotensin II Type I Receptor and peroxisome proliferators-activated receptors γ(PPARγ). Previous study thought that mechanism of action of ARBs was only through Angiotensin II Type I Receptor that inhibited the excessive activation of Renin-Angiotensin-System to make antihypertensive efficacy. The present study shows that ARBs can also make antihypertension efficacy. The pharmacological effects such as improvement of lipid glucose metabolism, stroke, cognition through activating PPARγ, and the activation of PPARγ accompanied make a wider ARBs clinical application.8 kinds of sartan drugs are mainly used in the market among ARBs at home and abroad. Telmisartan owns high bioavailability and long half-life, it is also the only sartan that could activate PPARγ under therapeutic dose. Based on the diverse biological activity of telmisartan, we study new-type ARBs, improve telmisartan’s physical and chemical properties and increase its lipid solubility by structural modification. The research group preliminarily got chemical compound Tek-1 through telmisartan remodel and studied relative Pharmacology and molecular mechanisms of the compound. The study found that the compound Tek-1 has a similar level as telmisartan that antagonizes AT1 receptor and partly activates PPARγ, while its lipid solubility is higher than that of telmisartan. Based on above study, we firstly assessed the in vivo antihypertensive efficacy of Tek-1, proving that is has good in vivo activity; and then, taking Tek-1 as leading compound, designeda series of compounds with completely new structure that had high PPARγ activating activity. Aiming at new-structure compounds Tek-2, Tek-3 possessing AT1 receptor antagonizing activity, the present firstly analyzed its activating activity and selectivity to PPARγ, then conducted pharmacological studies on compounds in vivo and vitro. It is expected that gained a candidate compound of multifunctional new ARBs that had good antihypertensive efficacy and higher activating activity to PPARγ.Main findings of present thesis include:1 Research on antihypertensive efficacy of leading compound Tek-1 in vivoTo prove that leading compound Tek-1 owning activity in vivo, we firstly determined the antihypertensive efficacy of the compound in vivo. Spontaneously hypertensive rats(SHRs)were administrated by compounds orally, and then the blood pressure of SHRs of different groups for 1-4 weeks were investigated. The result showed that, compared to the basic blood pressure of pretreatment of SHRs,telmisartan(5, 10mg/kg) and Tek-1(1, 5, 10mg/kg) both could significantly decrease the systolic and diastolic blood pressure of SHRs(P<0.01, P<0.01, P<0.05, P<0.05, P<0.01); the relationship between antihypertensive efficacy and dose showed in a dependent pattern,that is the more dose acquired, the lower the blood pressure decreased; telmisartan of 10mg/kg had the highest antihypertensive efficacy, Tek-1 of 10mg/kg and telmisartan of 5mg/kg had the similar antihypertensive efficacy.2. Research on in vitro pharmacological effects of new compounds Tek-2, Tek-3Determined by systematic pharmacology research of leading compound Tek-1in vivo and vitro.The research of pharmacological properties of new compounds Tek-2, Tek-3 were conduced in vitro.2.1 Research on PPAR selectivity and PPARγ activating activity of new compounds Tek-2 and Tek-3Establishing PPARγ-responsive element- lucifarase reporter system, using the system to assessed the PPARγ activating efficacy of Tek-2 and Tek-3, the result showed that activating folds of telmisartan with concentration of 1, 10μM to PPARγ were 1.30±0.07(P<0.05) and 1.27±0.01(P<0.05), respectively; Tek-2 of 0.1, 1, 10μM also could concentration-dependently activate PPARγ receptor, with corresponding concentration, the activating folds of Tek-2 were 1.34±0.12(P<0.01), 1.46±0.08(P<0.01) and 1.44±0.04(P<0.01), respectively; while new compound Tek-3 showed no significant activation to PPARγ receptor under various concentrations. Tek-2, with various concentrations, had higher activating activity than that of positive drug telmisartan, and its activating percentage with concentration of 10μMwas 22.7±4.2%, which was significantly different from 11.9±2.4% of telmisartan(P<0.05). Research on activating activity of new compound Tek-2 and Tek-3 to PPARα was furtherly investigated. Establishing PPARα-responsive element- lucifarase reporter, using the system to assess the PPARα activating efficacy of telmisartan, Tek-2 and Tek-3, and the results showed that there was no significant activating efficacy of telmisartan, Tek-2 and Tek-3 with various concentrations to PPARα receptor.2.2 Research on the affinity of new compound Tek-2 to AT1 receptorThrough [125I]-AngⅡradioligand binding assay on rat vascular smooth muscle cells(VSMCs), the affinity of telmisartan and Tek-2 to AT1 receptor was investigated. Telmisartan and Tek-2 had stronger competitive inhibition effect to AT1 receptor of primary VSMCs, Ki values are 9.25±1.08 n Mand 3.3±0.7n M, respectively. The results showed that Tek-2 had high affinity to AT1 receptorsimilar to that of telmisartan.2.3 Research on antagonizing activity of new compound Tek-2 to AT1 receptorUsing HEK293-Gα15 cell of stable AT1 receptor expression, the antagonistic effect of compound Tek-2 on AT1 receptor was explored using intracellular calcium mobilization detection assay. The result showed that telmisartan and Tek-2 with an concentration of 0.0128-1000 n M, the compound itself had no effect on calcium release in cells; while telmisartan and Tek-2 had significant antagonizing effect on calcium release in Ang II-induced cells, IC50 values were 0.7±0.11 n M and 2.28±0.24 n M, respectively. Thus it could be seen that Tek-2was antagonist for AT1 receptor whose antagonizing activity was slightly lower than that of telmisartan.3. Research on pharmacological effect of new compound Tek-2 in vivo3.1 Tek-2 could significantly decrease blood pressure of SHRsSimilarly, SHRs was orally administrated by Tek-2 and the effect of Tek-2 on blood pressure of rats of 1-4 weeks old was observed. The results showed that telmisartan(5, 10mg/kg) and Tek-2(2.5, 5, 10mg/kg)both could decrease SHRs systolic and diastolic blood pressurein the dose-dependent pattern; the antihypertensive efficacy of compound behaved positively correlated with dosage; telmisartan of 10mg/kg had the highest antihypertensive efficacy, and Tek-2 of 10mg/kg had similar antihypertensive efficacy as that of telmisartan of 5mg/kg.3.2 Tek-2 could significantly decrease infarct volume of t MCAO mice and exerted anti-inflammatoryeffect in brainIn comparison to sham-operated group, infarct volume of t MACO mice increased 13.02±1.15%(P<0.01), which indicated the successful establishment of t MCAO model in mice.In comparison to t MCAO model group, using TTC staining, it was found that groups of telmisartan of 10mg/kg、Tek-2 of 5mg/kg and Tek-2 of 10mg/kg could all significantly decrease brain infarct volume,which were 7.32±1.07%,8.45±0.57%, 5.95±0.52%(P<0.01, P<0.01, P<0.01), respectively.Given the same dose of 10mg/kg, the effect of telmisartan on the decreasement of brain infarct volumewas lower than that of Tek-2(P<0.05).While given 5mg/kg Tek-2,the extent of brain infarct volume decreasementwas similar as that of 10mg/kg of telmisartan.Immediately treated by 10mg/kg of telmisartan and Tek-2 following GW9662 ventricle administration, the infarct volumes of telmisartan and Tek-2 group were 8.45±1.03% and 9.06±1.05% respectively. Compared to the group thatwas not treated by GW9662, infarct volume of group of 10mg/kg of Tek-2 increased,which had significant difference(P<0.05), infarct volume of group of 10mg/kg of telmisartanhad no significant difference. Through neurologic severity score, it was found that groups of 10mg/kg of telmisartan, 5mg/kg of Tek-2 and 10mg/kg of Tek-2 could significantly lower neurologic severity score(P<0.01, P<0.01, P<0.01). In comparison to group of 10mg/kg of Tek-2, neurologic severity score of group of GW9662+Tek-2 10mg/kg was significantly increased(P<0.05). All these results showed that effect of Tek-2 on lowering infarct volume was related to inhibition of PPARγ.Using real-time quantitative PCR technology, Investigation of the changes of expression levels involving proinflammatory factors TNF-α, IL-6 and i NOS m RNA in penumbra on infract side of cerebral cortex was conducted on each treated group furtherly. it was found that expression level of TNF-α m RNA of telmisartantreated group significantly decreased(P<0.05); 10mg/kg Tek-2 could significantly inhibit the up-regulation of TNF-α, IL-6 and i NOS on m RNA level(P<0.01, P<0.05, P<0.01).The results indicatedthatthe effect of Tek-2 on inhibition of brain inflammation may be important mechanism for improving stroke.In conclusion, we obtained new-type antihypertensive compound Tek-2 with high PPARγ agonistic activity. Through the systematic research in vivo and vitro, we found that Tek-2 had clear antihypertensive efficacy in vivo; and on t MCAO model, we found that it had strong effect of protecting cerebral infarction and post-infraction nerve injury, whose mechanism was related to the improvement of cerebral inflammation. New-structural Tek-2 had pharmacological feature of AT1 receptor blocking and high PPARγ activation, which was expected to be new generation of ARBs with multiple pharmacological efficacies. |
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