Pharmacological Study On Dual Actions Of Novel Thienopyridines On Anti-platelet Aggregation And Endothelial Protection

At present, the incidence and mortality rates of cardiovascular andcerebrovascular diseases has continued to rise in China. Since2010, cardiovascularand cerebrovascular diseases have become the leading cause of deaths each year,accounting for about3.5million cases of deaths were reported. AS (atherosclerosis) isone of the most important pathological basis underlying cardiovascular andcerebrovascular diseases. The main features of AS are endothelial injuries, lipidaccumulation, inflammation, reconstruction of the blood vessel walls etc.Dyslipidemia, diabetes, hypertension, obesity, smoking and metabolic syndrome areclosely associated with atherogenesis.It is widely regarded that endothelial dysfunction plays an initial and crucial rolein atherogenesis. Therefore, drugs targeted at endothelial protection might potentiallybe effective in the treatment against AS. However, except heparin, there is still feweffective anti-atherosclerotic drug available that specifically acts on endothelial cells.Endothelial dysfunction activates platelets and promotes atheroscleroticprogression through a variety of mechanismes. Thus, anti-platelet therapy plays an crucial role in the therapy against AS. Clinically, anti-inflammatory andanti-atherosclerotic effects of clopidogrel and prasugrel have been well documented.Based on previous findings, Yang Rifang, associate professor of academy ofMilitary Medical Sciences, synthesized novel thienopyridines by integrating thefunctional groups of thienopyridines and α-aminobenzylphosphonates (synthesized byYang Rifang, research demonstrated their protective effects on endothelial cells andAS), to obtain independently developed novel drugs that possess anti-plateletaggregation and endothelial protective effects, for the prevention and therapy of AS.These drugs are postulated to possess fewer side effects and better metabolicprocesses than α-aminobenzylphosphonates and the existing thienopyridines.This study aims to elucidate at least one novel thienopyridine with dual effects onanti-platelet aggregation and endothelial protection, preliminary studies on theunderlying mechanisms of endothelial protection were investugated at the same time.PART I Screening of novel thienopyridine compounds foranti-platelet aggregating effects and preliminary studieson metabolic processes in vitroAIM: To screen novel thienopyridines possessing anti-platelet effects, and tostudy their metabolic process.METHODS: Platelet aggregation experiments (ex-vivo) were applied to screencandidate compounds which can inhibit platelets aggregation. Drug efficacy over thecourse of time of different-structured compounds, YX-TP03and YX-TP16, werestudied as well. Platelet aggregation experiments (in vitro) were carried out to investigate the possible metabolic processes. The impacts of the candidatecompounds on bleeding time (BT) were explored in mice. Further studies onanti-thrombosis effect were performed on carrageenan-induced caudal arterythrombosis in KM mice.RESULTS:1. Five compounds were screened out performing anti-platelet aggregationaction: compound YX-TP03, YX-TP07, YX-TP08, YX-TP10and YX-TP16All five compounds showed similar efficacy as that of asprin under the samedose, but less effective compared with clopidogrel.2. Compound YX-TP03and YX-TP16showed time-related activitiesCompound YX-TP03(15mg/kg) showed signs of platelet inhibition2h afteradministration, acquired anti-platelet activity4h later, with maximum effect at8hafter administration. Compound YX-TP16(15mg/kg) shows signs of plateletinhibition1h after administration, acquired anti-platelet activity2h later with themaximum effect after8h of administration.3. Compound YX-TP10displayed anti-platelet effect in vitro, while theothers didn’tCompound YX-TP03(300μM), YX-TP07(300μM), YX-TP08(300μM),YX-TP16(300μM) failed to inhibit platelet aggregation (in vitro), indicating thatthese four compounds are prodrugs. YX-TP10(300μM) was found to inhibit plateletsaggregation under in vitro conditions.4. Compound YX-TP03can significantly prolong BTCompound YX-TP03(21mg/kg) can significantly prolong bleeding time (BT);YX-TP07(21mg/kg) and YX-TP10(21mg/kg) showed tendency of BT prolongation;while YX-TP08(21mg/kg) and YX-TP16(21mg/kg) have no effect on BT. 5. Compound YX-TP03and YX-TP10performed anti-thrombosis effectsCompound YX-TP03(21mg/kg) and YX-TP10(21mg/kg) significantlyshortened the length of caudal artery thrombus induced by carrageenan in KM mice,suggesting that both compounds possess antithrombotic properties. Whenadministered at the same dosage as Clopidogrel, both drugs showed lower efficacy.Compared with Asprin, compound YX-TP03and YX-TP10increased the incidenceof plaque formation, but reduced plaque size. Dose-dependent effect was observed forcompound YX-TP03: at7mg/kg, antithrombotic effects was seen, with strongesteffects at63mg/kg; at higher doses, however, thrombosis was observed. In addition,YX-TP03(21mg/kg) administered1h before or24h after carrageenan injectiondisplayed absolute antithrombotic effect, with no antithrombotic effect24h beforecarrageenan injection.CONCLUSIONS:1. Five compounds with anti-platelet aggregation were identified: compoundYX-TP03, YX-TP07, YX-TP08, YX-TP10and YX-TP16.2. Compound YX-TP03displayed a certain extent of anti-platelet aggregation，BT prolongation and anti-thrombosis effects. Besides that, compound YX-TP03didn’t perform anti-platelet aggregation effect (in vitro), indicating that thiscompound is a prodrug.3. To our surprise, compound YX-TP10may inhibit platelet aggregation under invitro conditions, further studies are warranted to study its mechanisms andmetabolism processes in vivo to resolve this issue. PART II Screening of novel thienopyridine compoundswith endothelial protective effect and preliminary studieson mechanismsAIM: To screen novel thienopyridines possessing endothelial protective effects,and investigate the underlying mechanisms.METHODS: Cellular models of HUVEC injuries induced by ox-LDL or highconcentration of glucose were established to evaluate the protective actions of novelthienopyridines. Cell Counting Kit-8(CCK-8) assay was used to observe the viabilityof HUVEC. The expressions of ICAM-1(intercellular cell adhesion molecule-1),VCAM-1(vascular cell adhesion molecule-1), MCP-1(monocyte chemotacticprotein-1) mRNA in HUVEC were examined by QuantiTect SYBR Green PCR kit.The concentration of released NO (nitric oxide) in the medium were acquired byNitric Oxide Assay Kit (Flunorometric).4) Cell proliferation was detected withCCK-8assay.RESULTS:1. Compound YX-TP03, YX-TP12and YX-TP13performed inhibitoryeffects on high concentration of glucose or ox-LDL-induced cell deaths inHUVEC.Glucose (45mM) or ox-LDL(150μg/ml) can injuried HUVEC, threecompounds, compound YX-TP03, YX-TP12and YX-TP13, were screened outperforming endothelial protective effect on high concentration of glucose orox-LDL-induced cell deaths in HUVEC. 2. All the three compounds can significantly inhibited the upregulation ofICAM-1, VCAM-1, MCP-1mRNA in HUVECGlucose (45mM) or ox-LDL(100μg/ml) can upregulate ICAM-1, VCAM-1,MCP-1mRNA in HUVEC, over-expressed ICAM-1, VCAM-1, MCP-1mRNA inHUVEC were down-regulated by compound YX-TP03(100μM), YX-TP12(300μM)or YX-TP13(100μM). Their effects were equivalent to that’s of Vitamin C (Vc).3. Compound YX-TP03、YX-TP12、YX-TP13can significantly inhibited thereduction of NO released by high concentration of glucose or ox-LDL damagedHUVEC.Glucose (45mM) or ox-LDL (75μg/ml) can reduce NO release of HUVEC,compound YX-TP03(100μM), YX-TP12(300μM) or YX-TP13(100μM)significantly inhibited the reduction of released NO induced by ox-LDL or highconcentration of glucose in HUVEC.4. Compound YX-TP03、YX-TP12、YX-TP13can significantly promoteHUVEC proliferation.All the three compounds, compound YX-TP03(100μM), YX-TP12(300μM) orYX-TP13(100μM) promoted HUVEC proliferation.CONCLUSIONS:1. Novel thienopyridines, compound YX-TP03, YX-TP12or YX-TP13displayed protective effects on HUVEC against ox-LDL or high glucose-inducedinjuries. Their proliferation effects were evidenced at the same time.2. Their regulation on ICAM-1, VCAM-1, MCP-1mRNA expressions maycontribute to their anti-inflammatory and anti-atherosclerotic effects.3. This study also reveals that endothelial protection might be associated withregulation of NO release. SUMMARIES：In summary, this study identified one independently developed novelthienopyridine compound possessing dual activities on anti-platelet aggregation andendothelial protection: compound YX-TP03. It’s dual activities on anti-palateletaggregation and endothelial protection may make it be an effective drug for AS.In addition, compound YX-TP10can directly work on platelets in vitro,acquiring anti-platelet and antithrombotic effects, which may make it be a noveleffective anti-platelet drug.