Potent Antithrombotic Agents Research Articles

Design, green synthesis and pharmacological evaluation of novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine moiety as potential antithrombotic agents

The aim of this research work was to investigate a series of novel 5,6-diaryl-1,2,4-triazines (3a–3q) containing 3-morpholinoethylamine side chain, and to address their antiplatelet activity by in vitro, ex vivo and in vivo methods. All compounds were synthesized by environment benign route and their structures were unambiguously confirmed by spectral data. Compounds (3l) and (3m) were confirmed by their single crystal X-ray structures. Out of all the synthesized compounds, 10 were found to be more potent in vitro than aspirin; six of them were found to be prominent in ex vivo assays and one compound (3d) was found to have the most promising antithrombotic profile in vivo. Moreover, compound (3d) demonstrated less ulcerogenicity in rats as compared to aspirin. The selectivity of the most promising compound (3d) for COX-1 and COX-2 enzymes was determined with the help of molecular docking studies and the results were correlated with the biological activity.

Progress in the research of GPIIb/IIIa antagonists.

This review covers the recent advances in the development of small RGD (Arg-Gly-Asp sequence) containing peptides and their mimetics as potential antithrombotic agents. Glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists include monoclonal antibodies, RGD peptides, peptide hybrids and nonpeptide mimetics. The current trend in the development of nonpeptide mimetics is clearly directed toward orally active and safe antithrombotic drug candidates. But several nonpeptide mimetics, being evaluated for their oral activity in human clinical trials, are currently not approved for clinical use due to poor safety profile. It is expected that newer and more effective nonpeptide mimetics will be developed in the near future.

Pharmacological actions of nobiletin in the modulation of platelet function.

Background and PurposeThe discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin, in the modulation of platelet function.Experimental ApproachThe ability of nobiletin to modulate platelet function was explored by using a range of in vitro and in vivo experimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. Fibrinogen binding, α-granule secretion and calcium mobilization assays were performed using platelet-rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice.Key ResultsNobiletin was shown to suppress a range of well-established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilization and thrombus formation. Nobiletin extended bleeding time in mice and reduced the phosphorylation of PKB (Akt) and PLCγ2 within the collagen receptor (glycoprotein VI)-stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of vasodilator-stimulated phosphoprotein, a protein whose activity is associated with inhibitory cyclic nucleotide signalling.Conclusions and ImplicationsThis study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore, nobiletin may represent a potential antithrombotic agent of dietary origins.

Pharmacokinetics, dose proportionality and permeability of S002-333 and its enantiomers, a potent antithrombotic agent, in rabbits

1. S002-333 [(2-(4′-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-pyrido (3,4-b) indole-3-carboxylic acid amide)] is a novel and potent antithrombotic active agent. The present work investigates the pharmacokinetics, bioavailability, dose proportionality and permeability of the racemate, S002-333 in male New Zealand White (NZW) rabbits.2. Rabbits were administered single intravenous (i.v.) (2 mg/kg) and three oral doses of 10, 20 and 40 mg/kg of S002-333, respectively, at different occasions to evaluate dose proportionality. Serial blood samples were collected and analyzed by a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Since S002-333 is a racemate consisting of S004-1032 (R) and S007-1558 (S), same samples were analyzed using a chiralcel column so as to evaluate the respective enantiomers.3. The peak plasma concentration, after oral administration, occurred at ∼10 h post-dose. The clearance (CL) and volume of distribution (Vd) after i.v. dose were found to be 3.05 ± 0.09 l/h/kg and 6.73 ± 1.16 l/kg, respectively. The absolute oral bioavailability of S002-333 was 16.32%, whereas it was 6.62 and 5.90% for R- and S-enantiomers, respectively. The absolute bioavailability of 10, 20 and 40 mg/kg doses were found to be 27.91, 14.39 and 16.91%, respectively. The PAMPA (parallel artificial membrane permeability assay) assay shows that S002-333 has a low-passive permeability at gastric and intestinal environment.4. In conclusion, S002-333 has low-passive permeability, low CL and large Vd. The R-enantiomer has a “slightly” greater bioavailability than the S-enantiomer.

The role of the novel oral anticoagulants in the secondary prevention of acute coronary syndromes.

Acute coronary syndromes continue to be a dominant cause of death worldwide. Acute therapies with potent antithrombotic agents have improved in-hospital outcomes; however, despite the use of these therapies, recurrent events continue to occur long term. Statins, renin-angiotensin inhibitors, beta-blockers, and dual antiplatelet therapy all have roles in mitigating long-term risk, but given recurrent thrombotic events on contemporary pharmacologic agents, novel therapies inhibiting thrombin formation have been explored. The intent of the current article is to review recent phase II/III clinical trials of oral anticoagulants used for the prevention of acute coronary syndromes.

Spirulan from blue-green algae inhibits fibrin and blood clots: its potent antithrombotic effects.

We investigated in vitro and in vivo fibrinolytic and antithrombotic activity of spirulan and analyzed its partial biochemical properties. Spirulan, a sulfated polysaccharide from the blue-green alga Arthrospira platensis, exhibits antithrombotic potency. Spirulan showed a strong fibrin zymogram lysis band corresponding to its molecular mass. It specifically cleaved Aα and Bβ, the major chains of fibrinogen. Spirulan directly decreased the activity of thrombin and factor X activated (FXa), procoagulant proteins. In vitro assays using human fibrin and mouse blood clots showed fibrinolytic and hemolytic activities of spirulan. Spirulan (2 mg/kg) showed antithrombotic effects in the ferric chloride (FeCl3 )-induced carotid arterial thrombus model and collagen and epinephrine-induced pulmonary thromboembolism mouse model. These results may be attributable to the prevention of thrombus formation and partial lysis of thrombus. Therefore, we suggest that spirulan may be a potential antithrombotic agent for thrombosis-related diseases.

Abstract 499: Development of a Novel Antithrombotic Therapy Targeting Platelet GPIbα: Assessment of Anfibatide In Vitro and in Phase I Clinical Trial

Platelet adhesion and aggregation are critical for hemostasis and atherothrombosis. Interaction between the GPIb complex and VWF initiates platelet adhesion and contributes to complete vessel occlusion, particularly at sites of stenosis. However, no therapeutic targeting this pathway has been developed. Previously, we demonstrated in murine thrombosis models that Anfibatide, a snake venom-derived GPIbα antagonist, inhibited botrocetin-induced VWF-GPIbα binding and inhibited thrombus formation in vitro and in vivo, without significantly activating platelets or changing bleeding. We subsequently hypothesized that Anfibatide would be a safe and potent anti-thrombotic agent in humans. We tested Anfibatide with human platelets in vitro and conducted the first anti-GPIbα phase I clinical trial with 94 healthy volunteers. In vitro, Anfibatide inhibited ristocetin-induced and low dose thrombin-induced human platelet aggregation. Surface plasmon resonance studies showed that Anfibatide binds to GPIbα and blocks binding of both VWF A1 domain and thrombin. In ex vivo perfusion chambers, Anfibatide strongly inhibited human platelet adhesion, aggregation, and thrombus formation on a collagen-coated surface, especially at high shear flow conditions. Importantly, Anfibatide effectively dissolved the preformed thrombi in perfusion chambers. Anfibatide incubation with human whole blood did not significantly change coagulation parameters measured by thromboelastography. After treating volunteers with Anfibatide, no serious adverse events, premature discontinuations due to adverse events, or deaths occurred during the study. Anfibatide bound to 95% of GPIb and inhibited 90% of platelet aggregation without prolonging activated partial thromboplastin time, prothrombin time, thrombin time, or bleeding time. There was no bleeding tendency, no alteration of platelet count, and no detectable anti-Anfibatide antibodies. In conclusion, Anfibatide provides anti-thrombotic effects through inhibition of both GPIbα-VWF and GPIbα-thrombin interactions. These results demonstrate that Anfibatide is a safe and potent anti-platelet reagent with great potential for future anti-thrombotic therapy.

Antithrombotic activity of new 1‐ethylxanthine cyclohexylammonium salt (1054.5)

The previous findings in vitro show high antiaggregation activity of newly synthesized cyclohexilammonium salt of 2‐[1‐ethil‐3‐methyl‐7‐(dioxotiethanyl‐3)xantinyl‐8‐thio]acetic acid. The current findings are received in vivo on the model of collagen‐adrenaline thrombosis according to G.S. Di Minno subject to the FASEB Statement of Principles for the use of animals in research and education. Efficacy endpoint is the number of survived animals in comparison with the test group and comparator agents since the injection into the tail vein suspended matter of collagen and adrenalin upto the moment of death or during 14 days of monitoring. The survival analysis was carried out with the help of Kaplan‐Meier method. The difference in survival between the groups was assessed with the help of Wilkonson criterion.Intraperinoneal injection of the new secondary 1‐ethylxantin reduced mortality in lab mice by 1,3 times (P1<0,01) in comparison with the camparator agents. Thus, cyclohexilammonium salt of 2‐[1‐ethil‐3‐methyl‐7‐(dioxotiethanyl‐3)xantinyl‐8‐thio]acetic acid is a potential antithrombotic agent.

De Novo Design of Self-Assembling Foldamers That Inhibit Heparin–Protein Interactions

A series of self-associating foldamers have been designed as heparin reversal agents, as antidotes to prevent bleeding due to this potent antithrombotic agent. The foldamers have a repeating sequence of Lys-Sal, in which Sal is 5-amino-2-methoxy-benzoic acid. These foldamers are designed to self-associate along one face of an extended chain in a β-sheet-like interaction. The methoxy groups were included to form intramolecular hydrogen bonds that preclude the formation of very large amyloid-like aggregates, while the positively charged Lys side chains were introduced to interact electrostatically with the highly anionic heparin polymer. The prototype compound (Lys-Sal)4 carboxamide weakly associates in aqueous solution at physiological salt concentration in a monomer-dimer-hexamer equilibrium. The association is greatly enhanced at either high ionic strength or in the presence of a heparin derivative, which is bound tightly. Variants of this foldamer are active in an antithrombin III–factor Xa assay, showing their potential as heparin reversal agents.

Construction and characterization of a novel chimeric antibody c3C7 specific for the integrin αIIbβ3 complex

A murine monoclonal antibody (mAb) 3C7 against integrin αIIbβ3 was previously obtained as a potential antithrombotic agent in our laboratory. The epitope of 3C7 is a specific conformation of the αIIbβ3 complex, but not either of the two subunits, which makes it different from abciximab, a supplementary antibody drug used in percutaneous coronary intervention which has a cross-reaction with other integrins sharing the β3 subunit. To reduce the human anti-mouse antibody reactions of 3C7, the variable regions of this antibody were cloned and fused with the constant counterparts of human IgG1. Two vectors of light and heavy chains were constructed and co-transfected into CHO-dhfr(-) cells. The chimeric antibody c3C7 was purified and the properties of c3C7 were compared with 3C7. Identical to its parent antibody 3C7, c3C7 binds to the αIIbβ3 complex, but not to either of the subunits. The K(d) value of c3C7 was in the same order of magnitude as 3C7 (1.570 ± 0.326 vs 0.780 ± 0.182 nmol/L). Human platelet aggregation induced by adenosine diphosphate was effectively inhibited by c3C7 in a dose-dependent manner. In conclusion, after the modification, c3C7 retained the properties of its parent mAb with no loss of its biological activity. Therefore, c3C7 has the potential to become a novel agent for the treatment of thrombosis.

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P.3.c.008 Concentration-dependent contribution of human cytochrome P450 isoenzymes to the metabolism of levomepromazine

A significant number of new chemical entities (NCEs) fail in drug discovery due to inhibition of Cytochrome P450 (CYP) enzymes. Therefore, to avert costly drug failure at the clinical phase it becomes indispensable to evaluate the CYP inhibition profile of NCEs early in drug discovery. In light of these concerns, we envisioned to investigate the inhibitory effects of S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-carboxylic acid amide], a novel and potent antithrombotic agent, on nine major CYP enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4) of human liver microsomes (HLM). S002-333 exists as racemic mixture of S004-1032 (R-isomer) and S007-1558 (S-isomer), consequently, we further examined the enantioselective differences of S002-333 in the inhibition of human CYP enzymes. Of the CYP enzymes tested, CYP2B6-catalyzed bupropion 6-hydroxylation was inhibited by S002-333 (IC50 ∼ 9.25 ± 2.46 μM) in a stereoselective manner with (S)-isomer showing potent inhibition (IC50 ∼ 5.28 ± 1.25 μM) in contrast to (R)-isomer which showed negligible inhibition on CYP2B6 activity (IC50 > 50 μM). S002-333 and its (S)-isomer inhibited CYP2B6 activity in a non-competitive fashion with estimated Ki values of 10.1 ± 3.4 μM and 5.09 ± 1.05 μM, respectively. No shift in the IC50 value was observed for S002-333 and its isomers when preincubated for 30 min in the presence of NADPH suggesting that neither S002-333 nor its enantiomers are time-dependent inhibitors. Thus, the present findings signified that S002-333 is a potent stereoselective inhibitor of CYP2B6, whereas, inhibition for other CYPs was substantially negligible. These in vitro findings would be useful in deciding the development of S002-333 as a single-enantiomer or as a racemic mixture.

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P-Terphenyl curtisian E inhibits in vitro platelet aggregation via cAMP elevation and VASP phosphorylation

Mushrooms possess untapped source of enormous natural compounds showing anti-inflammatory, antioxidant and anti-platelet activities. Paxillus curtisii, wild mushroom, is a rich source of curtisian E (CE) reported for neuroprotective effects; however, its anti-platelet effect was unknown. Here, therefore, we investigated the anti-platelet activity of CE in rat platelets. Curtisian E (12.5–200μM) attenuated collagen (2.5μg/ml), thrombin (0.1U/ml) and ADP (10μM) induced platelet aggregation in vitro. Likewise, CE diminished intracellular calcium and adenosine triphosphate (ATP) release in collagen activated platelets. Fibrinogen binding and fibronectin adhesion to platelets were also inhibited. While CE downregulated c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38, and Akt dose dependently in collagen stimulated platelets, it upregulated intraplatelet cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated-phosphoprotein (VASP) phosphorylation. Protein kinase A inhibitor (H-89) markedly inhibited p-VASP157 protein expression, suggesting that cAMP-PKA-VASP157 pathway may mediate its anti-platelet effect and thus CE could be considered as a potential anti-thrombotic agent.

Abstract 19436: cAMP Regulation of Vascular Endothelial Ectoenzyme ENTPD1

Ectonucleoside triphosphate diphosphohydrolase 1 (or CD39), an enzyme expressed on the plasmalemma of leukocytes and endothelial cells, serves as a potent antithrombotic agent by sequentially phosphohydrolyzing ATP and ADP, to generate AMP. CD39 is thus able to locally deplete levels of extracellular ATP and ADP, which would otherwise promote inflammation and platelet aggregation by binding P2X and P2Y classes of purinergic receptors found on leukocytes and platelets. The only known pathway for the transcriptional regulation of CD39 was described in recent work by this lab, which demonstrated that increased cAMP levels led to CD39 upregulation in murine macrophages through binding to the cAMP response elements (CRE) in the CD39 promoter with a consequent increase in CD39 transcription. Here, in contrast, we show that human endothelial cells (from umbilical vein; HUVEC) express less CD39 in response to elevated cAMP levels. Furthermore, we demonstrate that blockade of a major cAMP effector protein, protein kinase A (PKA), restores CD39 expression. Treatment of HUVEC cells with 250 μM 8-Br-cAMP, a cell permeable analogue of cAMP, led to a 2-fold decrease in CD39 protein from whole cell lysates in Western blots (18 hrs, p&lt;0.001, n=5), and this pattern persisted in time course assays from 8 hours to 24 hours. This result was confirmed with immunoprecipitation for CD39 in HUVEC supernatants, where 8 hours of 8-Br-cAMP treatment at 250 μM led to a 2.2-fold decrease in CD39 protein. ELISA measurements of HUVEC treated with 20 μM of the PKA inhibitor H89 led to a 2.8-fold increase compared to control of CD39 protein from HUVEC supernatants (p=0.0021, n=6). This was confirmed by Western blot, where co-treatment with 250 μM 8-Br-cAMP and 20 μM H89 for 8 hours led to a 50% increase measured by Western blot in CD39 protein compared to control in whole cell lysates (p&lt;0.05, n=3). Together, these data demonstrate that endothelial expression of CD39, a potent antithrombotic and anti-inflammatory enzyme, is modulated by the cAMP/PKA pathway, and contributes to basic understanding of the regulatory pathways governing CD39.

Novel 2-aminobenzamides as potential orally active antithrombotic agents.

In an effort to develop potent antithrombotic agents, a series of novel 2-aminobenzamide derivatives were synthesized and screened for their in vivo antithrombotic activity. Among the 23 compounds tested, compound (8g) showed the most promising antithrombotic activity, which was comparable with clinically used aspirin or warfarin, but at variance with these standard drugs, 8g did not exhibit the increased bleeding time, suggesting its potential as a novel antithrombotic agent.

Managing Risk of Complications at Femoral Vascular Access Sites in Percutaneous Coronary Intervention

Percutaneous coronary intervention for acute coronary syndrome or non-ST-elevation myocardial infarction requires the use of potent oral and intravenous anti-platelet and antithrombin medications. Although these potent antithrombotic agents and regimens may increase the effectiveness of percutaneous coronary intervention, they are also generally associated with an increased risk of vascular access complications such as hematoma, retroperitoneal hematoma, pseudoaneurysm, arterial occlusion, and arteriovenous fistula, which in turn are associated with increased morbidity, mortality, and costs. Risk factors predisposing patients to these complications are both modifiable (procedure technique, medications, hemostasis method) and nonmodifiable (sex, age, body mass index, blood pressure, renal function). Patients' risks can be reduced by nurses who are knowledgeable about these risk factors and identify complications before they become problematic.

YC-1, a potent antithrombotic agent, induces lipolysis through the PKA pathway in rat visceral fat cells

This study investigated the effects of 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), a soluble guanylyl cyclase (sGC) activator and potential antithrombotic agent, on lipolysis in isolated visceral fat cells of the rat. Visceral fat cells were isolated from epididymal fat pads of rats and treated with YC-1 at different doses and times. Glycerol release, and intracellular cAMP and cGMP levels were analyzed by specific kits. Moreover, several inhibitors or drugs were used to examine the signal transduction pathways of YC-1-induced lipolysis in adipocytes. Herein we report that YC-1 stimulated glycerol release in dose- and time-dependent manners. Intracellular cAMP and cGMP levels of adipocytes both increased in time-dependent manners, but elevation of the cGMP level was faster and higher than that of the cAMP level after YC-1 treatment. An sGC inhibitor (ODQ) inhibited YC-1-induced glycerol release, indicating the involvement of sGC in YC-1-induced lipolysis. Administration of insulin, an activator of type-3B phosphodiesterase (PDE-3B), attenuated YC-1-induced lipolysis, indicating that elevation of the cAMP level is an important step in the lipolytic effect of YC-1. In addition, YC-1-induced lipolysis was inhibited by a protein kinase A (PKA) inhibitor (KT5720) but not by a PKG inhibitor (KT5823), indicating that YC-1-induced lipolysis occurs through a PKA-dependent pathway. A Western blot analysis showed that extracellular signal-regulated kinase was not phosphorylated by YC-1 treatment. In conclusion, our results suggest that YC-1 might stimulate lipolysis via activation of sGC/cGMP and then activation of the cAMP/PKA signaling cascade in isolated rat visceral adipocytes.

Inhibition of Platelet Glycoprotein β3 by Chimeric Monoclonal Antibody Prevents Arterial Thrombosis in Beagle Dogs,

Abstract 3357In this present study in beagle dogs, we evaluated the antithrombotic efficacy of Pulaimab, the chimeric monoclonal antibodies SZ21-F(ab)2 fragments against platelet glycoprotein (GP) β3, in a modified Folts model. The Folts model is widely accepted to be effective and clinically relevant for testing potential anti-thrombotic agents, in this model the cyclic flow reductions (CFRs) are caused by platelet dependent thrombi that form under high-shear conditions at injured stenosed sites of an artery. Thirty beagle dogs of either sex, weighing 7.5 to 14 kg, were randomly divided into five groups of six (three females and three males), the details were following: negative control group (injected normal saline), positive control group (injected 0.2mg/Kg of Reopro), and three experimental groups [injected 0.2, 0.4, 0.8 mg/Kg of SZ21-F(ab)2, respectively]. A dose range from 0.2 to 0.8 mg/kg of Pulaimab significantly reduced the CFRs by 21–73%, without reduction of platelet numbers and prolongation of the bleeding time. Ex vivo ADP-induced platelet aggregation was equally reduced. The present study demonstrates that the inhibition of platelet GP αIIbβ3function by SZ21-F(ab)2 is a powerful intervention to prevent platelet thrombus formation in injured arteries without thrombocytopenia and prolongation of the bleeding time. We therefore conclude that F(ab)2 fragments of inhibitory anti-GPβ3 antibodies may be useful compounds to prevent thrombosis. Disclosures:No relevant conflicts of interest to declare.

Left main coronary artery transradial rescue percutaneous coronary intervention for acute myocardial infarction complicated by cardiogenic shock with Impella ventricular mechanical support

Mechanical ventricular assist support and especially Impella device that is implanted via femoral access are considered a cornerstone in the therapeutic arsenal of the management of cardiogenic shock. Indeed, the potent antithrombotic agents administered during acute coronary syndromes constitute potential bleeding risk factors. Transradial interventions are nowadays widely used in ST-elevation myocardial infarction patients. However, some operators feel uncomfortable with the transradial approach when facing cardiogenic shock. We report a case of transradial rescue percutaneous intervention for cardiogenic shock in a young man with support of an Impella device via femoral access.