Development and assessment of immediate-release tablets containing clopidogrel bisulphate & aspirin-strategy for optimizing the combination formulation.

The main goal of the study was to improve the compliance and convenience of patients by designing and development of an immediate release (IR) fixed-dose combination (Clopidogrel bisulphate and Aspirin) tablets. The proposed combination product utilizes Clopidogrel to protect the moisture-sensitive aspirin component, enhancing its stability against atmospheric conditions. Response-surface approach (Design Expert vs. 13) was used to generate this IR tablet by calculating the right composition of independent variables such as Microcrystalline cellulose 102, pregelatinized starch and Hydroxypropyl cellulose. 32 factorial design was used to estimate the effects of these independent variables on the responses of dependent variables (disintegration & friability) and constructed a total of nine (9) formulations. Pre and Post formulation, quality control parameters were investigated as per pharmacopeia. A systematic approach was used for the optimization process and a prototype checkpoint batch (CPB) based on the better contrast of independent variables was prepared. In vitro analysis of formulations was carried out to estimate the responses. Friability was found in the range of 0.088-1.076%w/w, except F1 = 1.076 all are within limits (NMT 1.0%). Disintegration time was recorded 7.3 ± 1.20 as lower and 24.5 ± 1.63 min was the highest. The release of drugs from their dosage form was fast and rapid, for clopidogrel after 15min was 70.42-96.82% with SD ± 8.71 and aspirin was 69.88-91.49% in 15 min with SD ± 6.41, all the tablets were released more than 80% in 20 min. The stability outcomes of CPB tablets after 15 days of stress study (60 ± 2°C and 75 ± 5%) indicated good compatibility and stability of APIs with excipients. It was concluded that the direct compression method can be preferred to prepare a combination product with cost-effectiveness. It was also concluded that the proposed methodology could increase Aspirin's stability and allow for an aqueous coating system to finish the product with a film coating. By using Design Expert software, the best composition of the formulation can be selected and optimized in a short period of time with minimum trial and errors. The results also demonstrated that the use of a fixed-dose combination tablet instead of the individual is expected to be more convenient to patients and thus improves patient compliance and decreases the occurrence of adverse effects and side effects.

Linking triphenylphosphonium cation to a bicyclic hydroquinone improves their antiplatelet effect via the regulation of mitochondrial function.

Platelets are the critical target for preventing and treating pathological thrombus formation. However, despite current antiplatelet therapy, cardiovascular mortality remains high, and cardiovascular events continue in prescribed patients. In this study, first results were obtained with ortho-carbonyl hydroquinones as antiplatelet agents; we found that linking triphenylphosphonium cation to a bicyclic ortho-carbonyl hydroquinone moiety by a short alkyl chain significantly improved their antiplatelet effect by affecting the mitochondrial functioning. The mechanism of action involves uncoupling OXPHOS, which leads to an increase in mitochondrial ROS production and a decrease in the mitochondrial membrane potential and OCR. This alteration disrupts the energy production by mitochondrial function necessary for the platelet activation process. These effects are responsive to the complete structure of the compounds and not to isolated parts of the compounds tested. The results obtained in this research can be used as the basis for developing new antiplatelet agents that target mitochondria.

Discovery of Novel Hybrids of Edaravone and 6-Phenyl-4,5-dihydropyridazin-3(2H)-one with Antiplatelet Aggregation and Neuroprotection for Ischemic Stroke Treatment.

Drugs with anti-platelet aggregation and neuroprotection are of great significance for the treatment of ischemic stroke. A series of edaravone and 6-phenyl-4,5-dihydropyridazin-3(2H)-one hybrids were designed and synthesized. Among them, 6g showed the most effective cytoprotective effect against oxygen-glucose deprivation/reoxygenation-induced damage in BV2 cells and an excellent inhibitory effect on platelet aggregation induced by adenosine diphosphate and arachidonic acid. Additionally, 6g could prevent thrombosis caused by ferric chloride in rats and pose a lower risk of causing bleeding compared with aspirin. It provides better protection against ischemia/reperfusion injury in rats compared with edaravone and alleviates the oxidative stress related to cerebral ischemia/reperfusion by increasing the GSH and SOD levels and decreasing the MDA concentration. Finally, molecular docking results showed that 6g probably acts on PDE3 A and plays an anti-platelet aggregation effect. Overall, 6g could be a potential candidate compound for the treatment of ischemic stroke.

Antiplatelet and Anticoagulant Effects of Two New Phenylpropanoid Sucrose Esters and Other Secondary Metabolites from the Aerial Part of Canna edulis.

This study isolated pure compounds from Canna edulis aerial parts and assessed their antiplatelet and anticoagulant potential. Structural elucidation resulted in the identification of two new compounds: caneduloside A (1) and caneduloside B (2), and eleven known compounds: 6'-acetyl-3,6,2'-tri-p-coumaroyl sucrose (3), 6'-acetyl-3,6,2'-triferuloyl sucrose (4), tiliroside (5), afzelin (6), quercitrin (7), 2-hydroxycinnamaldehyde (8), cinnamic acid (9), 3,4-dimethoxycinnamic acid (10), dehydrovomifoliol (11), 4-hydroxy-3,5-dimethoxybenzaldehyde (12), and (S)-(-)-rosmarinic acid (13). Compounds 3, 4, 6-9, 13 were previously reported for antithrombotic properties. Hence, antithrombotic tests were conducted for 1, 2, 5, 10-12. All tested compounds demonstrated a dose-dependent antiaggregatory effect, and 10 and 12 were the most potent for both ADP and collagen activators. Additionally, 10 and 12 showed anticoagulant effects, with prolonged prothrombin time and activated partial thromboplastin time. The new compound 1 displayed antiplatelet and anticoagulant activity, while 2 mildly inhibited platelet aggregation. C. edulis is a potential source for developing antithrombotic agents.

Ginkgolides with anti-PAF activity from Ginkgo biloba L.

Four undescribed ginkgolides, including two rare sesquiterpene ginkgolides (compounds 1 and 2) and two diterpenoid ginkgolides (compounds 3 and 4), were isolated from Ginkgo biloba L. The structures of these four ginkgolides were identified based on extensive spectroscopic analysis, DP4+ probability analysis and X-ray diffraction. Compounds 1 and 2 exhibited excellent antiplatelet aggregation activities with IC50 values of 1.20 ± 0.25 and 4.11 ± 0.34 µM, respectively.

Design, Synthesis, In vitro and In vivo Evaluation of New Imidazo[1,2-a]pyridine Derivatives as Cyclooxygenase-2 Inhibitors.

BACKGROUND: Cyclooxygenase-2 (COX-2), the key enzyme in the arachidonic acid conversion to prostaglandins, is one of the enzymes associated with different pathophysiological conditions, such as inflammation, cancers, Alzheimer's, and Parkinson's disease. Therefore, COX-2 inhibitors have emerged as potential therapeutic agents in these diseases. OBJECTIVE: The objective of this study was to design and synthesize novel imidazo[1,2-a]pyridine derivatives utilizing rational design methods with the specific aim of developing new potent COX-2 inhibitors. Additionally, we sought to investigate the biological activities of these compounds, focusing on their COX-2 inhibitory effects, analgesic activity, and antiplatelet potential. We aimed to contribute to the development of selective COX-2 inhibitors with enhanced therapeutic benefits. METHODS: Docking investigations were carried out using AutoDock Vina software to analyze the interaction of designed compounds. A total of 15 synthesized derivatives were obtained through a series of five reaction steps. The COX-2 inhibitory activities were assessed using the fluorescent Cayman kit, while analgesic effects were determined through writing tests, and Born's method was employed to evaluate antiplatelet activities. RESULTS: The findings indicated that the majority of the tested compounds exhibited significant and specific inhibitory effects on COX-2, with a selectivity index ranging from 51.3 to 897.1 and IC50 values of 0.13 to 0.05 µM. Among the studied compounds, derivatives 5e, 5f, and 5j demonstrated the highest potency with IC50 value of 0.05 µM, while compound 5i exhibited the highest selectivity with a selectivity index of 897.19. In vivo analgesic activity of the most potent COX-2 inhibitors revealed that 3-(4-chlorophenoxy)-2-[4-(methylsulfonyl) phenyl] imidazo[1,2-a]pyridine (5j) possessed the most notable analgesic activity with ED50 value of 12.38 mg/kg. Moreover, evaluating the antiplatelet activity showed compound 5a as the most potent for inhibiting arachidonic acidinduced platelet aggregation. In molecular modeling studies, methylsulfonyl pharmacophore was found to be inserted in the secondary pocket of the COX-2 active site, where it formed hydrogen bonds with Arg-513 and His-90. CONCLUSION: The majority of the compounds examined demonstrated selectivity and potency as inhibitors of COX-2. Furthermore, the analgesic effects observed of potent compounds can be attributed to the inhibition of the cyclooxygenase enzyme.

Potential antithrombotic effect of two new phenylpropanoid sucrose esters and other secondary metabolites of Canna indica L. rhizome.

Canna indica L. has been traditionally used to treat various diseases. Based on previously reported antithrombotic effect for this plant, two new phenylpropanoid sucrose esters (canindicoside A (1) and canindicoside B (2)) and seven known compounds: nepetoidin B (3), caffeic acid (4), ferulic acid (5), (R)-(+)-rosmarinic acid (6), isorinic acid (7), (S)-(-)-rosmarinic acid (8) and (S)-(-)-rosmarinic acid methyl ester (9) were isolated from the ethyl acetate extract. Compounds were elucidated by NMR and MS spectroscopic methods. The antiplatelet effect was evaluated using turbidimetric method. Anticoagulant activity was examined by measuring activated partial thromboplastine time (APTT), prothrombin time, and thrombine time (TT). It was shown for the first time that both new phenylpropanoid sucrose esters 1 and 2, 7 and 9 displayed dose-dependent antiplatelet effects. 2 and 9 had the highest inhibitory activity on both adenosine diphosphate (ADP)- and collagen-induced platelet aggregation. Moreover, 1, 7 and 9 also exhibited anticoagulant activity. At 0.4 mg/mL, both 1 and 7 prolonged APTT compared to the negative control (p < 0.05), suggesting the possible inhibitory impact on the intrinsic coagulation pathway. Moreover, 9 at 0.4 mg/mL exerted higher TT values than the negative control (p < 0.05). C. indica and its bioactive phytochemicals are potential candidates for development of anti-thrombosis therapy.

Anti-thrombotic Effects Mediated by a Novel Dual-Target Peptide Inhibiting Both Platelet Aggregation and Thrombin Activity without Causing Bleeding.

BACKGROUND: Classical anticoagulants and antiplatelets are associated with high frequencies of bleeding complications or treatment failure when used as single agents. Thrombin plays an important role in the blood coagulation system. GP IIb/IIIa is the central receptor of platelets, which can recognize the Arg-Gly-Asp (RGD) sequence and activate platelets. MATERIAL AND METHODS: Molecular simulation and homology modeling were performed to design a novel dual-target anticoagulant short peptide (PTIP ). The activities of PTIP on coagulation and platelet in vitro were analyzed. The antithrombotic activity of PTIP was determined by pulmonary thromboembolism model, ferric chloride injury model and arteriovenous bypass thrombosis model. Bleeding effect and toxicity of PTIP were evaluated. RESULTS: We have constructed a novel dual-target peptide (PTIP) based on the direct thrombin inhibitor peptide (DTIP). PTIP was expressed at high levels in Pichia pastoris. PTIP interfered with thrombin-mediated coagulation and ADP-induced platelet aggregation in vitro. When injected intravenously or subcutaneously, PTIP showed potent and dose-dependent extension of aPTT and PT which were similar to DTIP; but only PTIP was capable of inhibiting platelet aggregation. PTIP (1.0 mg/kg) decelerated thrombosis formation in venous and arterial vessels induced by FeCl3 injury. PTIP (1.0 mg/kg) also prevented deep venous thrombosis and increased the survival rate associated with pulmonary thromboembolism. And PTIP effectively reduced thrombus length in arteriovenous bypass thrombosis model. Moreover, the antithrombotic dose of PTIP could not induce bleeding. CONCLUSION: These data establish that PTIP represents a novel antithrombotic agent whose effects involve both inhibition of platelet activation and reduction of fibrin generation. And PTIP not only can be used in venous thrombosis and arterial thrombosis, it can also replace the combined treatment of antiplatelet and anticoagulant drugs in thrombotic diseases.

Synthesis and Assessment of Antiplatelet and Antithrombotic Activity of 4-Amino-Substituted 5-Oxoproline Amides and Peptides.

Venous thromboembolism is a serious problem because it significantly increases the risk of developing vascular complications in elderly patients with obesity or immobilization, cancer, and many other diseases. Thus, there is a need to study new therapeutic strategies, including new medicinal agents for the efficient and safe correction of thrombus disorders. In this work, we have synthesized a number of new amides and peptides of 4-amino-5-oxoprolines and studied their antiplatelet and antithrombotic activity in experiments in vitro and in vivo. It has been found that the newly obtained compounds slow down the process of thrombus formation in a model of arterial and venous thrombosis, without affecting plasma hemostasis parameters. (2S,4S)-4-Amino-1-(4-fluorophenyl)-5-oxoprolyl-(S)-phenylalanine proved to be the most efficient among the studied derivatives. The results obtained indicate the advisability of further studies on 5-oxoproline derivatives in order to design pharmaceutical agents for the prevention and treatment of the consequences of thrombosis.

11,12-seco-Abietane-type diterpene lactones with potential antiplatelet activity from Salvia prattii.

Eleven new abietane-type diterpene lactones, salpratlactones D-N (1-11), including five 11,12-seco-11-nor-abietane diterpenes (1-5), four 11,12-seco-abietane diterpenes (6-9), two 20(10 â†’ 5)-abeo-4,5;11,12-bis-seco-abietane diterpenes (10-11), and two known analogues (12-13), were characterized from Salvia prattii. Notably, compounds 1-3 were characterized by a unique linear 6/6/6 tricyclic skeleton. The structures were established by spectroscopic data interpretation, calculated NMR-DP4+ and electronic circular dichroism analysis, as well as single-crystal X-ray diffraction. A bioactivity study showed that 1, 2, 5, 11, and 12 can potently inhibit platelet aggregation induced by arachidonic acid (AA), with IC50 values of 5.66-16.10 µg/ml, stronger than aspirin. In addition, the lactate dehydrogenase assay showed that they had no effect on platelet integrity. Structurally, the same 1,2-benzopyrone fragments of 1, 2, and 5 should be the important pharmacophore for antiplatelet activity.

rJararacin, a recombinant disintegrin from Bothrops jararaca venom: Exploring its effects on hemostasis and thrombosis.

Integrins are a family of heterodimeric transmembrane receptors which link the extracellular matrix to the cell cytoskeleton. These receptors play a role in many cellular processes: adhesion, proliferation, migration, apoptosis, and platelet aggregation, thus modulating a wide range of scenarios in health and disease. Therefore, integrins have been the target of new antithrombotic drugs. Disintegrins from snake venoms are recognized by the ability to modulate the activity of integrins, such as integrin αIIbß3, a fundamental platelet glycoprotein, and αvß3 expressed on tumor cells. For this reason, disintegrins are unique and potential tools for examining integrin-matrix interaction and the development of novel antithrombotic agents. The present study aims to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis. rJararacin was expressed in the Pichia pastoris (P. pastoris) expression system and purified the recombinant protein with a yield of 40 mg/L of culture. The molecular mass (7722 Da) and internal sequence were confirmed by mass spectrometry. Structure and folding analysis were obtained by Circular Dichroism and 1H Nuclear Magnetic Resonance spectra. Disintegrin structure reveals properly folded with the presence of ß-sheet structure. rJararacin significantly demonstrated inhibition of the adhesion of B16F10 cells and platelets to the fibronectin matrix under static conditions. rJararacin inhibited platelet aggregation induced by ADP (IC50 95 nM), collagen (IC50 57 nM), and thrombin (IC50 22 nM) in a dose-dependent manner. This disintegrin also inhibited 81% and 94% of the adhesion of platelets to fibrinogen and collagen under continuous flow, respectively. In addition, rjararacin efficaciously prevents platelet aggregation in vitro and ex vivo with rat platelets and thrombus occlusion at an effective dose (5 mg/kg). The data here provides evidence that rjararacin possesses the potential as an αIIbß3 antagonist, capable of preventing arterial thrombosis.

How snake venom disintegrins affect platelet aggregation and cancer proliferation.

Disintegrins are small peptides possessing a tripeptide motif capable of binding to integrins. These were first isolated from viper venoms and are now also found in many other hematophagous organisms. Many integrins have been studied for their role in the onset of disease and the interaction of disintegrins with these receptors makes them potential therapeutic molecules. Disintegrins are also used as molecular scaffolds to design effective drugs for cardiovascular diseases and cancer. Even the gene and protein sequencing data of disintegrins have provided insights into understanding the molecular complexity of disintegrins. In this review, we try to summarize the structural and functional importance of disintegrins in identifying the biological targets and triggering various signaling pathways involved in platelet aggregation and cancer. Also, we have tried to elucidate a possible molecular mechanism behind the action of disintegrins on platelet aggregation and cancer. This understanding will help us to design and to explore more of these integrin-binding molecules.

Synthesis of Novel 3-Butylphthalide Derivatives Containing Isopentenylphenol Moiety as Potential Antiplatelet Agents for the Treatment of Ischemic Stroke.

In order to find novel antiplatelet drugs for the treatment of ischemic stroke, a series of 3-butylphthalide derivatives containing isopentenylphenol moiety were designed, synthesized and characterized with spectroscopic analyses. The in vitro antiplatelet activity results indicated that compound 3 better inhibited the arachidonic acid (AA) induced platelet aggregation than aspirin (ASP) and 3-butylphthalide (NBP). Additionally, compared with precursor NBP, compound 3 possessed outstanding antithrombotic activity in the animal experiment model, which could effectively alleviate the formation of tail thrombus and carotid artery thrombus in mice. More importantly, intraperitoneal administration of compound 3 can well protected the rats against ischemia/reperfusion-induced brain injury. Further pharmacokinetic (PK) assay indicated that compound 3 had good absorption characteristics and metabolic stability in vivo. Overall, the present research provides a new candidate compound for the treatment of ischemic stroke caused by platelet aggregation.

(1R,3S)-THCCA-Asn: To show the discovery of selective inhibitor of thrombin by successfully combining virtual screening and biological assay.

Thrombin is the most potent platelet aggregator. To discover the selective inhibitor of thrombin that is important to curing platelet aggregation-related diseases, docking experiments were performed to dock (1R,3S)-2,3,4,9-tetrahydro-ß-carboline-3- carboxylic acid, [(1R,3S)-THCCA], and (1S,3S)-2,3,4,9-tetrahydro-ß-carboline-3- carboxylic acid, [(1S,3S)-THCCA], into the p pocket of bovine thrombin. The ideal match supported that (1R,3S)-THCCA could be used as a potential lead compound. In this case 20 natural amino acids were theoretically introduced into the 3-carboxyl of (1R,3S)-THCCA and 20 derivatives, (1R,3S)-THCCA-amino acids, were docked into p pocket of bovine thrombin to perform virtual screening. The screening revealed that comparing to (1R,3S)-THCCA itself the DockScores of 16 derivatives were higher, and (1R,3S)-THCCA-Asn (4j) got the highest DockScore. Thus, 16 derivatives were synthesized for experimental study. The in vitro anti-platelet aggregation assay showed that at 100 µM of concentration the 16 derivatives failed to inhibit the platelet aggregation induced by both adenosine diphosphate and arachidonic acid. On the other hand, however, the IC50 value of the 16 derivatives inhibiting the platelet aggregation induced by platelet activating factor and thrombin ranged from 9.44 µM to 194.64 µM and from 0.07 µM to 9.56 µM, respectively. The in vitro anti-platelet aggregation assay suggested that the 16 derivatives selectively inhibited the platelet aggregation induced by thrombin. In particular, the IC50 of (1R,3S)-THCCA-Asn (4j) had the lowest value. On rat model at 1 nmol/kg of dosage the 16 derivatives effectively prevented thrombus formation. It is worth pointing out that even at 0.01 nmol/kg of dosage, 4j still effectively prevented thrombus formation. 4j hardly has effects on the proliferation of mammalian cells and rat tail bleeding time. In conclusion, the combination of virtual screening and biological assays successfully lead to the discovery of 4j as a promising candidate of selective inhibitor of thrombin.

The recombinant disintegrin, jarastatin, inhibits platelet adhesion and endothelial cell migration.

Integrins are transmembrane heterodimeric glycoproteins, present in most cell types that act as mechanoreceptors, connecting extracellular matrix proteins to the cytoskeleton of the cell, mediating several physiological and pathological processes. The disintegrins are peptides capable of modulating the activity of integrins, such as αIIbß3, responsible for the platelet aggregation and αvß3, related to angiogenesis. The aim of this study was to produce the recombinant disintegrin jarastatin (rJast), to evaluate its secondary structure and biological activity. rJast was expressed in the yeast Komagataella phaffii (earlier Pichia pastoris) purified using molecular exclusion chromatography and the internal sequence and molecular mass were confirmed by mass spectrometry. The yield was approximately 40 mg/L of culture. rJast inhibited platelet aggregation induced by 2-4 µM ADP, 10 nM thrombin, and 1 µg/mL collagen (IC50 of 244.8 nM, 166.3 nM and 223.5 nM, respectively). It also blocked the adhesion of platelets to collagen under continuous flow in approximately 60% when used 1 µM. We also evaluated the effect of rJast on HMEC-1 cells. rJast significantly inhibited the adhesion of these cells to vitronectin, as well as cell migration (IC50 1.77 µM) without changing the viability. Conclusions: rJast was successfully expressed with activity in human platelets aggregation identical to the native molecule. Also, rJast inhibits adhesion and migration of endothelial cells. Thus, being relevant for the development of anti-thrombotic and anti-angiogenic drugs.

Effective components and mechanism analysis of anti-platelet aggregation effect of Justicia procumbens L.

ETHNOPHARMACOLOGICAL RELEVANCE: Justicia procumbens L. is a traditional Chinese medicine, first recorded in "Shen Nong's Herbal Classic", for the treatment of lumbar pain and fever. As a widely distributed herb, it has also been documented in India, Nepal, and Malaysia. In "Tang Materia Medica", a famous medicinal book of Tang Dynasty in ancient China, it was first used to treat diseases associated with blood stasis. Blood stasis syndrome is closely related to thrombus formation and platelet aggregation. Although some compounds isolated from this plant have anti-platelet aggregation effects, the main chemical components and mechanism of J. procumbens in terms of these effects are little known. AIMS OF THE STUDY: Through in vivo and in vitro experiments, this studsy revealed the characteristic components and action mechanism of anti-platelet aggregation by J. procumbens from an overall perspective. MATERIALS AND METHODS: The effective crude extracts of the whole plant were screened via an in vitro anti-platelet aggregation test. After incubating these extracts with apheresis platelets, high affinity compounds were detected by HPLC-MS and regulatory genes were detected using gene chips. The effective components and potential target proteins were analyzed using computational docking technology. Furthermore, the compound with the strongest predicted activity was evaluated in vivo via an anti-thrombotic test. RESULTS: Integrin aⅡbß3, PKCα, PI3Kγ, and mitogen-activated protein kinase 14 were found to be potential targets. Justicidin B, tuberculatin, chinensinaphthol methyl ether, and neojusticin B were effective compounds that inhibited human platelet aggregation by suppressing Gq-PLC-PKC and Gi-PI3K-MAPK signaling pathways. Among the compounds that bind to platelets, justicidin B showed the strongest virtual binding force. The test of carotid artery thrombosis induced by ferric chloride in SD rats confirmed that justicidin B inhibited thrombus formation. CONCLUSION: Experimental investigation showed that arylnaphthalene lignan aglycones with one methylenedioxy group and two methoxy groups are effective components for anti-platelet aggregation by J. procumbens. These compounds inhibit Gq-PLC-PKC and Gi-PI3K-MAPK signaling pathways by suppressing the expression of genes such as ITGB3, PRKCA, PIK3CG, and MAPK14. These results reflected the characteristics of multi-component and multi-target synergistic treatment of Chinese medicine.

Diindolylmethane ameliorates platelet aggregation and thrombosis: In silico, in vitro, and in vivo studies.

Diindolylmethane (DIM), a major metabolite of indole-3-carbinol (I3C), plays a vital role in the pharmacological actions of I3C. The role of DIM in the inhibition of platelet aggregation and thrombus generation is yet to be revealed. However, how DIM and I3C modulate the interaction of platelets with the glycoproteinVI (GPVI) and purinergic receptor Y12 (P2Y12) receptors is unknown. In silico studies revealed that the indole group of DIM and indole and the hydroxyl group of I3C are responsible for modulating platelet interaction with GPVI and P2Y12 receptors. In silico studies further predicted that DIM more superiorly modulates platelet interaction with GPVI and P2Y12 receptors than I3C. In vitro studies identified that DIM significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), collagen, thrombin, and arachidonic acid, increasing the thrombin-induced clot retraction size and clot retraction weight. Moreover, in vivo results of ferric chloride (FeCl3) induced carotid artery thrombus generation indicate that DIM significantly reduced the reactive oxygen species (ROS), hydrogen peroxide (H2O2), thromboxane 2 (TXB2), cyclooxygenase 1 (COX-1), prostaglandin E2 (PGE2), thrombus weight, increased the cyclic adenosine monophosphate (cAMP), and extended the time to occlusion (TTO). Furthermore, DIM did not show thrombolytic activity. Therefore, DIM acts as an antiplatelet aggregation and antithrombotic agent. Moreover, DIM is responsible for the antiplatelet aggregation and antithrombotic activity of I3C. Therefore, DIM could be used to treat thrombotic diseases.

Flavonoids: Antiplatelet Effect as Inhibitors of COX-1.

Flavonoids are compounds with a benzopyranic structure that exhibits multiple pharmacological activities. They are known for their venotonic activity, but their mechanism of action remains unclear. It is thought that, as this mechanism is mediated by prostaglandins, these compounds may interfere with the arachidonic acid (AA) cascade. These assays are designed to measure the antiplatelet aggregation capacity of quercetin, rutin, diosmetin, diosmin, and hidrosmin, as well as to evaluate a potential structure-activity ratio. In this paper, several studies on platelet aggregation at different concentrations (from 0.33 mM to 1.5 mM) of different flavone compounds are conducted, measuring platelet aggregation by impedance aggregometry, and the cyclooxygenase (COX) activity by metabolites generated, including the activity of the pure recombinant enzyme in the presence of these polyphenols. The results obtained showed that quercetin and diosmetin aglycones have a greater antiplatelet effect and inhibit the COX enzyme activity to a greater extent than their heterosides; however, the fact that greater inhibition of the pure recombinant enzyme was achieved by heterosides suggests that these compounds may have difficulty in crossing biological membranes. In any case, in view of the results obtained, it can be concluded that flavonoids could be useful as coadjuvants in the treatment of cardiovascular pathologies.

Inhibitors of protease-activated receptor 4 (PAR4): a review of recent patents (2013-2021).

INTRODUCTION: Protease-activated receptor 4 (PAR4), belonging to a subfamily of G-protein-coupled receptors (GPCR), is expressed on the surface of Human platelets, and the activation of it can lead to platelets aggregation. Studies demonstrated that PAR4 inhibition protect mice from arterial/arteriolar thrombosis, pulmonary embolism and cerebral infarct, while do not affect the hemostatic responses integrity. Therefore, PAR4 has been a promising target for the development of anti-thrombotic agents. AREAS COVERED: This review covers recent patents and literature on PAR4 and their application published between 2013 and 2021. EXPERT OPINION: PAR4 is a promising anti-thrombotic target and PAR4 inhibitors are important biologically active compounds for the treatment of thrombosis. Most the recent patents and literature focus on PAR4 selective inhibitors, and BMS-986120 and BMS-986141, which were developed by BMS, have entered clinical trials. With the deep understanding of the crystal structures and biological functions of PAR4, we believe that many other novel types of molecules targeting PAR4 would enter the clinical studies or the market.

Synthesis of 3,3-dimethyl-6-oxopyrano[3,4-c]pyridines and their antiplatelet and vasodilatory activity.

OBJECTIVES: Both pyridine and pyrano derivatives have been previously shown to possess biologically relevant activity. In this study, we report the incorporation of these two scaffolds into one molecule. METHODS: The designed 3,3-dimethyl-6-oxopyrano[3,4-c]pyridines were synthesized by the acylation of enamine under Stork conditions followed by condensation of formed ß-diketones with 2-cyanoacetamide. The structures of these compounds were confirmed by using a wide spectrum of physico-chemical methods. Their antiplatelet, anticoagulant and vasodilatory activity together with toxicity were evaluated. KEY FINDINGS: A series of 6-oxopyrano[3,4-c]pyridines 3a-j was obtained. Four of these compounds were reported for the first time. None of the tested compounds demonstrated anticoagulant effect but 8-methyl derivative (3a) was a potent antiplatelet compound with IC50 numerically twice as low as the clinically used acetylsalicylic acid. A series of further mechanistic tests showed that 3a interferes with calcium signaling. The compound is also not toxic and in addition possesses vasodilatory activity as well. CONCLUSIONS: Compound 3a is a promising inhibitor of platelet aggregation, whose mechanism of action should be studied in detail.