Cangrelor increases the magnitude of platelet inhibition and reduces interindividual variability in clopidogrel-pretreated subjects

BackgroundInadequate platelet inhibition despite aspirin and clopidogrel therapy during and after a percutaneous coronary intervention is associated with an impaired clinical outcome. Cangrelor, a direct and reversible P2Y₁₂ inhibitor that is currently in development, has the potential to achieve higher levels of inhibition of ADP-induced platelet aggregation than clopidogrel. The aim of the present study was to compare the magnitude of platelet inhibition in clopidogrel-pretreated patients before and after the in vitro addition of a subtherapeutic dose of cangrelor. MethodsBlood samples were drawn from patients pretreated with clopidogrel and aspirin who were undergoing elective percutaneous coronary intervention (n=39). Platelet function analysis with ‘classical’ light transmittance aggregometry (both peak and late aggregation [at 6 min]) was performed before and after the in vitro addition of cangrelor (0.25 μmol/l) to platelet-rich plasma (PRP). After an incubation period of five minutes, platelet aggregation was induced by 5 and 20 μmol/l ADP. ResultsThe in vitro addition of 0.25μmol/l cangrelor to the PRP from clopidogrel-treated subjects resulted in an additional reduction in ADP-induced platelet aggregation. For ADP concentrations of 5 and 20 μmol/l, peak aggregation showed a decrease of 75 and 85%, respectively (p<0.001 for both), while late aggregation was almost completely diminished (p=0.003 and p<0.001, respectively). Furthermore, the interindividual variation in inhibition of ADP-induced platelet aggregation by clopidogrel was greatly reduced after the addition of cangrelor. ConclusionWe demonstrate that the in vitro addition of even a subtherapeutic dose of cangrelor to the PRP of clopidogrel-pretreated patients results in an additional reduction of ADP-induced platelet aggregation. Moreover, cangrelor was able to diminish the interindividual variation observed in clopidogrel-inhibited platelet aggregation. (Neth Heart J 2009;17:195–8.)     

The effect of cilostazol on the expression of matrix metalloproteinase-1 and type I procollagen in ultraviolet-irradiated human dermal fibroblasts

AimCilostazol is a selective inhibitor of type III phosphodiesterase that inhibits platelet aggregation. Cilostazol is a useful vasodilator, antithrombotic, and cardiotonic agent. Ultraviolet B (UVB) irradiation increases the production of matrix metalloproteinase-1 (MMP-1) during skin photoaging. The UVB-induced increase of MMP-1 results in connective tissue damage, and the skin becomes wrinkled and aged. Here, we investigated the capacity of cilostazol to inhibit MMP-1 expression in UVB-irradiated human dermal fibroblasts.Main methodsCultured human dermal fibroblasts were irradiated with UVB, followed by the addition of cilostazol to the culture medium.Key findingsPost-treatment with cilostazol attenuated UVB-induced production of MMP-1 and prevented the reduction of type I procollagen. Cilostazol inhibited UVB irradiation-induced phosphorylation of the mitogen-activated protein kinase (MAPK) signaling molecules Jun-N-terminal kinase (JNK) and p38 kinase, as well as activator protein-1 (AP-1) in dermal fibroblasts.SignificanceOverall, these results demonstrate that cilostazol regulates UVB-induced MMP-1 expression and type I procollagen synthesis by inhibiting MAPK signaling and AP-1 activity. Therefore, we suggest that cilostazol may be useful for the prevention and treatment of skin photodamage caused by UVB-irradiation.     

Synthesis,and In Vitro and In Silico α-Glucosidase Inhibitory Studies of 5-Chloro-2-Aryl Benzo[d]thiazoles

Twenty-five derivatives of 5-chloro-2-aryl benzo[d]thiazole (1–25) were synthesized and evaluated for their α-glucosidase (S. cerevisiae EC 3.2.1.20) inhibitory activity in vitro. Among them eight compounds showed potent activity with IC₅₀ values between 22.1 ± 0.9 and 136.2 ± 5.7 μM, when compared with standard acarbose (IC₅₀ = 840 ± 1.73 μM). The most potent compounds 4, 9, and 10 showed IC₅₀ values in the range of 22.1 ± 0.9 to 25.6 ± 1.5 μM. Compounds 2, 5, 11, and 19 showed IC₅₀ values within the range of 40.2 ± 0.5 to 60.9 ± 2.0 μM. Compounds 1 and 3 were also found to be good inhibitors with IC₅₀ values 136.2 ± 5.7 and 104.8 ± 9.9 μM, respectively. Their activities were compared with α-glucosidase inhibitor drug acarbose (standard) (IC₅₀ = 840 ± 1.73 μM). The remaining compounds were inactive. Structure-activity relationships (SAR) have also been established. Kinetics studies indicated compounds 2, 3, 10, 19, and 25 to be non-competitive, while 1, 5, 9, and 11 as competitive inhibitors of α-glucosidase enzyme. All the active compounds (1–5, 9–11, and 19) were also found to be non-cytotoxic, in comparison to the standard drug i.e., doxorubicin (IC₅₀ = 0.80 ± 0.12 μM) in MTT assay. Furthermore, molecular interactions of active compounds with the enzyme binding sites were predicted through molecular modeling studies.     

Synthesis of CC,CN coupled novel substituted dibutyl benzothiazepinone derivatives and evaluation of their thrombin inhibitory activity

The formation of a thrombus is a key event in thromboembolic disorders, that contribute to high mortality and morbidity in affected patients. In the present study, we synthesized a library of novel substituted 3,3-dibutyl-8-methoxy-2,3-dihydrobenzo [b] [1,4] thiazepin-4(5H)-one derivatives which were tested for their platelet aggregation and thrombin inhibitory activity. Among the tested compounds, 3,3-dibutyl-7-(2-chlorophenyl)-8-methoxy-2,3-dihydrobenzo[b] [1,4]thiazepin-4(5H)-one (DCT) displayed the maximum thrombin inhibition with an IC₅₀ value of 3.85 μM and thus DCT was chosen for further studies. Next, the effect of DCT on primary hemostasis was evaluated using agonist-induced platelet aggregation model. The lead compound inhibited the collagen- or ADP- or thrombin-induced platelet aggregation in a dose-dependent manner. Furthermore, DCT prolonged the process of clot formation when evaluating plasma re-calcification time (320 ± 11 sec at 5 µg DCT), activated partial thromboplastin time (58.0 ± 0.01 sec at 2 µg), and prothrombin time (14.7 ± 0.01 sec at 5 µg). Molecular docking studies suggested that the benzothiazepinones evaluated here consistently display hydrogen bonding with Ser214 of thrombin, which is similar to that of the co-crystallized ligand (1-(2R)-2-amino-3-phenyl-propanoyl-N-(2,5dichlorophenyl)methylpyrrolidine-2-carboxamide). DCT displayed additional hydrogen bonding to Ser195 and π-π interactions between its methoxyphenyl groups and Trp60, thereby providing a structural rationale for the observed biological effect.     

Inhibition of dopamine β-hydroxylase by bidentate chelating agents

1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-car☐ylic acid (QCA), 1-isoquinolinecar☐ylic acid (IQCA), 2,2′-bi-1H-imidazole (2,2′-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine β-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine β-hydroxylase with respect to ascorbate (K_is = 5.7(±0.9) μM, 0.14(±0.03) mM, 0.80(±0.20) mM; n_H= 1.4(±0.1), 1.8(±0.4), 2.8(±0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine β-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (K_is = 1.9(±0.3) μM, 21(±6) μM, and 0.9(±0.3) μM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine β-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine β-hydroxylase.     

Bioactive chemical constituents from the root bark of Morus australis

Two new pyranoflavonoids, morustralins A (1) and B (2), a new natural benzene derivative, one benzenoid (Z)-1-hydroxy-4-(2-nitroethenyl)benzene (3), and thirty known compounds were isolated and characterized from the root bark of Morus australis. The structures of the new compounds were established from spectroscopic and spectrometric analyses. Ten isolates (1–10) were examined for inhibitory effects on adenosine diphosphate (ADP)-, arachidonic acid (AA)-, and platelet-aggregating factor (PAF)-induced platelet aggregation. Among the tested compounds, compound 3 displayed the most significant inhibition of ADP- and AA-induced platelet aggregation with IC₅₀ values of 9.76 ± 5.54 and 9.81 ± 2.7 μM, respectively. In addition, eight purified compounds (3–10) were examined for inhibition of nitric oxide (NO) production in RAW 264.7 cells and six compounds (3–8) displayed significant inhibitory effects with IC₅₀ values ranging from 2.1 ± 0.3 to 6.3 ± 0.6 μM.     

Cilostazol inhibits accumulation of triglyceride in aorta and platelet aggregation in cholesterol-fed rabbits

Cilostazol is clinically used for the treatment of ischemic symptoms in patients with chronic peripheral arterial obstruction and for the secondary prevention of brain infarction. Recently, it has been reported that cilostazol has preventive effects on atherogenesis and decreased serum triglyceride in rodent models. There are, however, few reports on the evaluation of cilostazol using atherosclerotic rabbits, which have similar lipid metabolism to humans, and are used for investigating the lipid content in aorta and platelet aggregation under conditions of hyperlipidemia. Therefore, we evaluated the effect of cilostazol on the atherosclerosis and platelet aggregation in rabbits fed a normal diet or a cholesterol-containing diet supplemented with or without cilostazol. We evaluated the effects of cilostazol on the atherogenesis by measuring serum and aortic lipid content, and the lesion area after a 10-week treatment and the effect on platelet aggregation after 1- and 10-week treatment. From the lipid analyses, cilostazol significantly reduced the total cholesterol, triglyceride and phospholipids in serum, and moreover, the triglyceride content in the atherosclerotic aorta. Cilostazol significantly reduced the intimal atherosclerotic area. Platelet aggregation was enhanced in cholesterol-fed rabbits. Cilostazol significantly inhibited the platelet aggregation in rabbits fed both a normal diet and a high cholesterol diet. Cilostazol showed anti-atherosclerotic and anti-platelet effects in cholesterol-fed rabbits possibly due to the improvement of lipid metabolism and the attenuation of platelet activation. The results suggest that cilostazol is useful for prevention and treatment of atherothrombotic diseases with the lipid abnormalities.     

Protective effect of the phosphodiesterase III inhibitor cilostazol on amyloid β-induced cognitive deficits associated with decreased amyloid β accumulation

Alzheimer’s disease (AD), which is characterized by progressive cognitive impairment, is the most common neurodegenerative disease. Here, we investigated the preventive effect of a phosphodiesterase III inhibitor, cilostazol against cognitive decline in AD mouse model. In vitro studies using N2a cells stably expressing human amyloid precursor protein Swedish mutation (N2aSwe) showed that cilostazol decreased the amyloid β (Aβ) levels in the conditioned medium and cell lysates. Cilostazol attenuated the expression of ApoE, which is responsible for Aβ aggregation, in N2aSwe. Intracerebroventricular injection of Aβ_25–35 in C57BL/6J mice resulted in increased immunoreactivity of Aβ and p-Tau, and microglia activation in the brain. Oral administration of cilostazol for 2 weeks before Aβ administration and once a day for 4 weeks post-surgery almost completely prevented the Aβ-induced increases of Aβ and p-Tau immunoreactivity, as well as CD11b immunoreactivity. However, post-treatment with cilostazol 4 weeks after Aβ administration, when Aβ was already accumulated, did not prevent the Aβ-induced neuropathological responses. Furthermore, cilostazol did not affect the neprilysin and insulin degrading enzymes involved in the degradation of the Aβ peptide, but decreased ApoE levels in Aβ-injected brain. In addition, cilostazol significantly improved spatial learning and memory in Aβ-injected mice. The findings suggest that a phosphodiesterase III inhibitor, cilostazol significantly decreased Aβ accumulation and improved memory impairment induced by Aβ_25–35. The beneficial effects of cilostazol might be explained by the reduction of Aβ accumulation and tau phosphorylation, not through an increase in Aβ degradation but via a significant decrease in ApoE-mediated Aβ aggregation. Cilostazol may be the basis of a novel strategy for the therapy of AD.     

Bis-coumarins; non-cytotoxic selective urease inhibitors and antiglycation agents

The current study is concerned with the identification of lead molecules based on the bis-coumarin scaffold having selective urease inhibitory and antiglycation activities. For that purpose, bis-coumarins (1-44) were synthesized and structurally characterized by different spectroscopic techniques. Eight derivatives 4, 8-10, 14, 17, 34, and 40 demonstrated urease inhibition in the range of IC₅₀ = 4.4 ± 0.21–115.6 ± 2.13 μM, as compared to standard thiourea (IC₅₀ = 21.3 ± 1.3 μM). Especially, compound 17 (IC₅₀ = 4.4 ± 0.21 μM) was found to be five-fold more potent than the standard. Kinetic studies were also performed on compound 17 in order to identify the mechanism of inhibition. Kinetic studies revealed that compound 17 is a competitive inhibitor. Antiglycation activity was evaluated using glycation of bovine serum albumin by methylglyoxal in vitro. Compounds 2, 11-13, 16, 17, 19–22, 35, 37, and 42 showed good to moderate antiglycation activities with IC₅₀ values of 333.63–919.72 μM, as compared to the standard rutin (IC₅₀ = 294.46 ± 1.5 μM). Results of both assays showed that the compounds with urease inhibitory activity did not show any antiglycation potential, and vice versa. Only compound 17 showed dual inhibition potential. All compounds were also evaluated for cytotoxicity. Compounds 17, 19, and 37 showed a weak toxicity towards 3 T3 mouse fibroblast cell line. All other compounds were found to be non-cytotoxic. Urease inhibition is an approach to treat infections caused by ureolytic bacteria whereas inhibition of glycation of proteins is a strategy to avoid late diabetic complications. Therefore, these compounds may serve as leads for further research.     

Effect of OKY-046, a selective thromboxane inhibitor,on prostaglandin production and function of pregnant rat uteri and platelets

The effect of OKY-046 (ONO, Japan), a selective TX inhibitor, was studied for its effect on uterine and platelet activity. On day 21 of pregnancy, rats were injected with either 0, 1, or 5 mg/kg OKY-046 via the tail vein. One hour following injections, in vitro activity of uteri and platelets was assessed. A decrease (P<.01) in uterine TXB₂ production (measured by RIA) occurred with increasing OKY-046 dose ( 104 ± 31 vs 44 ± 6 vs 24 ± 2 ng TXB₂/g tissue/45 min). OKY-046 treatment had no effect on other prostanoids. Contractile activity was not affected by OKY-046. The amount of TXB₂ produced in platelets from OKY-046 (5 mg/Kg) treated rats was 45.5% less than that from controls (P<.001). Likewise, arachidonate-induced aggregation of platelets from OKY-046 treated rats was 46.1% less (P<.05) than that of controls. In summary, in vivo administration of OKY-046 selectively reduced uterine TXB₂ without altering other prostanoids, or affecting uterine contractions. In contrast, both platelet TXB₂ production and platelet function (aggregation) was decreased.     

2,5-Disubstituted thiadiazoles as potent β-glucuronidase inhibitors; Synthesis,in vitro and in silico studies

Twenty-five thiadiazole derivatives 1–25 were synthesized from methyl 4-methoxybenzoate via hydrazide and thio-hydrazide intermediates, and evaluated for their potential against β-glucuronidase enzyme. Most of the compounds including 1 (IC₅₀ = 26.05 ± 0.60 μM), 2 (IC₅₀ = 42.53 ± 0.80 μM), 4 (IC₅₀ = 38.74 ± 0.70 μM), 5 (IC₅₀ = 9.30 ± 0.29 μM), 6 (IC₅₀ = 6.74 ± 0.26 μM), 7 (IC₅₀ = 18.40 ± 0.66 μM), and 15 (IC₅₀ = 18.10 ± 0.53 μM) exhibited superior activity potential than the standard d-saccharic acid-1,4-lactone (IC₅₀ = 48.4 ± 1.25 μM). Molecular docking studies were conducted to correlate the in vitro results and to identify possible mode of interaction with enzyme active site.     

A new entry into the portfolio of α-glucosidase inhibitors as potent therapeutics for type 2 diabetes: Design,bioevaluation and one-pot multi-component synthesis of diamine-bridged coumarinyl oxadiazole conjugates

Diabetes mellitus (DM), a chronic multifarious metabolic disorder resulting from impaired glucose homeostasis has become one of the most challenging diseases with severe life threat to public health. The inhibition of α-glucosidase, a key carbohydrate hydrolyzing enzyme, could serve as one of the effective methodology in both preventing and treating diabetes through controlling the postprandial glucose levels and suppressing postprandial hyperglycemia. In this context, three series of diamine-bridged bis-coumarinyl oxadiazole conjugates were designed and synthesized by one-pot multi-component methodology. The synthesized conjugates (4a–j, 5a–j, 6a–j) were evaluated as potential inhibitors of glucosidases. Compound 6f containing 4,4′-oxydianiline linker was identified as the lead and selective inhibitor of α-glucosidase enzyme with an IC₅₀ value of 0.07 ± 0.001 μM (acarbose: IC₅₀ = 38.2 ± 0.12 μM). This inhibition efficacy was ∼545-fold higher compared to the standard drug. Compound 6f was also emerged as the lead molecule against intestinal maltase-glucoamylase with good inhibition strength (IC₅₀ = 0.04 ± 0.02 μM) compared to acarbose (IC₅₀ = 0.06 ± 0.01 μM). Against β-glucosidase enzyme, compound 6 g was noted as the lead inhibitor with IC₅₀ value of 0.08 ± 0.002 μM. Michaelis–Menten kinetic experiments were performed to explore the mechanism of inhibition. Molecular docking studies of the synthesized library of hybrid structures against glucosidase enzyme were performed to describe ligand-protein interactions at molecular level that provided an insight into the biological properties of the analyzed compounds. The results suggested that the inhibitors could be stabilized in the active site through the formation of multiple interactions with catalytic residues in a cooperative fashion. In addition, strong binding interactions of the compounds with the amino acid residues were effective for the successful identification of α-glucosidase inhibitors.     

Effects of α,β-methylene-adenosine-5′-diphosphate on blood platelet aggregation

The effects on platelet aggregation of α,β-methylene-adenosine-5′-diphosphate (Ado-PCP) have been investigated. Using human citrated platelet-rich plasma it has been shown that: (i) at concentrations of 10^?3 M or higher Ado-PCP is able to induce platelet aggregation; (ii) the rate of Ado-PCP-induced aggregation increases on raising the pH of platelet-rich plasma above the pK_a for the secondary phosphonyl dissociation of Ado-PCP; (iii) at concentrations from 1 · 10^?4 to 5 · 10^?4 M Ado-PCP does not cause platelet aggregation itself, but it inhibits ADP-induced aggregation. This inhibition is also observed in washed platelet suspensions. The data suggest that Ado-PCP acts at the same site on the platelet membrane as does ADP and that ADP to AMP transformation is not a prerequisite for the process of aggregation. The observed effect of pH on the rate of Ado-PCP induced aggregation suggests that the ionization state of a nucleotide terminal acid group is important in the process of aggregation.     

Synthesis,biological evaluation,and molecular docking study of sulfonate derivatives as nucleotide pyrophosphatase/phosphodiesterase (NPP) inhibitors

A new series of sulfonate derivatives 1a–zk were synthesized and evaluated as inhibitors of nucleotide pyrophosphatases. Most of the compounds exhibited good to moderate inhibition towards NPP1, NPP2, and NPP3 isozymes. Compound 1m was a potent and selective inhibitor of NPP1 with an IC₅₀ value of 0.387 ± 0.007 µM. However, the most potent inhibitor of NPP3 was found as 1x with an IC₅₀ value of 0.214 ± 0.012 µM. In addition, compound 1e was the most active inhibitor of NPP2 with an IC₅₀ value of 0.659 ± 0.007 µM. Docking studies of the most potent compounds were carried out, and the computational results supported the in vitro results.     

Inhibition effect of flavonoid compounds against neuraminidase expressed in Pichia pastoris

Neuraminidase (NA) is one of the two glycoproteins on the surface of influenza virus, which cleaves terminal sialic acid residues and facilitates the release of virions from infected cells. The recombinant NA from H5N1 influenza virus strain A/Vietnam/1203/04 was expressed in Pichia pastoris X33 as a 45 kDa protein that displayed a K _m of 9.96 ± 1.26 μM with fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetyl neuraminic acid. Partially purified NA was used for the inhibition and kinetic assays with eight flavonoid compounds and gallic acid. Among them, gallocatechin gallate (GCG) showed the best inhibition against NA with the IC₅₀ of 8.98 ± 0.46 μM and showed a competitive inhibition pattern with K _i value of 8.34 ± 0.25 μM. In molecular docking experiments, GCG displayed a binding energy of ?13.71 kcal/mol to the active site of NA and the galloyl moiety was required for NA inhibition activity.     

Cilostazol Strengthens Barrier Integrity in Brain Endothelial Cells

We studied the effect of cilostazol, a selective inhibitor of phosphodiesterase 3, on barrier functions of blood–brain barrier (BBB)-related endothelial cells, primary rat brain capillary endothelial cells (RBEC), and the immortalized human brain endothelial cell line hCMEC/D3. The pharmacological potency of cilostazol was also evaluated on ischemia-related BBB dysfunction using a triple co-culture BBB model (BBB Kit?) subjected to 6-h oxygen glucose deprivation (OGD) and 3-h reoxygenation. There was expression of phosphodiesterase 3B mRNA in RBEC, and a significant increase in intracellular cyclic AMP (cAMP) content was detected in RBEC treated with both 1 and 10 μM cilostazol. Cilostazol increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), and decreased the endothelial permeability of sodium fluorescein through the RBEC monolayer. The effects on these barrier functions were significantly reduced in the presence of protein kinase A (PKA) inhibitor H-89. Microscopic observation revealed smooth and even localization of occludin immunostaining at TJs and F-actin fibers at the cell borders in cilostazol-treated RBEC. In hCMEC/D3 cells treated with 1 and 10 μM cilostazol for 24 and 96 h, P-glycoprotein transporter activity was increased, as assessed by rhodamine 123 accumulation. Cilostazol improved the TEER in our triple co-culture BBB model with 6-h OGD and 3-h reoxygenation. As cilostazol stabilized barrier integrity in BBB-related endothelial cells, probably via cAMP/PKA signaling, the possibility that cilostazol acts as a BBB-protective drug against cerebral ischemic insults to neurons has to be considered.     

Synthesis and Biological Evaluation of 1,3,5-Trisubstituted 2-Pyrazolines as Novel Cyclooxygenase-2 Inhibitors with Antiproliferative Activity

A new series of 1,3,5-trisubstituted 2-pyrazolines for the inhibition of cyclooxygenase-2 (COX-2) were synthesized. The designed structures include a COX-2 pharmacophore SO₂CH₃ at the para-position of the phenyl ring located at C-5 of a pyrazoline scaffold. The synthesized compounds were tested for in vitro COX-1/COX-2 inhibition and cell toxicity against human colorectal adenocarcinoma cell lines HT-29. The lead compound (4-chlorophenyl){5-[4-(methanesulfonyl)phenyl]-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl}methanone ( 16 ) showed significant COX-2 inhibition (IC₅₀=0.05±0.01 μM), and antiproliferative activity (IC₅₀=5.46±4.71 μM). Molecular docking studies showed that new pyrazoline-based compounds interact via multiple hydrophobic and hydrogen-bond interactions with key binding site residues of the COX-2 enzyme.     

Calpain activation in shear-induced platelet aggregation

Fluid shear stress has been known to activate platelet reaction such as aggregation, but the exact mechanism of shear-induced platelet aggregation (SIPA) has not been fully understood. Calpain, an intracellular calcium-activated cysteine protease, is abundant in platelets and is considered to be activated and involved in the proteolytic processes during platelet activation. A possible activation of calpain in SIPA was investigated, employing a newly developed aggregometer and specific monoclonal antibodies to detect activation of calpain. When a shear stress gradient varying between 6 and 108 dyn/cm² was applied to platelets, activation of μ-calpain was observed only in high-shear-stressed platelets, resulting in the proteolysis of talin. At 1 min after the onset of constant high shear stress of 108 dyn/cm², μ-calpain activation and proteolysis of talin were detected and increased in a time-dependent manner. Constant shear stress more than 50 dyn/cm², applied for 5 min, caused μ-calpain activation and proteolysis of talin, which were increased in a shear-force-dependent manner. Calpeptin, a calpain-specific peptide antagonist, caused the complete inhibition of both μ-calpain activation and proteolysis of talin, while SIPA profiles with calpeptin showed almost no change compared to those without calpeptin. These results suggest the possibility of calpain involvement in late phases of shear-induced platelet activation such as cytoskeletal reorganization. J. Cell. Biochem. 66:54–64, 1997. © 1997 Wiley-Liss, Inc.     

Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase

Sesamol is a potent phenolic antioxidant which possesses antimutagenic, antihepatotoxic and antiaging properties. Platelet activation is relevant to a variety of acute thrombotic events and coronary heart diseases. There have been few studies on the effect of sesamol on platelets. Therefore, the aim of this study was to systematically examine the detailed mechanisms of sesamol in preventing platelet activation in vitro and in vivo. Sesamol (2.5?5 μM) exhibited more potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists. Sesamol inhibited collagen-stimulated platelet activation accompanied by [Ca²⁺]_i mobilization, thromboxane A₂ (TxA₂) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) phosphorylation in washed platelets. Sesamol markedly increased cAMP and cGMP levels, endothelial nitric oxide synthase (eNOS) expression and NO release, as well as vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the sesamol-mediated inhibitory effects on platelet aggregation and p38 MAPK phosphorylation, and sesamol-mediated stimulatory effects on VASP and eNOS phosphorylation, and NO release. Sesamol also reduced hydroxyl radical (OH^●) formation in platelets. In an in vivo study, sesamol (5 mg/kg) significantly prolonged platelet plug formation in mice. The most important findings of this study demonstrate for the first time that sesamol possesses potent antiplatelet activity, which may involve activation of the cAMP-eNOS/NO-cGMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA₂ cascade, and, finally, inhibition of platelet aggregation. Sesamol treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders.     

One-pot four-component synthesis of thiazolidin-2-imines using CuI/ZnII dual catalysis: A new class of acetylcholinesterase inhibitors

An efficient one-pot four-component strategy involving aldehydes, amines, alkynes and isothiocyanates has been developed to access a novel series of thiazolidine-2-imines (5a-x). This process operates under the action of a cooperative catalysis composed of Cu(I) and Zn(II) delivering the desired five-membered heterocyclic compounds in good to excellent yields. Notably, this transformation avoids the use of pre-formed imines or propargylamines and proceeds via an intramolecular 5-exo-dig hydrothiolation reaction of the in situ formed propargyl thiourea. Furthermore, the biological application of these motifs was demonstrated in terms of their strong acetylcholinesterase (AChE) inhibitory activity where compound 5s was identified as the lead AChE inhibitor with an IC₅₀ value of 0.0023 ± 0.0002 μM, 88-folds stronger inhibition than standard drug (neostigmine methyl sulphate; IC₅₀ = 0.203 ± 0.004 μM). Molecular docking analysis reinforced the in vitro biological activity results revealing the formation of several useful interactions of the potent lead with amino acid residues in the active site of the enzyme.