Publications (181) View all
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Article: Mechanisms of aggregation inhibition by aspirin and nitrate-aspirin prodrugs in human platelets.
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ABSTRACT: Aspirin is the mainstay of anti-platelet therapy in the secondary prevention of cardiovascular disease. However, problems with aspirin safety and resistance demand clinical strategies based on multiple pharmacological approaches. Prodrugs of aspirin may offer beneficial effects in terms of gastro-intestinal safety and multiple pharmacological approaches. However, the pharmacological profile of aspirin prodrugs in human platelets has not been completed yet. We aimed to compare the effects of aspirin and prodrugs of aspirin (1-5) on human platelet aggregation stimulated by ADP and collagen and associated receptor expression (GPIIb/IIIa and P-selectin) in platelet-rich plasma (PRP) and washed platelets (WP). As aspirin is released from prodrugs following esterase hydrolysis we studied the expression and activity of butyrylcholineterase (BuChE) and carboxyesterase (CE) in plasma and platelets. The mechanism of prodrug-induced platelet aggregation inhibition was explored by studying the effects of plasma and purified human BuChE on aggregation. Finally, the relative contribution of nitric oxide (NO) bioactivity to nitrate-containing prodrugs of aspirin-induced inhibition of aggregation was determined using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ,) a selective inhibitor of the soluble guanylyl cyclase. ST0702, 2, a nicotinic acid-aspirin codrug was equipotent with aspirin with respect to inhibition of collagen-induced platelet aggregation. Compound 4, a NO releasing aspirin was the most potent inhibitor of ADP-induced platelet aggregation, an effect partially reversed by ODQ. The platelet inhibitory effects of aspirin prodrugs were time-dependent as the maximal inhibitory effects against collagen-induced aggregation were achieved by aspirin at 2 min, 1 at 5 min and ST0702 at 15 min. The aspirin prodrugs were significantly less potent in WP than in PRP and the reverse was true of aspirin. In the presence of complete BuChE inhibition in PRP, there was almost complete loss of aspirin prodrug, but not aspirin anti-aggregatory activity. Interestingly, CE activity was observed in WP and platelet lysate with pNPA substrate. Accordingly, 1 and ST0702 retained 50% and 100% anti-aggregatory activity at maximal concentrations in WP, which was attenuated in the presence of esterase inhibitor phenylmethylsulphonyl fluoride. The inhibitory effect of aspirin prodrugs in PRP is due to prodrug activation by BuChE. In contrast, the platelet-inhibitory effects of aspirin prodrugs in WP may be mediated through the activity of platelet CE. Compound 4, a NO-containing aspirin prodrug, may exert dual inhibitory effects in platelets. Thus, aspirin prodrugs effectively inhibit human platelet aggregation and as such may be an alternative to conventional aspirin.The Journal of pharmacy and pharmacology. 01/2012; 64(1):77-89. -
Article: Fisetin prevents fluoride- and dexamethasone-induced oxidative damage in osteoblast and hippocampal cells.
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ABSTRACT: Fluoride intoxication and dexamethasone treatment produce deleterious effects in bone and brain. The aim of this study was to evaluate the effect of fluoride (F) and dexamethasone (Dex) co-exposure on oxidative stress and apoptosis in osteoblast-like MC3T3-E1 and hippocampal HT22 cell lines. Co-exposure to F and Dex resulted in a concentration-dependent decrease in cell viability, induction of apoptosis and increased generation of reactive oxygen species (ROS) and nitric oxide (NO) following 72 h of incubation. Fluoride-induced apoptosis in MC3T3-E1 and HT22 cells was attenuated by catalase and L-NNMA, indicating a role for H2O2 and NO as mediators of cytotoxicity. Dexamethasone-induced apoptosis was associated with H2O2 generation in both cell lines and it was attenuated during co-incubation with catalase. These data indicate that co-exposure to F and Dex amplifies their respective cytotoxicity in H2O2- and NO-dependent manner. As flavonoid fisetin prevented F- and Dex-induced cytotoxicity the potential role of this product in pharmacology and diet may be considered.Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 12/2011; 50(3-4):583-9. · 2.99 Impact Factor -
Article: Differential inhibition of tumour cell-induced platelet aggregation by the nicotinate aspirin prodrug (ST0702) and aspirin.
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ABSTRACT: Tumour cell-induced platelet aggregation (TCIPA) facilitates cancer cell invasion, angiogenesis and the formation of metastatic foci. TCIPA can be modulated by pharmacological inhibitors of MMP-2 and ADP; however, the COX inhibitor aspirin did not prevent TCIPA. In this study, we have tested the pharmacological effects of a new group of isosorbide-based aspirin prodrugs on TCIPA. TCIPA was induced in human platelets by mixing with human adenocarcinoma or fibrosarcoma cells under no flow and flow conditions. The release of gelatinases and P-selectin expression during TCIPA were studied by zymography and flow cytometry respectively. Tumour cells caused platelet aggregation. This aggregation resulted in the release of MMP-2 and a significant up-regulation of P-selectin on platelets, indicative of platelet activation. Pharmacological modulation of TCIPA revealed that ST0702, one of the aspirin prodrugs, down-regulated TCIPA while aspirin was ineffective. The deacetylated metabolite of ST0702, 5-nicotinate salicylate (ST0702 salicylate), down-regulated both ADP-stimulated platelet aggregation and TCIPA. Our results show that ST0702 was an effective inhibitor of TCIPA in vitro. Its deacetylated metabolite may contribute to the effects of ST0702 by inhibiting ADP-mediated TCIPA.British Journal of Pharmacology 11/2011; 166(3):938-49. · 4.41 Impact Factor -
Chapter: Isolation of Exosomes for Subsequent mRNA, MicroRNA, and Protein Profiling
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ABSTRACT: Exosomes are nano-sized, cell membrane surrounded structures that are released from many cell types. These exosomes are believed to transport a range of molecules, including mRNAs, miRNAs, and proteins; the contents depending on their cell of origin. The physiological and pathological relevance of exosomes has yet to be fully elucidated. Exosomes have been implicated in cell-to-cell communication. For example, in relation to the immune system, such exosomes may enable exchange of antigen or major histocompatibility complex–peptide complexes between antigen-bearing cells and antigen-presenting cells; in cancer, they may contain molecules that not only have relevance as biomarkers, but may also be taken up and cause adverse effects on secondary cells. Furthermore, exosomes have been proposed as autologous delivery systems that could be exploited for personalised delivery of therapeutics. In order to explore the contents and functional relevance of exosomes from medium conditioned by culture cells or from other biological fluids, prior to extensive molecular profiling, they must be isolated and purified. Here, we describe differential centrifugation methods suitable for isolating exosomes from conditioned medium and from other biological fluids, including serum, saliva, tumour ascites, and urine. We also detail Western blotting and transmission electron microscopy methods suitable for basic assessment of their presence, size, and purity, prior to progressing to global mRNA, miRNA, or protein profiling. Key wordsExosomes-Multivesicular bodies-Extracellular-Cell line-Conditioned medium-Serum-Plasma-Urine-Saliva09/2011: pages 181-195; -
Article: N-substituted homopiperazine barbiturates as gelatinase inhibitors.
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ABSTRACT: Matrix metalloproteinases are implicated in a wide range of pathophysiological processes and potent selective inhibitors for these enzymes continue to be eagerly sought. 5,5-Disubstituted barbiturates hold promise as inhibitor types being stable in vivo and relatively selective for the gelatinases (MMP-2 and MMP-9). In this paper we describe the synthesis of 5-piperazine and -homopiperazine substituted barbiturates. The activity of these compounds as gelatinase inhibitors was evaluated using supernatants from 12-O-tetradecanoylphorbol-13-acetate (PMA)-stimulated HT-1080 cells as well as using recombinant human MMPs. N-Acyl homopiperazine compounds were found to be potent inhibitors of the gelatinases (range in nM) and generally more potent than the corresponding piperazine analogues. The panel of N-acyl homopiperazines was enlarged in order to exploit differences between the gelatinases at the S2' site in order to design MMP-2- or MMP-9-selective inhibitors. Compounds in this group exhibited single digit nano-molar potency and some selectivity between the two enzymes. Representative potent compounds were effective inhibitors of cancer cell migration.Bioorganic & medicinal chemistry 08/2011; 19(16):4985-99. · 2.82 Impact Factor
