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In an effort to develop potent antiplatelet agents, 12 O-prenylated (2-13) and 10 O-allylated (14-23) chalcones were synthesized and screened for in vitro inhibitory effects on aggregation of washed rabbit platelets induced by ADP (20 μM) and collagen (10 μg/mL). In addition, the platelet aggregation activity of previously synthesized Mannich bases of heterocyclic chalcones (MBHC) (24-62) was evaluated. The preliminary structure-activity relationships suggested that the antiplatelet activity was governed to a great extent by the presence of a pyridyl ring-B and a hydroxy group at position C-3' in ring-A of the MBHC templates., (Copyright © 2011. Published by Elsevier Ltd.)

Two series of 4-benzylideneamino- and 4-phenyliminomethyl-benzenesulfonamide derivatives were designed and synthesized for the evaluation as selective cyclooxygenase-2 (COX-2) inhibitors in a cellular assay using human whole blood (HWB). Extensive structure-activity relationships (SAR) were studied within these series. Several compounds were found to be novel and selective COX-2 inhibitors. Among them, the most potent and selective was 4-(3-carboxy-4-hydroxy-benzylideneamino)benzenesulfonamide (20, LA2135), (IC(50)'s for COX-1: 85.13 microM; COX-2: 0.74 microM; SI: 114.5), being more active COX-2 selective than celecoxib.

Accelerating thrombolysis using plasminogen activators (PAs) encapsulated liposomes or PEG microparticles by pressure-driven permeation have been demonstrated in vitro and in vivo in animal models. However, designing and delivering PA-encapsulated nanoparticles (NPs) to enhance thrombolysis by applying electrostatic forces or ligand-receptor interactions between the NPs and blood clots has not been proposed. Therefore, without a pressure-driving factor, tissue-plasminogen activator (t-PA) encapsulated in PLGA NPs with chitosan (CS) and CS-GRGD coating and their thrombolysis capabilities in a blood clot-occluded tube model were evaluated by determining clot lysis times and the masses of the digested clots. The characteristics and release profiles of t-PA-encapsulated PLGA, PLGA/CS and PLGA/CS-GRGD NPs are determined by FT-IR, a laser particle/zeta potential analyzer and HPLC. Additionally, the permeation capacities of the NPs into flat blood clots were examined. For example, the mean particle sizes and encapsulation efficacies of t-PA for the NPs are in the ranges 260-320 nm and 65.5-70.5%, respectively. The results reveal that the NPs for the shortest clot lysis time and the highest weight percentages of digested clot are PLGA/CS (20.7 +/- 0.7 min) and PLGA/CS-GRGD (25.7 +/- 1.3 wt%), respectively. Compared with t-PA solution, the NPs can significantly shorten clot lysis times in the following order: PLGA/CS NPs (38.8 +/- 1.5%) > PLGA/CS-GRGD NPs (16.3 +/- 1.0%) > PLGA NPs (7.7 +/- 1.2%). Compared with t-PA solution, the NPs significantly increase the weight of digested clots in the order, PLGA/CS-GRGD (40.9 +/- 1.5%) > PLGA/CS (27.8 +/- 1.2%) > PLGA (8.6 +/- 0.6%). The highest release rate of t-PA in the fast release phase and the highest permeability into intra-clots of PLGA/CS and PLGA/CS-GRGD NPs, respectively, correspond to the shortest clot lysis time and the largest increase in weight of the digested clots among the NP system. In conclusion, the NPs designed based on new concepts significantly accelerate thrombolysis in vitro in this model, and may be useful in clinical study.

T cell immune responses play important roles in the pathogenesis of systemic lupus erythematosus (SLE). (S)-Armepavine (C19H23O3N; MW313) from Nelumbo nucifera suppresses T cells proliferation. To study its potential benefit on SLE, we examined effects of (S)-armepavine on MRL/MpJ-lpr/lpr mice, which have similar disease features to human SLE. MRL/MpJ-lpr/lpr mice were treated orally with (S)-armepavine for 6 weeks and their SLE characteristics were evaluated. The results revealed that (S)-armepavine prevented lymphadenopathy and elongated life span of MRL/MpJ-lpr/lpr mice. It seemed to be mediated by inhibition of splenocytes proliferation, suppression of interleukin-2 (IL-2), interleukin-4, interleukin-10, and interferon-gamma (IFN-gamma) gene expressions, reduction of glomerular hypercellularity and immune complexes deposition, and decrease of urinary protein and anti-double stranded DNA autoantibody production. Furthermore, the data demonstrated (S)-armepavine impaired IL-2 and IFN-gamma transcripts in human peripheral blood mononuclear cells. We suggest that (S)-armepavine may be an immunomodulator for the management of autoimmune diseases like SLE.

A series of amides of caffeic acid were synthesized and evaluated for their anti-platelet and anti-oxidative activities. N-(2-Bromo-phenyl)-3-(3,4-dihydroxy-phenyl)-acrylamide (12) and N-(3-Bromo-phenyl)-3-(3,4-dihydroxy-phenyl)-acrylamide (13) exhibited potent inhibitory activity (IC(50)=5.8 and 6.7 microM, respectively) against arachidonic acid-induced (AA) platelet aggregation, comparable with invalid caffeic acid. Most of the synthesized caffeic acid anilides exhibited the promising anti-platelet aggregation in AA-induced assay and anti-oxidative activities. This study also exhibited that caffeic anilides displayed more potent anti-oxidative activity in the radical scavenging activity assay than trolox and vitamin E.

We elucidate the roles of various protein kinases involved in complement 5a (C5a)-induced cell migration. Results showed that extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (P13K) were necessary for C5a-induced migration, whereas protein kinase C and c-Jun N-terminal kinase (JNK) were nonessential. C5a-induced migration was also suppresses by phospholipase C (PLC) inhibitor U73122 and pertussis toxin (PTX). We found that C5a-induced, time-dependent (1) ERK1/2 phosphorylation was markedly diminished by PTX, U73122, P13K inhibitors wortmannin and LY294002 and ERK1/2 inhibitor PD98059; (2) Akt phosphorylation was also attenuated by the above inhibitors except PD98059; (3) p38 MAPK phosphorylation was only affected by PTX. Furthermore, C5a also stimulated PLCbeta(2) membrane translocation in a time-dependent manner that occurred early prior to Akt phosphorylation and could be abolished only by PTX and U73122. These results suggest that C5a, through the activation of PTX-sensitive G protein, to differentially stimulate ERK1/2 and p38 MAPK phosphorylation and evoke cell migration. That is, ERK1/2 but not p38 MAPK phosphorylation is down stream of P13K/Akt and modulated by PLC. Additionally, beta(2) isoform may be one of the participates in C5a signal and acts more upstream of P13K/Akt.

We now evaluated the anti-inflammatory mechanisms of andrographolide on complement 5a (C5a)-induced macrophage recruitment in vitro. Andrographolide concentration dependently inhibited cell migration toward C5a with an IC50 of 5.6+/-0.7 microM. With relatively specific kinase inhibitors (PD98059, SB203580, SP600125, wortmannin and LY294002, respectively) the results showed that extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol-3-kinase (PI3K) were necessary for C5a-induced migration, whereas c-Jun N-terminal kinase (JNK) was nonessential. Andrographolide significantly attenuated C5a-stimulated phosphorylation of ERK1/2, and of its upstream activator, MAP kinase-ERK kinase (MEK1/2). C5a-activated ERK1/2 phosphorylation was 86+/-9% inhibited by 30 microM andrographolide. Under the same conditions, however, andrographolide failed to affect C5a-stimulated p38 MAPK and JNK phosphorylation. Andrographolide also strongly abolished C5a-stimulated Akt phosphorylation, a downstream target protein for PI3K. These results indicate that inhibition of cell migration by interfering with ERK1/2 and PI3K/Akt signal pathways may contribute to the anti-inflammatory activity of andrographolide.

Organophosphate poisonings are not uncommon, and are the leading cause of death in suicide patients in Taiwan. Acute cholinergic crisis caused by the inhibition of synaptic acetylcholinesterase is the major manifestation of organophosphate poisoning and may cause death within minutes. Delayed neurotoxicities include intermediate syndrome and delayed polyneuropathy have also been described. However, these symptoms may not characterize the complete picture of organophosphate poisoning. Among the 633 patients ever admitted to our hospital with organophosphate poisoning, three patients were found exhibiting impermanent neuromuscular dysfunction, including blepharoclonus, oculogyric crisis, intermittent dystonia, rigidity, and tremor, with two of them developing mask face, dyskinesia and akathisia later, following acute cholinergic crisis. The symptoms appeared within 4 days with the duration ranging from 25 days to 2 months. Other causes of the extrapyramidal syndrome noted on these patients have been excluded, and we consider the extrapyramidal syndrome a possible neurotoxic manifestation of organophosphate poisoning, which is transient, needs no treatment, and may be missed because of the critical condition, in a minority of patients. The mechanism remains to be identified, but may be related to the impediment of the function of acetylcholinesterase to modify nigrostriatal dopaminergic system, which is independent of hydrolyzing acetylcholine. More detailed observation for organophosphate poisoned patients and more studies for the biological functions of acetylcholinesterase including the influence on the nigrostriatal dopaminergic system are needed.

(2S)-5-methoxy-6-methylflavan-7-ol (MMF) was purified from Draconis Resina and its in vitro effects on various aspects of platelet reactivity were examined. Results indicated that MMF dose dependently inhibited aggregation of washed rabbit platelets induced by collagen, arachidonic acid, ADP, U46619 or platelet-activating factor (PAF), with IC50) values of 17.2, 49.8, 179.8, 109.6, and 189.2 microM, respectively. Concomitantly, MMF also dose dependently suppressed ATP release by platelets activated by these stimulants. The increase in intracellular free calcium ([Ca2+]i), elicited by these activating agents, was inhibited by MMF as reflected by fura-2 fluorescence measurements. However, MMF had no effects on the cyclic AMP level of platelets. In addition, MMF inhibited the arachidonic acid-induced thromboxane B2 and prostaglandin D2 formation in intact platelet suspensions or homogenized platelet lysates. This study provided evidence that MMF is an antiplatelet agent whose activity is likely related to cyclooxygenase inhibition and suppression of [Ca2+]i increase.