A novel and simple dynamic coating capillary electrophoresis method for the chiral separation and quantification of mitiglinide enantiomers using hydroxyethyl cellulose as a dynamic coating agent.

A capillary electrophoretic method for the chiral separation and quantification of mitiglinide (MTG) enantiomers is described (less than 9.5 min) with resolution value Rs = 5.25 and with excellent peak shapes after performing the dynamically coating for the bare fused capillary. The study aims to develop and validate a novel and simple method for the separation and quantification of MTG enantiomers using CE after dynamic coating the capillary wall using the hydroxyethyl cellulose (HEC) coating agent. Dynamic coating procedure of the capillary inner surface is conducted via rapid flushes using 0.1 M sodium hydroxide, water, and aqueous solution containing HEC, and hydroxypropyl-γ-cyclodextrin (HP-γ-CD). Besides buffer was used for the dynamic coating process in addition to its use as the separation medium. When the dynamic coating was used, peak symmetry was improved. A bare fused-silica capillary was used throughout the separation after being coated using HEC dissolved in the background electrolyte (BGE) of 50 mM Na2HPO4 - 1 M H3PO4 solution; pH 8.5; containing 25 25 mg mL-1 of each HP-γ-CD and HEC. The dynamic coating procedure achieved an improvement in migration time as well as peak area precision. The adsorbed coating agent showed slight interactions with MTG, providing efficient separation with outstanding durability and reproducibility at slightly alkaline conditions (pH 8.5). Acceptable validation criteria for selectivity, linearity, precision, and accuracy were also studied. The newly developed method was effectively applied to the assay of enantiomers of MTG in pharmaceutical formulations. Additionally, it was proven to have the advantages of being simple, rapid, and accurate.

Chemopotentiating effects of a novel NAD biosynthesis inhibitor, FK866, in combination with antineoplastic agents.

FK866 is a novel anticancer agent that was previously shown to interfere with NAD superset+ biosynthesis by inhibition of nicotinamide phosphoribosyltransferase and to initiate apoptosis in cancer cells. As NAD superset+ is involved in cellular DNA repair processes, the present in vitro study on THP-1 and K562 leukemia cells was conducted to investigate the cytotoxicity of FK866 combination treatment with various cytotoxic agents: the antimetabolite Ara-C, the DNA-intercalating agent daunorubicin and the alkylating compounds 1-methyl-3-nitro-1-nitrosoguanidinium (MNNG) and melphalan. Cell viability after drug exposure was assessed by propidium iodide (PI) staining. Non-cytotoxic concentrations of FK866 (10 superset-9 M or less), applied simultaneously or 24 hours before adding cytotoxic agents, caused a depletion in the intracellular NAD superset+ and--to a lesser extent-- NADH levels in THP-1 cells. After 48 and 72 hours treatment with daunorubicin and Ara-C, respectively, increased cell death was observed in THP-1 cells that were pretreated with FK866, as compared to cells exposed to antineoplastic drugs alone. However, this effect was transient, and there was no difference in cell survival after 72 hours incubation with daunorubicin or 96 hours with Ara-C. - Non-toxic concentrations of FK866 added 8, 16, or 24 hours before starting treatment with the PARP-activating agent MNNG synergistically decreased intracellular NAD superset+ contents, and increased MNNG-induced cytotoxicity both in THP-1 and K562 cells for at least 72 hours. This effect was less pronounced when FK866 was used in combination with another alkylating agent, melphalan. The PARP inhibitor 3-aminobenzamide delayed MNNG-induced cytotoxicity by 24 hours both in cells that were pretreated with FK866 and in non-pretreated cells. 48 hours later, the protective effect of 3-aminobenzamide could no longer be observed, but FK866-pretreated cells retained increased sensitivity to MNNG. - In conclusion, the chemosensitizing effect of FK866 on cell death induced by antineoplastic drugs was particularly obvious in combination with substances like MNNG that cause NAD superset+ depletion per se. It was less pronounced and only transiently measurable in combination with daunorubicin, Ara-C, and melphalan, respectively. These results may indicate different levels of DNA damage implicated in the action of the cytotoxic agents used.

Key role for scavenger receptor B-I in the integrative physiology of host defense during bacterial pneumonia.

Scavenger receptor B-I (SR-BI) is a multirecognition receptor that regulates cholesterol trafficking and cardiovascular inflammation. Although it is expressed by neutrophils (PMNs) and lung-resident cells, no role for SR-BI has been defined in pulmonary immunity. Herein, we report that, compared with SR-BI(+/+) counterparts, SR-BI(-/-) mice suffer markedly increased mortality during bacterial pneumonia associated with higher bacterial burden in the lung and blood, deficient induction of the stress glucocorticoid corticosterone, higher serum cytokines, and increased organ injury. SR-BI(-/-) mice had significantly increased PMN recruitment and cytokine production in the infected airspace. This was associated with defective hematopoietic cell-dependent clearance of lipopolysaccharide from the airspace and increased cytokine production by SR-BI(-/-) macrophages. Corticosterone replacement normalized alveolar neutrophilia but not alveolar cytokines, bacterial burden, or mortality, suggesting that adrenal insufficiency derepresses PMN trafficking to the SR-BI(-/-) airway in a cytokine-independent manner. Despite enhanced alveolar neutrophilia, SR-BI(-/-) mice displayed impaired phagocytic killing. Bone marrow chimeras revealed this defect to be independent of the dyslipidemia and adrenal insufficiency of SR-BI(-/-) mice. During infection, SR-BI(-/-) PMNs displayed deficient oxidant production and CD11b externalization, and increased surface L-selectin, suggesting defective activation. Taken together, SR-BI coordinates several steps in the integrated neutrophilic host defense response to pneumonia.

The antithrombotic effect of aurin tricarboxylic acid in the guinea pig is not solely due to its interaction with the von Willebrand factor-GPIb axis.

Commercial aurin tricarboxylic acid (ATA) has been reported to interfere specifically with von Willebrand factor-glycoprotein Ib (vWF-GPIb) axis. This study was designed to explore the antithrombotic effects of ATA by examining its effects on guinea pig platelet function in vitro, in vivo and ex vivo. In vitro, addition of various concentrations of ATA to platelet-rich guinea pig plasma totally inhibited ristocetin-induced platelet aggregation, as expected. Unexpectedly, however, ATA similarly inhibited the aggregation induced by ADP, PAF, collagen, I-BOP (a thromboxane A2/prostaglandin H2 analogue) and arachidonic acid. In vivo, the antithrombotic action of ATA was assessed in a model of acute platelet-dependent guinea pig mesenteric artery thrombosis triggered by laser-induced intimal injury. As the thrombotic response of arteries to such injury is a spontaneous cyclic recurrent process, 5 arteries in each animal were consecutively studied for 15 min each after i.v. bolus injection of 5, 7.5 or 10 mg/kg of ATA, which reduced the number of recurrent thrombi per artery in a dose-dependent manner. The highest dose of 10 mg/kg induced maximal inhibition of thrombus formation (72%, p < 0.001) 5 min after injection. Ex vivo, platelet aggregation was assessed in blood samples taken before and after i.v. bolus injection of 10 or 15 mg/kg ATA. Ten mg/kg significantly inhibited collagen-induced aggregation, and 15 mg/kg, the aggregation induced by ristocetin, ADP, PAF, collagen, I-BOP and arachidonic acid. The results of the in vivo studies confirmed that ATA is an effective antithrombotic agent. In the in vitro and ex vivo studies, ristocetin-induced platelet aggregation confirmed that ATA interacts with the vWF-GPIb axis, and suggests that the final common pathway of the aggregation induced by other agents tested consists of fibrinogen binding to the platelet GPIIb/IIIa receptor. We conclude that ATA interferes with vWF binding to GPIb, that it may interact with fibrinogen binding to GPIIb/IIIa, and that it might possess potent antithrombotic properties in platelet-mediated thrombosis.

Antithrombotic effect of a recombinant von Willebrand factor, VCL, on nitrogen laser-induced thrombus formation in guinea pig mesenteric arteries.

To assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded. In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i.v. injection inhibits risocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time.(ABSTRACT TRUNCATED AT 250 WORDS)

[What are the criteria for choosing a model for evaluating arterial antithrombotic therapy?]. / Sur quels critères choisir un modèle pour évaluer une thérapeutique antithrombotique artérielle?

The use of models of experimental arterial thrombosis both in vivo and ex vivo in animals and ex vivo in humans is an obligatory step to the understanding of mechanisms involved in thrombogenesis as well as in the evaluation of anti-thrombotic therapeutics. Arterial thrombogenesis is a complex phenomenon which involves multiple systems, mechanisms and parameters. Therefore studies of thrombogenesis from a pathological as well as a therapeutic point are necessary for understanding this problem in its entirety. For these reasons, it is necessary to use models as representative as possible of the human pathological condition. Besides these theoretical requirements, practical needs have also to be fulfilled (accessibility of the models, adaptation to the type of the technique to different animal model and/or of the size of the animal to the amount of molecule available, cost ...) which necessary lead to some promises. In this review we have tried to underline the criteria for the choice, characteristics, advantages and disadvantages of the major models commonly accepted and used, in such a form that the reader who may not be an expert in the field would be led either to choose a particular model for a specific purpose or to appreciate a paper or a report based on an experimental model of arterial thrombosis. In vitro models of arterial thrombosis are so far removed from reality and due to their nature can generate so much artifacts thus we have omitted their discussion from this paper.

Which experimental model to choose to study arterial thrombosis and evaluate potentially useful therapeutics?

The use of experimental models of arterial thrombosis both in vivo and ex vivo in animals and ex vivo in humans is an obligatory step for the understanding of mechanisms involved in thrombogenesis as well as in the evaluation of anti-thrombotic therapeutics. Arterial thrombogenesis is a complex phenomenon which involves multiple systems, mechanisms and parameters. Therefore studies of thrombogenesis from a pathological as well as a therapeutic point are necessary for understanding this problem in its entirety. For these reasons, it is necessary to use models as representative as possible of the human pathological condition. Besides these theoretical requirements, practical needs have also to be fulfilled (accessibility of the models, adaptation to the type of the technique to different animal model and/or of the size of the animal to the amount of molecule available, cost...) which necessarily lead to some compromises. In this review we have tried to underline the criteria for the choice, characteristics, advantages and disadvantages of the major models commonly accepted and used, in such a form that the reader who may not be an expert in the field would be led either to a choice of a particular model for a specific purpose or to appreciate a paper or a report based on an experimental model of arterial thrombosis. In vitro models of arterial thrombosis are so far removed from reality and due to their nature can generate so much artifacts that we have omitted their discussion from this paper.