Endogenous serum erythropoietin and no-reflow in patients with ST-elevation myocardial infarction.

BACKGROUND: In models of acute ischaemia, erythropoietin (EPO) administration has been found to attenuate vascular injury largely through reduced apoptosis, suppressed inflammation and increased nitric oxide availability. We studied the association between circulating endogenous EPO and no-reflow in patients with first ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). METHODS: Blood sampling was performed before PPCI. Consecutive patients with (n = 24) or without (n = 24) evidence of angiographic no-reflow after PPCI were enrolled. Angiographic no-reflow was defined as Thrombolysis in Myocardial Infarction (TIMI) flow ≤ 2 or as TIMI flow = 3 but with myocardial blush grade < 2. We also assessed electrocardiographic (ECG) no-reflow as ≤ 50% resolution of maximal ST elevation 60 min after PPCI. RESULTS: Baseline characteristics did not correlate significantly with EPO concentrations. In contrast, both angiographic and ECG no-reflow correlated with lower EPO levels at univariate analysis [median (interquartile): 4·2 (0·6-9·5) vs. 12·2 (5·2-20·3) mIU mL(-1), P = 0·001, and 4·0 (0·6-7·1) vs. 9·3 (1·0-12·6) mIU mL(-1), P = 0·01, respectively]. At multivariable analysis, decreasing EPO tertiles and left anterior descending as the infarct-related artery were the only factors that predicted both angiographic and ECG no-reflow (P = 0·017 and P = 0·02 for EPO; P < 0·005 and P > 0·05 for left anterior descending artery, respectively). CONCLUSIONS: We found an independent, graded, inverse relation between endogenous EPO levels and angiographic and ECG no-reflow following PPCI. In animal models of ischaemia, EPO has been found to be protective. In humans, endogenous EPO may contribute to offset the mechanisms responsible for no-reflow.

Effect of cholesterol on the formation and hydration behavior of solid-supported niosomal membranes.

The effect of cholesterol on the formation and hydration behavior of solid-supported polysorbate 20 (Tween 20)/cholesterol self-assemblies was investigated by means of in situ energy-dispersive X-ray diffraction in a wide range of relative humidity (0.4 < RH < 1). At low hydration, Tween 20 and cholesterol were found to demix, with the latter molecules forming crystallites with a pseudobilayer structure (d approximately = 34 A). Water adsorption promoted the progressive solubilization of cholesterol crystallites. When in the presence of enough cholesterol, the formation of niosomal bilayer membranes rich in Tween 20 occurred (RH approximately = 0.985). Upon further hydration, two distinct regimes associated with remarkable changes in the niosomal membrane structure were identified. In the first regime (0.985 < RH < 0.988), the swelling of the lamellar d spacing was due to the enlargement of the membrane thickness. In the second regime, the structure of Tween 20/cholesterol membranes was quite insensitive to hydration, and the thickness of the intermembrane water layer increased substantially. Remarkably, the curve of the calculated number of waters per surfactant molecule showed a distinct break at RH approximately 0.988, suggesting that the observed structural change in niosomal membranes was most likely due to the completion of the filling of the Tween 20 hydration shell. At full hydration, niosomal membranes exhibited the same lamellar d spacing of niosomes vesicles in aqueous solution. The process completely reversed upon dehydration.

Potential dopamine prodrug-loaded liposomes: preparation, characterization, and in vitro stability studies.

Dopamine delivery to the central nervous system (CNS) undergoes the permeability limitations of the blood-brain barrier (BBB). Condensation of dopamine with neutral amino acids could afford potential prodrugs able to interact with the BBB endogenous transporters and easily enter the brain. To improve the bioavailability of the dopamine prodrug, 2-amino-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (DOPH), it was encapsulated in unilamellar liposomes of dimiristoylphosphatidylcholine (DMPC) and cholesterol. Vesicles were characterized by dynamic light scattering in order to evaluate their dimensions and vesicle stability, by zeta-potential measurements, by means of electronic microscopy after freeze-fracture and differential scanning calorimetry. The influence of vesicle composition on DOPH chemical and enzymatic stability was also evaluated. The formulated liposome suspensions were found to be stable, monodisperse systems with a negative zeta potential. From the obtained results, it is possible to conclude that, in studied samples, DOPH inclusion in liposomes offers the possibility of preventing photodegradation and of enhancing in vitro plasma stability. These studies suggest the potential of these formulations as a method to prevent DOPH chemical degradation and enzymatic metabolism.

Asymmetric dimethylarginine and impaired cardiovascular healing.

Asymmetric dimethylarginine (ADMA) typically accumulates in the plasma of patients with chronic renal failure. Moreover, its plasma levels are raised in the presence of virtually all of the traditional cardiovascular risk factors. ADMA inhibits the three isoforms of nitric oxide (NO) synthase, thereby blunting the known cardioprotective effects of NO. Through its NO inhibitor actions, ADMA also exerts pro-apoptotic effects and suppresses progenitor cell mobilization, differentiation and function. Among patients with ischemic heart disease, low progenitor cell bioavailability and kidney dysfunction are emerging as strong predictors of death and recurrent cardiovascular events. We propose that patients with ischemic heart disease, kidney dysfunction, and high risk factor burden exhibit adverse cardiovascular outcomes, at least in part, through ADMA-mediated NO depression, enhanced apoptotic signalling, and reduced progenitor cell bioavailability, with consequent blunting of cardiovascular healing. Further research into the mechanisms that regulate the NO/ADMA balance may advance our understanding of cardiovascular diseases.

Update on phase II studies of erythropoietin in acute myocardial infarction. Rationale and design of Exogenous erythroPoietin in Acute Myocardial Infarction: New Outlook aNd Dose Association Study (EPAMINONDAS).

Erythropoietin (Epo) is a hematopoietic hormone produced mainly by the kidneys in response to hypoxia. Recent acquisitions in the fields of hematology, neurology, cardiology, and experimental medicine show cytoprotective, angiogenetic and antiinflammatory effects of Epo. Exogenous erythroPoietin in Acute Myocardial Infarction: New Outlook aNd Dose Association Study (EPAMINONDAS, EudraCTno. 200500485386) is one of four ongoing randomized controlled trials, each testing the effects of Epo in >or=100 patients with STEMI. EPAMINONDAS is a multicenter, prospective, double-blind, placebo-controlled, dose-finding study assessing intravenous moderate doses of human recombinant Epo (epoietin-alpha, 100 or 200 IU/kg/die) versus placebo, given on the first 3 days, in 102 patients with first ST-segment elevation myocardial infarction. Initial dosing is within 12 h of primary percutaneous coronary revascularization. The primary endpoint is infarct size, quantified by CK-MB time-concentration curve, left ventricular wall motion score index, and pattern of contrast-enhanced magnetic resonance imaging. Secondary endpoints are ischemic recurrences, ventricular remodelling, and safety events, assessed in-hospital and at 12 months' follow-up. The results of current phase II studies will help define the safety/efficacy profile of Epo for patients with STEMI.

Span and Tween neutral and pH-sensitive vesicles: characterization and in vitro skin permeation.

The aim of this work was the preparation, characterization, and comparison of novel pH-sensitive nonphospholipid vesicles (niosomes) from two nonionic surfactants, with different hydrophilic-lipophilic balance values (Tween20-TW20 = 16.7 and Span60-SP60 = 4.7). Surfactants were mixed with cholesterol (CHOL) and its derivative, cholesteryl hemisuccinate (CHEMS), as a pH-sensitive molecule. Vesicles were characterized by dynamic light scattering, in order to evaluate their dimensions and vesicle stability, by zeta-potential measurements and by means of electronic microscopy after freeze-fracture. Ibuprofen (IBU) was used as the model drug, and high-performance liquid chromatography analyses were performed to evaluate drug-entrapment efficiency and release in a neutral, acidic environment. The influence of the vesicle composition on skin accumulation and transdermal permeation of IBU across excised hairless rat skin was investigated by using vertical Gummer diffusion cells. When niosomes with SP60 and CHEMS were prepared, there was a statistically significant increase of skin permeation of IBU, while TW20 niosomes did not show statistically significant differences in P(app) values without the influence of the vesicle size and charge.

[Body mass index and cardiovascular events: the "obesity paradox"]. / Recenti acquisizioni sui rapporti tra indice di massa corporea e danno cardiovascolare: il "paradosso dell'obesità".

Excessive body mass among healthy subjects carries an increased risk of subsequent cardiovascular events. Excess weight implies the presence of white, viscero-abdominal fat, that promotes insulin-resistance, is infiltrated by macrophages, and is less differentiated compared to subcutaneous or brown fat. Conversely, among patients with cardiovascular disease, slim patients have a greater risk of recurrent atherothrombotic events than fatter patients ("obesity paradox"). Lean patients with cardiovascular disease, on average, have more comorbidities and haemorrhagic complications than their heavier counteparts, and probably they conceal predisposing factors that are still unknown and therefore difficult to treat.

L-dopa- and dopamine-(R)-alpha-lipoic acid conjugates as multifunctional codrugs with antioxidant properties.

A series of multifunctional codrugs (1-4), obtained by joining L-Dopa (LD) and dopamine (DA) with (R)-alpha-lipoic acid (LA), was synthesized and evaluated as potential codrugs with antioxidant and iron-chelating properties. These multifunctional molecules were synthesized to overcome the pro-oxidant effect associated with LD therapy. The physicochemical properties, together with the chemical and enzymatic stabilities of synthesized compounds, were evaluated in order to determine both their stability in aqueous medium and their sensitivity in undergoing enzymatic cleavage by rat and human plasma to regenerate the original drugs. The new compounds were tested for their radical scavenging activities, using a test involving the Fe (II)-H2O2-induced degradation of deoxyribose, and to evaluate peripheral markers of oxidative stress such as plasmatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the plasma. Furthermore, we showed the central effects of compounds 1 and 2 on spontaneous locomotor activity of rats in comparison with LD-treated animals. From the results obtained, compounds 1-4 appeared stable at a pH of 1.3 and in 7.4 buffered solution; in 80% human plasma they were turned into DA and LD. Codrugs 1-4 possess good lipophilicity (log P > 2 for all tested compounds). Compounds 1 and 2 seem to protect partially against the oxidative stress deriving from auto-oxidation and MAO-mediated metabolism of DA. This evidence, together with the "in vivo" dopaminergic activity and a sustained release of the parent drug in human plasma, allowed us to point out the potential advantages of using 1 and 2 rather than LD in treating pathologies such as Parkinson's disease, characterized by an evident decrease of DA concentration in the brain.

Maleic- and fumaric-diamides of (O,O-diacetyl)-L-Dopa-methylester as anti-Parkinson prodrugs in liposomal formulation.

The maleic and fumaric diamides preparation of (O,O-diacetyl)-L-Dopa-methylester [(+)-4, (+)-5] are reported; they were synthesized in order to attenuate marked fluctuations of L-DOPA (LD) plasma levels and to overcome the problem of low bioavailability of LD. The new compounds were characterized evaluating solubility, chemical stability, apparent partition coefficient (log P) and comparing neostriatum dopamine (DA) levels in freely moving rats after i.p. administration of prodrugs [(+)-4, (+)-5] with prodrugs in liposomal formulations [(+)-4Lip, (+)-5Lip]. All the new compounds showed chemical stability in aqueous buffer solutions (pH 1.3 and 7.4). A relatively slow release of LD in human plasma was observed. Among the studied products, prodrug was able to induce sustained delivery of DA in rat striatal dialysate with respect to equimolar i.p admistration of LD. Furthermore, neostriatum DA concentration after administration of the synthesized prodrugs vs. prodrugs in liposomal formulations was compared (+)-4Lip, (+)-5Lip). The results suggest that cis dimeric prodrug (+)-4 and (+)-4Lip can improve the release of DA in rat brain and demonstrate the potential of these formulations for controlled delivery of antiparkinson agents.

Evaluation of rat striatal L-dopa and DA concentration after intraperitoneal administration of L-dopa prodrugs in liposomal formulations.

Parkinson's disease is a neurodegenerative disease and its symptoms are relieved by administration of L-dopa (LD), which is converted by neuronal aromatic L-aminoacid decarboxylase (AADC), restoring dopamine (DA) levels in surviving neurons. In order to minimize unfavourable side effects, we studied new dimeric LD derivatives, as potential prodrugs for Parkinson's therapeutic treatment. To improve the bioavailability of the synthesized prodrugs, they were encapsulated in unilamellar liposomes of dimiristoylphosphatidylcholine (DMPC) and cholesterol (CHOL). In vivo microdialysis was used to monitor the striatal LD and DA concentrations after i.p. administration of new delivery systems. Bioavailability evaluation was performed by means of the HPLC-EC method. The striatal levels of LD and DA were remarkably elevated after i.p. administration of liposomal formulation of prodrug (+)-1b ([(O,O-diacetyl)-L-dopa-methylester]-succinyldiamide). This formulation showed about 2.5-fold increase in the basal levels of DA in dialysate rat striatum, suggesting that liposomal formulation of (+)-1b significantly increases LD and DA concentrations with respect to equimolar administration of LD itself or free prodrug (+)-1b.

Structural and rheological characterization of Scleroglucan/borax hydrogel for drug delivery.

The polysaccharide Scleroglucan, one of the most rigid polymers found in nature, can form a chemical/physical gel, in the presence of borax. The obtained hydrogel was loaded with three different model molecules (Theophylline, Vitamin B12 and Myoglobin) and then, after freeze-drying, was used as a matrix for tablets. The release profiles of the substances from the dosage forms were evaluated; the matrix appeared capable to modulate the diffusion of the chosen molecules, and different diffusion rates were observed, according to the different radii of the tested molecules. Interestingly, in the dissolution medium the matrix undergoes an anisotropic swelling taking place only in the axial direction, while a negligible radial variation occurs. The water uptake of the matrix occurs according to a Fickian process. Samples at two different polymer concentrations (0.7 and 2.3%, w/v) were characterized in terms of rheological and mechanical parameters and the properties were interpreted in terms of the molecular structure obtained by conformational analysis. The flow curves acquired in the viscoelasticity interval, show the effect of the borate ion in improving the resistance of the gel in comparison to the polymer alone. The evaluation of the moduli indicates that the system is viscoelastic, with an appreciable liquid component that increases as the polymer concentration decreases. Also the cohesion of the gel is higher in comparison to the Scleroglucan and is strongly dependent on temperature. The combination of experimental and theoretical conformational analysis approaches, allowed us to propose a model for the structure of the macromolecular network and to give an explanation to the anomalous swelling that was observed. It came out that the polymer can built up a channel structure, mediated via borax ion interaction, that can accommodate guest molecules of different size.

Erythropoietin in heart and vessels: focus on transcription and signalling pathways.

Erythropoietin (Epo) is synthesized mainly under hypoxic conditions by renal and extrarenal tissues, including liver, spleen, brain, lung, bone marrow, and reproductive organs. Hypoxia abrogates the degradation of hypoxia-inducible factors (HIF)-1 and -2, that can then bind to the hypoxia response element within the Epo gene, activating its transcription. Receptors for Epo are expressed on cells known to synthesize Epo, but also on cardiomyocytes, cardiac fibroblasts, and endothelial, retinal, gastric, prostate and vascular smooth muscle cells. Epo-receptor binding triggers at least three intracellular signalling cascades: (1) janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5); (2) phosphatidylinositol-3 kinase (PI3K)/Akt, and (3) RAS/mitogen-activated protein kinase (MAPK). Epo also enhances nitric oxide (NO) bioavailability through endothelial NO synthase transcription and activation, and exerts antiapoptotic actions through Bcl-2 and Bcl-XL. NO is a powerful vasodilator, insulin-sensitizer, inhibitor of atherothrombosis and apoptosis, and essential for progenitor mobilization. This article is a concise review of recent advances regarding the molecular and cardiovascular effects of Epo.

Physicochemical and biological study of selected hydrophobic polyethylenimine-based polycationic liposomes and their complexes with DNA.

Non-viral gene therapy is based on the development of efficient and safe gene carrier systems able to transfer DNA into cells. Polyethylenimine (PEI), the most promising non-viral vector, with its high cationic-charge-density potential is able (1) to compact DNA in complexes (polyplexes) smaller than those formed by liposomes (lipoplexes) and (2) to destabilize the endosomal membrane by a 'proton sponge' effect. Several PEI's hydrophobic modifications were reported in the last several years but in some cases a reduced transfection efficiency was observed. The mechanism underlying this phenomenon is not well understood so far. In order to extensively investigate these mechanisms, we reported a physicochemical and biological study of selected hydrophobic PEI's derivatives grafted with chains of different length and percentages of substitution able to form vesicles (polycationic liposomes) and to bind DNA. Their properties were studied by means of dynamic light scattering, freeze-fracture microscopy, potentiometric titrations, gel retardation assays, polyanion exchange reactions, toxicity assays, in vitro transfections, and fluorescence microscopy. Our results indicate that even if polyplexes are able to pass through the cellular membrane, the stability of PEI's hydrophobic polyplexes likely explain their different transfection efficiency in vitro.