Dietary sodium and nocturnal blood pressure dipping in normotensive men and women.

Impaired nocturnal blood pressure (BP) dipping (i.e., <10% decline in nocturnal BP) is associated with an increased risk of cerebrovascular and cardiovascular diseases. Excess sodium has been shown to impair BP regulation and increase cardiovascular disease risk, yet few studies have assessed the influence of dietary sodium on nocturnal dipping in normotensive adults. The purpose of this study was to determine the effects of dietary sodium on BP dipping in normotensive men and women. Eighty healthy normotensive adults participated in a controlled feeding study (men: n=39, 34±2 years; women: n=41, 41±2 years). Participants consumed a standardized run-in 100 mmol sodium per day diet for 7 days, followed by 7 days of low-sodium (LS; 20 mmol per day) and high-sodium (HS; 300 mmol per day) diets in random order. On the final day of each diet, subjects wore a 24 h ambulatory BP monitor, collected a 24 h urine sample and provided a blood sample. During the run-in diet, 24 h urinary sodium excretion was 79.4±5.1 mmol per 24 h in men and 85.3±5.5 mmol per 24 h in women (P>0.05). Systolic BP dipping was not different between men (11.4±1.0%) and women (11.2±0.9%); (P>0.05). During the HS diet, 24 h urinary sodium excretion increased compared with the LS diet in men (LS=31.7±4.6 mmol per 24 h vs HS=235.0±13.9 mmol per 24 h, P<0.01) and women (LS=25.8±2.2 mmol per 24 h vs HS=234.7±13.8 mmol per 24 h, P<0.01). Despite this large increase in sodium intake and excretion, systolic BP dipping was not blunted in men (LS=8.9±1.0% vs HS=9.4±1.2%, P>0.05) or women (LS=10.3±0.8% vs HS=10.5±0.8%, P>0.05). Among normotensive men and women, HS does not blunt nocturnal BP dipping.

Solid lipid microparticles for the stability enhancement of the polar drug N6-cyclopentyladenosine.

The objective of this study was to prepare solid lipid microparticles (SLMs) loaded with the polar adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA). The microparticles were produced by the conventional hot emulsion technique, using different lipidic carriers (tristearin, glyceryl behenate and stearic acid) and hydrogenated phosphatidylcholine as the surfactant. The controlled release of CPA was achieved only with stearic acid microparticles. This phenomenon has been attributed to direct acid-base interactions between the basic nitrogen atoms of CPA and stearic acid. These SLMs were characterized by release studies, scanning electron microscopy and powder X-ray diffraction analyses. The obtained particles showed proper features in terms of morphology and size distribution (3.2-10.3microm), with a drug loading of 0.15+/-0.04%. The influence of the SLMs carrier system on CPA stability was investigated in vitro using human whole blood. The degradation kinetic of microparticle-entrapped CPA was significantly lower from that measured for the free CPA. The overall results indicate that it was possible to achieve the encapsulation and controlled release of a polar drug, such as CPA, within a lipid matrix without resorting to the complex methods generally used for the preparation of these systems.

Selection of reference genes for normalisation of specific gene quantification data of Aspergillus niger.

Aspergillus niger is a widely used expression host for homologous and heterologous protein production in biotechnological processes. In order to increase product yields, a thorough optimisation of these cultivation processes is necessary. Considering mRNA as the key molecule, which transports the genetic information between DNA and protein production side, the quantification of product specific gene expression provides useful information about product formation already on the level of transcription. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a powerful tool to obtain data about gene transcription. However, using this technique the choice of an appropriate reference system is a crucial aspect to provide optimal data normalisation. A prominent approach is the use of so called housekeeping genes as internal references. However, validation of the usability of these reference genes is the fundamental step before starting with qRT-PCR experiments. Adequate reference genes for A. niger have not been published so far. Therefore, 10 possible candidate genes from different functional classes were selected and their applicability as internal references validated. Transcript levels of these genes were compared in sets of 9, 41 and 19 samples from diverse cultivations of A. niger. Under the chosen experimental conditions, the genes act, sarA and cox5 have been identified as genes with the most stable gene expression. The three reference genes were used to normalise qRT-PCR data for glaA gene expression which showed a high correlation with glucoamylase production in continuous cultivations.

Expression and characterization of vitamin C transporter in the human trophoblast cell line HTR-8/SVneo: effect of steroids, flavonoids and NSAIDs.

Vitamin C plays an important role in embryogenesis and fetal growth as well as in the progression of pregnancy and delivery. Therefore, it is important to understand the mechanism that mediates its transport to the fetus as well as the possible influences by endogenous and exogenous substances on its placental uptake. The aim of this study was to investigate placental sodium-dependent vitamin C transporters (SVCT) 1 and 2. By means of RT-PCR, we found that SVCT2, but not SVCT1, mRNA is expressed in human trophoblast cell line HTR-8/SVneo. Our method was able to confirm SVCT2 mRNA expression in human first-trimester chorionic villi but not in term placental tissue. Cell line kinetic studies of [(14)C] ascorbic acid (AA) uptake indicated a one-site model and a saturable process. Fetal bovine serum (FBS) and epidermal growth factor (EGF) do not influence the transport properties, although they significantly increase the expression of SVCT2. Steroid hormones (17beta-estradiol, progesterone and cortisol), flavonoids (genistein and quercetin) and non-steroidal anti-inflammatory drugs (NSAIDs) (indomethacin and diclofenac) inhibit [(14)C]AA uptake in a dose-dependent and non-competitive manner. On the contrary, the process is not influenced by aspirin. Our study suggests the use of HTR-8/SVneo cells as a suitable model for trophoblast vitamin C transport investigation.

Molecular mechanism involved in the transport of a prodrug dopamine glycosyl conjugate.

We have previously demonstrated that dopamine conjugation to glucose allows it to induce therapeutic effects against Parkinson's disease after intravenous administration. In this paper we demonstrate that, unlike dopamine, the prodrug glu-dopamine is a transportable substrate of glucose transporters. Towards this, the effect of glucose-conjugation on the affinity and uptake of dopamine have been assessed in vitro, using human retinal pigment epithelium (HRPE) cells. Glucose transporter-mediated uptake was measured using [(3)H]3-O-methylglucose ([(3)H]3-O-MG) as the tracer. The uptake was found to be rapid and hyperbolically related to its concentrations (K(t)=7.8+/-1.2mM and V(max)=54+/-2 nmol/min mg protein). Inhibition experiments showed that dopamine was able to interact with glucose carriers only when conjugated to glucose (IC(50)=2.6+/-0.6mM). HPLC analysis of HRPE cell extracts showed that both dopamine and the prodrug permeate the cell, but only the uptake of the prodrug is inhibitable by glucose. This confirms that glucose transporters mediate the transport of the prodrug glu-dopamine, but not of dopamine. HRPE cells is therefore proposed as a promising model for in vitro studies involving the glucose transporter-mediated transport of drugs and their conjugates.

Nuclear retinoic acid receptor beta as a tool in chemoprevention trials.

Retinoic acid (RA) and its natural and synthetic derivatives (retinoids) are important dietary factors which regulate cellular differentiation and growth, so that they are thought to be particularly effective at preventing the development of several tumours. They play this role as ligands of the RAR and RXR nuclear retinoic acid receptors, including the RA receptor isoforms alpha, beta, and gamma. These ligand-activated nuclear receptors induce the transcription of target genes by binding to RA-responsive elements in the promoter regions. Among these target genes, the RARbeta gene is of great interest, being able to encode a potential tumour suppressor. It should be emphasized that most breast carcinomas and breast cancer cell lines show loss or down-regulation of RARbeta receptor expression, whereas RARalpha and gamma, as well as retinoid X receptors, appear to be variably expressed in both normal and tumour cells. It is also interesting to note that basal and RA-induced RARbeta mRNA levels tend to increase with senescence of normal cells. This information provides further support for the hypothesis that genetic events involved in cellular senescence may also play a significant role in tumour suppression in humans. The aim of this review is to clarify whether expression of RARbeta could be modulated by chemopreventive intervention and may therefore serve as an intermediate biomarker in chemoprevention trials for some cancers.

Cationic liposomes as potential carriers for ocular administration of peptides with anti-herpetic activity.

In the present study the preparation, characterization and activity of cationic liposomes containing the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) or two related polylysine rich peptides, namely DTK1 and DTK2, were described. The immunotherapeutic potential of these HSV antigens containing liposomes was examined with a rabbit ocular model of HSV-1 infection. Our study indicates that the liposomes (i) are able to encapsulate quantitatively gB1s and around 30% the DTK peptides, (ii) are characterized by dimensions compatible with ocular applications and (iii) can release the peptide comparably to the free solution. In addition, neutralization studies demonstrated that an anti-DTK specific polyclonal antiserum can inhibit HSV-1 infection, indicating that such peptides could be a good immunogen/antigen in an anti-HSV vaccine formulation. Although the vaccination protocol did not induce protection against the eye disease, a significative protection against a lethal ocular challenge was detectable together with the absence of reactivation episodes from latency on the survived animals. In this respect, the use of cationic liposomes coupled to gB1s and DTK peptides, as a local ocular vaccine, could represent an interesting approach in order to obtain a possible efficacy in protecting animals against a subsequent HSV-1 ocular challenge.

Nanoparticle formulation may affect the stabilization of an antiischemic prodrug.

The prodrug 5'-octanoyl-CPA (Oct-CPA) of the antiischemic N6-cyclopentyladenosine (CPA) has been encapsulated by nanoprecipitation in poly(lactic acid) nanoparticles, which have been recovered by gel-filtration, ultra-centrifugation or dialysis. We have analysed how different surfactants and purification methods can influence the nanoparticle characteristics. The particle sizes have been obtained by scanning electron microscope, whereas a SdFFF system was employed to detect their distributions. The Oct-CPA release from nanoparticles and stabilities in human blood of free and encapsulated prodrug have been analysed by HPLC techniques. The effects of nanoparticles on CPA interaction toward adenosine A1 receptor (its action site) have been analysed using radiolabelled drugs. The smallest nanoparticles and the best degree of homogeneity have been obtained using sodium cholate; the best recovery has been achieved by dialysis, whereas gel-filtration and ultra-centrifugation have induced the greatest removal of surfactants. The release of Oct-CPA was better controlled from the nanoparticles obtained using Pluronic F68 and purified by gel-filtration or ultra-centrifugation. Similarly, these nanoparticles better increased the stability of the prodrug in human blood. In particular, the nanoparticles purified by ultra-centrifugation induced a strong stability to a fraction of the encapsulated Oct-CPA. Any interference by unloaded nanoparticles has been registered for CPA-adenosine A1 receptor interaction.

Caffeine intake induces an alteration in human neutrophil A2A adenosine receptors.

Caffeine is the most widely used drug in the world and acts mainly through antagonism of the effects mediated by the adenosine receptor subtypes A1, A2A, A2B and A3. We determined whether repeated caffeine administration at different doses and for different periods of time (400 or 600 mg/day for 1 week and 400 mg/day for 2 weeks) alters human neutrophil A2A adenosine receptor density and function. Saturation binding assays showed an increase in affinity (K(D)) and density (B(max)) of A2A adenosine receptors after caffeine intake. These changes were accompanied by increases in cAMP accumulation and decreases in superoxide anion production after stimulation of the A2A receptor subtype using the agonist 5'-N-ethylcarboxamidoadenosine (NECA). Binding and functional changes of A2A receptors returned to baseline after 48 h of caffeine withdrawal. The findings are consistent with a potential anti-inflammatory effect of caffeine mediated by neutrophil A2A receptors.

Vitamin C and 6-amino-vitamin C conjugates of diclofenac: synthesis and evaluation.

Diclofenac (Diclo), its ascorbic acid (AA) or 6-amino-AA (AA-NH2) pro-drugs (AA-Diclo or AA-NH-Diclo) were prepared and evaluated on human retinal pigment epithelium (HRPE) cells to investigate their ability to interact with the vitamin C transporter SVCT2 and their cellular uptake. Furthermore, stabilities in physiological fluids of these compounds were investigated. For kinetic experiments, AA-Diclo was incubated in Tris-HCl buffer, human plasma or whole blood. The extracted samples were analysed by HPLC. AA-Diclo was hydrolysed following first order kinetics in buffer, plasma (t1/2 about 10 h) and whole blood (t1/2 about 3.5 h). Transport and inhibition assays were performed by adding [14C]AA and the above-mentioned unlabelled compounds to plated HRPE cells. Intracellular accumulation was measured incubating HRPE cells with increasing concentrations of unlabelled compounds, following by HPLC analysis. Diclo resulted as a non-competitive inhibitor of AA-transport, showing a Na+-dependent and ascorbate-independent uptake. AA-Diclo behaved as a competitive inhibitor, but it was not transported into cells, whereas its analogue AA-NH-Diclo showed a decreased inhibitory activity. Stability studies suggest AA-Diclo as a potential candidate to enhance the Diclo short half life in vivo. The discovery of a Na+-dependent transporter for Diclo on HRPE cells opens new perspectives for targeting diclofenac into the brain.

Studies on human neutrophil biological functions by means of formyl-peptide receptor agonists and antagonists.

Phagocytes are activated by several extracellular signals, including formyl-peptides derived from bacterial proteins or disrupted cells. The most intensely studied member of the formylpeptide family is the synthetic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), whose specific receptors have been identified on neutrophil plasma membrane and subsequently cloned. The fMLP-receptor interaction activates multiple transduction pathways responsible for various neutrophil functions such as adhesion, chemotaxis, exocytosis of secretory granules and superoxide anion production, which represent the physiological response to bacterial infection and tissue damage. An unresolved question is whether signaling requirements are identical or specific for each physiological function. The development of fMLP receptor agonists and antagonists has led to an improvement of our knowledge about the above issue. Of particular interest is the possibility that receptorial antagonists, able to transiently inhibit neutrophil responses to formylpeptides, could be therapeutic agents in the treatment of inflammation-related diseases. Aim of this review is, i) to summarise the current understanding of the series of events that begins at the level of formylpeptide-receptor interaction and is responsible for the activation of transduction pathways, which finally determine neutrophil response; ii) to define the state of art regarding the synthesis as well as the biological actions of fMLP receptor agonists and antagonists.

Poly(lactic acid) microspheres for the sustained release of antiischemic agents.

We report a preliminary study evaluating the encapsulation modalities in microparticles of the antiischemic drug N(6)-cyclopentyladenosine (CPA). The effects of release systems have been evaluated on the stability in human whole blood of CPA and its affinity toward human adenosine A(1) receptors. The microspheres were prepared by an emulsion-solvent evaporation method (different CPA amounts and two stirring rates were employed) using poly(lactic acid). Free and encapsulated CPA was incubated in human blood and the drug stability was analyzed. The affinity of CPA to human A(1) receptor was also obtained in the presence and in the absence of unloaded microspheres. The microspheres obtained using 1200 rpm showed a broad size distribution and a mean diameter value of 21+/-9 microm. Using 1700 rpm the mean diameter decreased to 5+/-2 microm and a more homogeneous size distribution was obtained. The CPA release changed with the particle size and the different amounts of drug employed during the preparation of the microspheres. The degradation in human whole blood of CPA encapsulated in the microspheres was negligible, with respect to that of free CPA. Affinity values of CPA obtained in the absence and in the presence of unloaded microspheres were the same.

Poly(lactic acid) microspheres for the sustained release of a selective A1 receptor agonist.

A study concerning the feasibility of microsphere use as sustained delivery systems for N(6)-cyclopentyladenosine (CPA) administration has been performed. The release of this drug and the related stability effects in human whole blood have been tested. Moreover, the impact of the delivery system on CPA interaction toward human adenosine A1 receptor and the related cellular responses has been analyzed. The microspheres were prepared by an emulsion-solvent evaporation method using poly(lactic acid). Free and encapsulated CPA was incubated in fresh blood and the drug stability was analyzed with HPLC. The affinity of CPA to human A1 receptor expressed by CHO cells was obtained by binding experiments. Activity was evaluated by measurements of the inhibition of forskolin-stimulated 3',5'-cyclic adenosine monophosphate (c-AMP) performing competitive binding assays. Encapsulated CPA was released within 72 h and its degradation in blood was negligible. Affinity and activity values of CPA obtained in the absence and in the presence of unloaded microspheres were the same. CPA encapsulation in microspheres allows its sustained release and its stabilization in human whole blood to be obtained. The presence of this release system does not interfere with the CPA activity at its action site.

Synthesis and study of 5'-ester prodrugs of N6-cyclopentyladenosine, a selective A1 receptor agonist.

PURPOSE: A series of 5'-esters of N6-cyclopentyladenosine (CPA) were prepared with the aim to improve stability and bioavailability of selective A1 agonists. Log P values, stability, affinity, and activity toward human adenosine A1 receptors were evaluated. METHODS: An appropriate synthetic procedure was adopted to avoid concomitant deamination at position 6. Log P values were obtained by the Mixxor system. The stability of CPA and its 5'-ester was evaluated in human plasma and whole blood and analyzed with high-performance liquid chromatography. The affinities to human A1 receptor expressed by N6-cyclohexyladenosine cells were obtained by binding experiments. The activities were evaluated by measurements of the inhibition of forskolin stimulated 3'-5'-cyclic adenosine monophosphate, performing competitive binding assays. RESULTS: All prodrugs were more lipophilic than CPA, and their hydrolysis, in whole blood and in plasma, was found related, respectively, to the length and hindrance of 5'-substituents. Affinity and activity values indicated a very weak interaction toward adenosine A1 receptor of the intact prodrugs. CONCLUSIONS: We propose 5'-esters of CPA, characterized by suitable lipophilicity and elevated degree of stability in physiological fluids, as possible candidates for CPA prodrugs.

Determination of the adenosine A(1) agonist N(6)-cyclopentyladenosine in rat blood by solid-phase extraction and HPLC.

A solid-phase extraction procedure has been developed for the isolation of the adenosine A1 receptor agonist N(6)-cyclopentyladenosine from rat blood. The biological samples were spiked with N(6)-cyclopentyladenosine and the analogue N(6)-cyclohexladenosine (internal standard), diluted with sodium hydroxide, loaded onto disposable cartridges with subsequent desorption with methanol and analysis by HPLC. The performance of columns pre-packed with different C18-bonded silica phases or with a polymeric reversed-phase sorbent (Oasis HLB) was assessed. The highest extraction efficiencies (recovery rates>83.3%) for the two N6-alkyl substituted adenosines were achieved by the Oasis HLB cartridges. In addition, the polymeric sorbent provided reproducible recoveries (relative standard deviation<4.8%), whereas large variations (relative standard deviation values, 9--16.3%) in the extraction yields were observed using the conventional silica-based C18 cartridges. The described sample preparation method is rapid, simple, selective and it is suitable for pharmacokinetic studies.

Met-Ile-Phe-Leu derivatives: full and partial agonists of human neutrophil formylpeptide receptors.

The biological action of a series of Met-Ile-Phe-Leu analogues was analyzed on human neutrophils, to evaluate their ability to interact with formylpeptide receptors and to induce the related neutrophil responses. Three in vitro assays were carried out: receptor binding, chemotaxis and superoxide anion release. Our results demonstrate that formyl-Met-Ile-Phe-Leu derivatives act as more potent full agonists than formyl-Met-Leu-Phe, the tripeptide normally used as a model chemoattractant for the study of cell functions. On the other hand, the presence of N-ureidoisopropyl substituent in tetrapeptides imparts weak partial agonist properties. It has furthermore been demonstrated that the C-terminal methyl esterification or amination weakly influences the properties of tetrapeptide homologues. Finally, t-Boc-Met-Ile-Phe-Leu derivatives do not appear able to interact with formylpeptide receptors.

Can thermodynamic measurements of receptor binding yield information on drug affinity and efficacy?

The present commentary surveys the methods for obtaining the thermodynamic parameters of the drug-receptor binding equilibrium, DeltaG degrees, DeltaH degrees, DeltaS degrees, and DeltaC degrees (p) (standard free energy, enthalpy, entropy, and heat capacity, respectively). Moreover, it reviews the available thermodynamic data for the binding of agonists and antagonists to several G-protein coupled receptors (GPCRs) and ligand-gated ion channel receptors (LGICRs). In particular, thermodynamic data for five GPCRs (beta-adrenergic, adenosine A(1), adenosine A(2A), dopamine D(2), and 5-HT(1A)) and four LGICRs (glycine, GABA(A), 5-HT(3), and nicotinic) have been collected and analyzed. Among these receptor systems, seven (three GPCRs and all LGICRs) show "thermodynamic agonist-antagonist discrimination": when the agonist binding to a given receptor is entropy-driven, the binding of its antagonist is enthalpy-driven, or vice versa. A scatter plot of all entropy versus enthalpy values of the database gives a regression line with the equation TDeltaS degrees (kJ mol(-1); T = 298.15 K) = 40.3 (+/- 0.7) + 1.00 (+/-0.01) DeltaH degrees (kJ mol(-1)); N = 184; r = 0.981; P < 0.0001 - which is of the form DeltaH degrees = beta. DeltaS degrees, revealing the presence of the "enthalpy-entropy compensation" phenomenon. This means that any decrease of binding enthalpy is compensated for by a parallel decrease of binding entropy, and vice versa, in such a manner that affinity constant values (K(A)) of drug-receptor equilibrium (DeltaG degrees = -RT ln K(A) = DeltaH degrees - TDeltaS degrees ) cannot be greater than 10(11) M(-1). According to the most recent hypotheses concerning drug-receptor interaction mechanisms, these thermodynamic phenomena appear to be a consequence of the rearrangement of solvent molecules that occurs during the binding.

Binding thermodynamics and intrinsic activity of adenosine A1 receptor ligands.

A thermodynamic analysis of the binding to rat cortex adenosine A1 receptors of 5'-deoxyribose-N6-cyclopentyladenosine (full agonist) and several 8-alkylamino homologues of N6-cyclopentyladenosine (partial agonists) was performed. The intrinsic activity of the compounds was also evaluated by measuring the inhibition of forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (c-AMP) levels in isolated epididymal rat adipocytes. Standard free energy (deltaG), enthalpy (deltaH ) and entropy (deltaS ) of the binding equilibrium were determined by affinity measurements carried out at different temperatures (0, 10, 20, 25, 30 degrees C). Affinity constants of drug-receptor interactions were obtained by displacement experiments in the presence of 1nM [3H]N6-cyclohexyladenosine. Levels of c-AMP were evaluated by performing competitive binding assays. As the affinity of the ligands was found to increase with temperature enhancement, the binding of full and partial agonists is therefore totally entropy-driven. Standard entropy values of a wide series of adenosine derivatives, including the compounds under examination, are strictly correlated to those of intrinsic activity. Similarly, deltaS values appear correlated to the in vivo ability of the adenosine derivatives to inhibit rat heart rate. Thermodymanic data of adenosine A1 receptor ligands are proposed as an indicator of their pharmacodynamics.

Conformational aspects of human formylpeptide receptor antagonists.

The conformation of several Phe-D-Leu-Phe-D-Leu-Phe analogues was analyzed using infrared absorption and circular dichroism. Their effect on human neutrophils was verified by receptor binding and chemotaxis assays. The results demonstrate that the compounds examined prefer an ordered conformation (beta-turn) in amphipatic environment, and that they are able to antagonize the neutrophil functions evoked by CHO-Met-Leu-Phe.