[Severe pneumococcal infections in the elderly - preventable by vaccination]. / Schwere Pneumokokkeninfektionen im Alter - impfpräventabel.

Elderly people are more susceptible to pneumococcal infections. Data in Germany from 2005-2010 shows that especially seniors are prone to develop serious complications such as sepsis. Women are obviously less affected than men. Most of the infections occurred during the winter months. The majority of isolates, i.e., about 80%, possess capsular polysaccharide antigens which are represented in the 23-valent vaccine. Consequently, it could be assumed that the severe complications ensuing long hospital stays and associated with a high mortality could have been avoided, if the elderly people would have been vaccinated, which, however, was only true in a small proportion (28%). Recently, a new conjugated vaccine was introduced to the market. In principle, several antibiotics are available for direct antibacterial treatment. All isolates are susceptible to cefotaxime as well as to ceftriaxone. Resistance to penicillin as well as ampicillin is very rare in Germany. The vast majority of isolates are susceptible to quinolones such as levofloxacin and moxifloxacin. Resistance to macrolides, for example to erythromycin, occurs to a certain extent but the percentage has been declining in recent years. Nevertheless, in many instances therapy is too late. Thus, prevention is of great importance.

Stabilization of reactive nitroxides using invasomes to allow prolonged electron paramagnetic resonance measurements.

The detection of the antioxidative capacity of the skin is of great practical relevance since free radicals are involved in many skin damaging processes, including aging and inflammation. The nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl) in combination with electron paramagnetic resonance spectroscopy was found suitable for measuring the antioxidative capacity since its reaction with reducing agents is considerably fast. Yet, in order to achieve longer measurement times, e.g. in inflammatory skin diseases, the stabilizing effect of an invasome (ultraflexible vesicle/liposome) suspension with TEMPO was investigated ex vivo on porcine skin and in vivo on human skin. Invasomes increased the measurement time ex vivo 2-fold and the reduction was significantly slowed down in vivo, which is due to membrane-associated and therefore protected TEMPO. Furthermore, TEMPO accumulation in the membrane phase as well as the decreasing polarity of the ultimate surroundings of TEMPO during skin penetration explains the stabilizing effect. Thus, an invasome suspension with TEMPO exhibits stabilizing effects ex vivo and in vivo.

Is binge-watching addictive? Effects of motives for TV series use on the relationship between excessive media consumption and problematic viewing habits.

This study investigates the circumstances under which binge-watching can become a problematic behavior. Applying a user-centered perspective, it demonstrates how different motivations to engage in high-dosage TV series consumption influence the occurrence of problematic viewing habits. A quantitative online survey of N = 415 media users with access to at least one streaming service was conducted. The questionnaire assessed current viewing habits, motivations to watch series, and indicators of problematic viewing habits. The results suggest that frequency of use, motives to engage in high dosage viewing sessions, as well as the combined effect of these two factors help to explain problematic viewing behaviors. Moreover, the results give cause to refrain from a generalizing problematization of binge-watching.

Polysaccharide conjugates surpass monosaccharide ligands in hepatospecific targeting - Synthesis and comparative in silico and in vitro assessment.

Ligands with the polysaccharide headgroups have been recently reported by our group to possess enhanced interaction with asialoglycoprotein receptor (ASGPR) in silico as compared to ligands having galactose moieties. This enhanced interaction is a result of the polymer's backbone support in anchoring the ligand in a specific orientation within the bilayer. In this paper, we have attempted to provide an in vitro proof of concept by performing a comparative evaluation of polysaccharide and monosaccharide-based ligands. Docking was performed to understand interaction with ASGPR in silico. Agarose and galactose conjugates with behenic acid were synthesized, purified, and characterized to yield biocompatible hepatospecific ligands which were incorporated into nanoliposomes. Cellular internalization of these targeted liposomes was studied using confocal microscopy and flow cytometry. The toxicity potential was assessed in vivo. Results indicated that the polysaccharide-based ligand increased cellular uptake due to better interaction with the receptor as compared to ligand bearing a single galactose group. In addition to developing novel liver targeting ligands, the study also established proof of concept that has been suggested by earlier in silico investigations. The approach can be used to design targeting ligands and develop formulations with improved targeting efficacy.

Evaluation of a new selective medium, BD BBL CHROMagar MRSA II, for detection of methicillin-resistant Staphylococcus aureus in different specimens.

The sensitivity of screening for methicillin-resistant Staphylococcus aureus (MRSA) can be improved by adding other specimen sites to nares. We describe an evaluation of a new selective medium, BBL CHROMagar MRSA II (CMRSAII), for its ability to detect MRSA from different specimen types. CMRSAII is a chromogenic medium which incorporates cefoxitin for the detection of MRSA. A study was performed at four clinical laboratories with the following specimens: 1,446 respiratory, 694 stool, 1,275 skin, and 948 wound specimens and 688 blood culture bottles containing Gram-positive cocci. The recovery of MRSA on traditional culture media was compared to results with CMRSAII. S. aureus was tested by cefoxitin disk diffusion. CMRSAII was interpreted as positive for MRSA at 24 h (range, 18 to 28 h) based solely on the visualization of mauve-colored colonies and at 48 h (range, 36 to 52 h) based on detection of mauve colonies with subsequent confirmation as S. aureus (by coagulase or latex agglutination testing). MRSA was recovered more frequently on CMRSAII (89.8% at 24 h and 95.6% at 48 h) than on traditional culture plates (83.1% at 24 h and 79.8% at 48 h) for all specimen types combined (P < 0.001). The percent sensitivities of CMRSAII at 24- and 48-h reads, respectively, were 85.5 and 92.4% for respiratory specimens, 87.9% and 98.3% for stool specimens, 88.4% and 96.1% for skin specimens, 92.1% and 94.6% for wound specimens, and 100% and 100% for positive blood cultures. The specificity was 99.8% for respiratory specimens and 100% for all others. In conclusion, CMRSAII is a reliable screening medium for multiple specimen types.

Lipid nanoconstructs for superior hepatoprotection: In vitro assessments as predictive tool for in vivo translation.

AIM: To investigate comparative in vitro and in vivo performance of lipid vesicular and particulate systems in escalating oral bioavailability for superior hepatoprotection. MATERIALS AND METHODS: Systems were fabricated using easy to scale up process and novel excipients to deliver Silibinin. In vitro characterization followed by pharmacokinetic and pharmacodynamic evaluation in rats was conducted to establish a correlation. RESULTS: Nanoformulations resulted in 20 fold increase in solubilisation and significant increase in permeation. 2.5 fold increase in bioavailability was evident in vivo. Vesicles demonstrated greatest hepatoprotective potential in efficacy study. CONCLUSION: The findings establish a link between in vitro and in vivo performance to rank order lipid nanoartchitects. Concurrently, a significant potential in therapeutic intervention of hepatotoxicity is envisaged as elucidated.

High interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry has emerged as a rapid, cost-effective alternative for bacterial species identification. Identifying 60 blind-coded nonfermenting bacteria samples, this international study (using eight laboratories) achieved 98.75% interlaboratory reproducibility. Only 6 of the 480 samples were misidentified due to interchanges (4 samples) or contamination (1 sample) or not identified because of insufficient signal intensity (1 sample).

Molecular cytogenetic characterization of the human cerebral microvessel endothelial cell line hCMEC/D3.

The immortalized human cerebral microvessel endothelial cell line hCMEC/D3 has been repeatedly used as a model of human blood-brain barrier (BBB). hCMEC/D3 cells between passage 25 and 35 are most often applied in research, remained phenotypically nontransformed, and cells maintained many characteristics of human brain endothelial cells. Also hCMEC/D3 was thought to have conserved a normal diploid karyotype over all these passages. Here we characterized the cell line using high-resolution multicolor fluorescence in situ hybridization (FISH) approaches and revealed a complex karyotype in the 30th passage. Clonal cryptic unbalanced structural rearrangements and numerical aberrations were discovered and described as follows: 45 approximately 48,XX, -X,del(5)(q11)[2],del(9)(q11)[3],+9[3],del(11)(q13 approximately 14)[2], der(14)t(14;21)(q32.33;q22.3)[28],der(15)t(9;15)(p11;p11)[13], dup(15)(p11q11)[5],der(21)t(17;21)(p12;q22)[9],-22[6][cp28]. In summary, a complex karyotype with clonal unbalanced chromosomal rearrangements is present in hCMEC/D3. Thus, we solicit to include molecular cytogenetics in the testing of all cell lines prior to application of their use in complex studies.

Stable carbamazepine colloidal systems using the cosolvent technique.

The aim of this paper was to prepare stable carbamazepine nanosuspensions containing 10mg/ml drug concentration by screening different polymers. Stable formulations were created by the cosolvent technique with polyethylene glycol (PEG-300) and water as the cosolvents. Rapid growth of long needle shaped CBZ crystals was observed in the absence of polymer. The presence of hydroxypropyl methylcellulose (HPMC) or methylcellulose (MC) inhibited crystal growth and the mean particle sizes were in the range 10-20 nm. Simultaneous presence of HPMC and polyvinylpyrrolidon (PVP PF17) polymers in CBZ suspensions enhanced the overall stability of the formulations. The additional stability improvement was attributed to the interaction between the polymers by the formation of hydrogen bonds. Suspension stability was evaluated over 5 months where the particle size remained constant. FT-Raman studies showed the existence of form I within the stable CBZ suspensions.

Enhanced dissolution of oxcarbazepine microcrystals using a static mixer process.

The purpose of this study was to form micronized powders of Oxcarbazepine (OXC), a poorly water-soluble drug, using a static mixer technique to enhance the dissolution rate. Controlled precipitation was achieved injecting the organic OXC solution rapidly into an aqueous methylcellulose (MC) protective solution by means of a static mixer thus providing turbulent and homogeneous mixing. Furthermore, a factorial design was implemented for data analysis. The physicochemical properties of the freeze-dried dispersions were evaluated by differential scanning calorimetry (DSC), infrared spectroscopy (FTIR) and X-ray diffraction (XRD). Drug microcrystals showed a narrow size distribution with approximately 2 microm mean particle size and high drug loading. DSC and FTIR studies revealed that the drug remained in crystalline state and no drug-polymer interaction occurred. The dissolution studies showed enhanced dissolution of OXC microcrystals compared to the pure drug. The static mixer technique was proved capable for micro-sized polymeric particles. This is an inexpensive, less time consuming and fully scalable process for development of poorly soluble drugs.

Uptake of phosphatidylserine-containing liposomes by liver sinusoidal endothelial cells in the serum-free perfused rat liver.

We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [3H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.

Direct detection of methicillin-resistant Staphylococcus aureus in clinical specimens by a nucleic acid-based hybridisation assay.

The occurrence of methicillin-resistant Staphylococcus aureus (MRSA) is still increasing worldwide and is associated with significant morbidity, mortality and hospital costs. Screening for MRSA plays a key role in limiting further nosocomial spread of this organism. Control measures require a rapid and sensitive test for direct detection of MRSA carriage. This study evaluated an easy-to-use PCR-hybridisation assay for the direct detection of MRSA in clinical swab specimens. In total, 508 pairs of swabs from 242 patients at risk for MRSA carriage were analysed by the standard culture method and the PCR assay. One swab was used for PCR and culture, while the second was used for culture only. Of the 508 pairs tested, 37 were positive by culture and 35 were positive by PCR. Among the 471 culture-negative specimens, 465 were negative by PCR and six were PCR-positive. The PCR assay had a sensitivity of 94.59%, a specificity of 98.73%, a positive predictive value of 85.37%, and a negative predictive value of 99.57%. The PCR-hybridisation assay enabled reliable detection of MRSA carriage in c. 4 h, thereby allowing its effective use in an MRSA control strategy.

Nano- and micro-particulate formulations of poorly water-soluble drugs by using a novel optimized technique.

A novel technique for the production of nano- and micro-particulate formulations of poorly water-soluble drugs has been developed. This technique involves the use of static mixer elements to provide fast precipitation by continuous turbulent mixing of two liquid flows, an aqueous phase and an organic phase, respectively. The objective of this study was to develop the mixer technique by investigating the influence of the element number on the particle size of the resulting dispersions. Four model active pharmaceutical ingredients (APIs) with a variety of polymers, lipids or surfactants underwent intensive mixing and the final suspensions showed a narrow size distribution. Parameters such as the flow rate and the temperature of the precipitated organic-aqueous phases were also significant in the reduction of particle size. Further development of the mixing technique led to reproducible and stable formulations with minimal excipient amounts. These formulations were spray- or freeze-dried to improve stability.

Photoaffinity labeling of acetylcholine receptor in millisecond time scale.

Photoaffinity labeling of acetylcholine receptors can be performed with a time resolution allowing to discriminate reaction sites within the receptor protein in its different functional states. This is achieved by a combination of a stopped-flow apparatus with a high energy pulse laser. The photoaffinity label used is the lipophilic cation [3H]TPMP+ which has been shown to be a non-competitive antagonist and a specific ion channel blocker. AChR in its resting (channel closed) and active (channel open) state incorporates the label mainly into the alpha-polypeptide chain of the receptor. Only several hundred milliseconds after mixing AChR with agonist labeling of delta-chains becomes significant.

Highly efficient transduction of endothelial cells by targeted artificial virus-like particles.

Targeting the tumor vasculature by gene therapy is a potentially powerful approach, but suitable vectors have not yet been described. We have designed a new type of liposomal vector, based on the composition of anionic retroviral envelopes, that is serum-resistant and nontoxic. These artificial virus-like envelopes (AVEs) were endowed with a cyclic RGD-containing peptide as a targeting device for the a(v)beta3-integrin on tumor endothelial cells (ECs). The packaging of plasmid DNA complexed with low-molecular-weight, nonlinear polyethyleneimine into these AVEs yielded artificial virus-like particles (AVPs) that transduced ECs with efficiencies of up to 99%. In contrast, transduction of a variety of other cell types by these RGD-AVPs was comparably inefficient under the same experimental conditions. This EC selectivity was mediated, in part, but not exclusively, by the RGD ligand, as suggested by the reduced, but still relatively high, transduction efficiency seen with AVPs lacking RGD. The interaction of anionic lipids of the AVPs with ECs may therefore contribute to the observed selective and highly efficient transduction of this cell type. These findings suggest that the targeted AVE technology is a useful approach to create highly efficient nonviral vectors.

Cyclosporin clinical pharmacokinetics.

Cyclosporin is a powerful immunosuppressive drug used in transplantation medicine and to treat autoimmune diseases. It is a lipophilic molecule, with its bioavailability dependent on food, bile and other interacting factors. Cyclosporin is extensively metabolised in the liver by the cytochrome P450 3A system, which is subject to considerable interindividual variation. Distribution of cyclosporin depends not only on physicochemical characteristics, but also on biological carriers such as lipoproteins and erythrocytes in blood. Cyclophilin, a binding protein for cyclosporin, influences distribution of cyclosporin in the body. Despite its lipophilicity, cyclosporin does not appear in the brain. The distribution of metabolites in the body can differ from that of cyclosporin itself. Elimination of the drug is mainly via the bile as metabolites, other routes not being very important. Pharmacokinetic parameters of cyclosporin are highly variable and depend on factors such as age, the physical condition of the patient, type of organ transplant or comedication. Renal side effects of cyclosporin are dose-related, but the influence of the dosage regimen has not been thoroughly investigated. An important factor in the reported variability is the different analytical methods used. Following the recommendations of recent consensus documents to monitor blood concentrations, this source of variability may diminish in the future. Several metabolites are reported as having less immunosuppressive activity than the parent drug. Metabolites with renal side effects have been reported. These and other effects of metabolites have not been clearly defined in the literature, presumably because of the highly variable activity of cyclosporin-metabolising liver enzymes and the paucity of data available on metabolite pharmacokinetics. The therapeutic range and dosage of cyclosporin are therefore highly dependent on many individual parameters in patients. Dosages of less than 5 mg/kg/day, however, rarely cause renal side effects. Further studies to correlate the clinical pharmacokinetics of metabolites with their activity and adverse effects are needed.

Blood pressure changes in spontaneously hypertensive rats correlate with aortic prostacyclin formation.

1 The relationship between the blood pressure fall, induced by antihypertensive drugs or bleeding, and the formation of prostacyclin (PGI2)-like activity in the thoracic aorta of spontaneously hypertensive rats has been investigated. Inhibition of ADP-induced platelet aggregation was used to assess PGI2-like activity. 2 The decreases in blood pressure produced by clonidine, dihydralazine and prazosin were associated with increases of PGI2-like activity of 50-80%. The increase in PGI2-like activity correlated well with the blood pressure decrease, independently of the mechanism of the fall in blood pressure.

Permeation enhancement of octreotide by specific bile salts in rats and human subjects: in vitro, in vivo correlations.

1. The potential of bile salts to improve the enteral absorption of octreotide, an orally active somatostatin analogue, was investigated by a combination of in vitro, in situ and in vivo experiments. 2. Incorporation of octreotide into lipid monolayers (as measured by area increase of the monolayer at constant surface pressure using a Langmuir-Blodgett trough set-up) depended on the type of bile salt used for monolayer pre-treatment. Addition of 20 microM octreotide to the subphase containing 20 microM of the dihydroxylated bile salt ursodeoxycholate (UDCA) causes a 9% increase in area, whereas addition of octreotide to the subphase containing the 7 alpha-enantiomer of UDCA, chenodeoxycholate (CDCA), resulted in an area increase of the lipid monolayer of 20%. Area increase by octreotide alone was not significantly different from the increase of octreotide and UDCA in combination. 3. CDCA and UDCA in combination with octreotide increased the permeability of liposomal membranes for rubidium ions, whereas octreotide alone did not significantly change the permeability. This indicates membrane distortion as a possible cause for the enhanced absorption of octreotide by bile salts. 4. In polarized Caco-2 cell monolayers octreotide exhibited a permeation coefficient of 0.008 +/- 0.004 cm h-1. Addition of 0.2-1% of UDCA to the apical incubation medium had no significant effect upon the permeation coefficient. In contrast, 0.2-1% CDCA in the incubation medium resulted in a significant increase (P < 0.05) of the monolayer permeability of octreotide (0.015-0.037 cm h-1). 5. Octreotide was absorbed as the intact peptide from the gastrointestinal tract in rats with an absorption efficiency of 0.26%. Coadministration of bile salt resulted in a dose-dependent increase in absorption efficiency of the peptide up to 20.2%. The observed effect was more pronounced for CDCA than for UDCA. 6. The effect of CDCA and UDCA on octreotide absorption in vivo was assessed in a pharmacokinetic study with healthy volunteers. After oral administration of 4 mg octreotide in the presence of 100 mg bile salt, an average bioavailability of the peptide of 1.26% was achieved in the presence of CDCA, whereas in the presence of UDCA a bioavailability of only 0.13% was reached. This difference was statistically significant (P < 0.01). 7. In conclusion, the co-administration of CDCA is able to enhance the enteral absorption of octreotide. The in vitro and in situ experiments were predictive for the observed effect in human subjects.

P-glycoprotein- and mrp2-mediated octreotide transport in renal proximal tubule.

1. Transepithelial transport of a fluorescent derivative of octreotide (NBD-octreotide) was studied in freshly isolated, functionally intact renal proximal tubules from killifish (Fundulus heteroclitus). 2. Drug accumulation in the tubular lumen was visualized by means of confocal microscopy and was measured by image analysis. Secretion of NBD-octreotide into the tubular lumen was demonstrated and exhibited the all characteristics of specific and energy-dependent transport. Steady state luminal fluorescence averaged about five times cellular fluorescence and was reduced to cellular levels when metabolism was inhibited by NaCN. 3. NBD-octreotide secretion was inhibited in a concentration-dependent manner by unlabelled octreotide, verapamil and leukotriene C(4) (LTC(4)). Conversely, unlabelled octreotide reduced in a concentration dependent manner the p-glycoprotein (Pgp)-mediated secretion of a fluorescent cyclosporin A derivative (NBDL-CS) and the mrp2-mediated secretion of fluorescein methotrexate (FL-MTX). 4. This inhibition was not due to impaired metabolism or toxicity since octreotide had no influence on the active transport of fluorescein (FL), a substrate for the classical renal organic anion transport system. 5. The data are consistent with octreotide being transported across the brush border membrane of proximal kidney tubules by both Pgp and mrp2.

Liposomal formulations of Cyclosporin A: a biophysical approach to pharmacokinetics and pharmacodynamics.

There are about 20 publications about liposomal formulations of Cyclosporin A (CyA) in the pharmaceutical and preclinical literature. Liposomal formulations were developed in order to reduce the nephrotoxicity of CyA and to increase pharmacological effects. However, conflicting results have been published as to the therapeutic properties of these formulations. This is also true for the change in pharmacokinetics and organ distribution of the liposomally encapsulated CyA as compared to conventionally formulated CyA. Using biophysical methods, it could be shown that CyA is not tightly entrapped in liposomal membranes, despite its high lipophilicity. CyA shows retardation only at high lipid concentrations in blood, following a massive injection of liposomes. This effect may diminish nephrotoxicity, as could be demonstrated by in vitro studies using a model tubule system. The results of these studies can be used to predict the formulation behavior in vivo and to optimize liposomal formulations. When applied in an early phase of the drug formulation process, these types of biophysical experiments can also help minimize animal experiments. However, these basic interaction studies cannot cover all physiological, pharmacological, and toxic effects in animals and humans.