Progress in nasal drug delivery systems.

Most of the available drugs are usually administered orally (e.g. in tablets or capsules) or by parenteral injection in the case of substances being destroyed in the gastric environment or not being absorbed. However, this bears disadvantages as many people have trouble swallowing tablets and parenteral injection requires trained personnel and/or a reasonably sterile environment to minimize the possibility of contamination. Thus, as an easy to use alternative nasal drug delivery was developed. Drug delivery systems are used to achieve a reproducible high drug concentration. These systems overcome various disadvantages leading to stabilization of the drug, advanced drug transport, improvement of the physicochemical properties of the drug like water solubility, and increase of drug uptake and bioavailability. In addition, properties such as bad taste or smell of the drug are masked. Nasal drug delivery systems are suitable for use both locally and systemically. In the last five years, the development and progression of nasal drug delivery systems has gained importance due to their numerous advantages. This work gives an overview of the basics, such as structure and function of the nose, as well as a short introduction to local and systemic application of drugs. Furthermore, selected drug delivery systems are explained with examples of active ingredients, as well as additional possibilities to increase nasal drug uptake and factors influencing the absorption.

Potential metal impurities in active pharmaceutical substances and finished medicinal products - A market surveillance study.

A market surveillance study has been established by using different atomic spectrometric methods for the determination of selected elemental impurities of particular interest, to gain an overview about the quality of presently marketed drug products and their bulk drug substances. The limit tests were carried out with respect to the existing EMA guideline on the specification limits for residuals of metal catalysts or metal reagents. Also attention was given to the future implementation of two new chapters of the United States Pharmacopoeia (USP) stating limit concentrations of elemental impurities. The methods used for determination of metal residues were inductively coupled plasma-mass spectrometry (ICP-MS), inductively coupled plasma-optical emission spectrometry (ICP-OES), and atomic absorption spectrometry technologies (GFAAS, CVAAS, HGAAS). This article presents the development and validation of the methods used for the determination of 21 selected metals in 113 samples from drug products and their active pharmaceutical ingredients.

Interactions between nano-TiO2 and the oral cavity: impact of nanomaterial surface hydrophilicity/hydrophobicity.

Titanium dioxide (TiO2) nanoparticles are available in a variety of oral applications, such as food additives and cosmetic products. Thus, questions about their potential impact on the oro-gastrointestinal route rise. The oral cavity represents the first portal of entry and is known to rapidly interact with nanoparticles. Surface charge and size contribute actively to the particle-cell interactions, but the influence of surface hydrophilicity/hydrophobicity has never been shown before. This study addresses the biological impact of hydrophilic (NM 103, rutile, 20 nm) and hydrophobic (NM 104, rutile, 20 nm) TiO2 particles within the buccal mucosa. Particle characterization was addressed with dynamic light scattering and laser diffraction. Despite a high agglomeration tendency, 10% of the particles/agglomerates were present in the nanosized range and penetrated into the mucosa, independent of the surface properties. However, significant differences were observed in intracellular particle localization. NM 104 particles were found freely distributed in the cytoplasm, whereas their hydrophobic counterparts were engulfed in vesicular structures. Although cell viability/membrane integrity was not affected negatively, screening assays demonstrated that NM 104 particles showed a higher potential to decrease the physiological mitochondrial membrane potential than NM 103, resulting in a pronounced generation of reactive oxygen species.

Differentiation of influenza B virus lineages Yamagata and Victoria by real-time PCR.

Since the 1970s, influenza B viruses have diverged into two antigenically distinct virus lineages called the Yamagata and Victoria lineages. We present the first real-time PCR assay for virus lineage differentiation to supplement classical antigenic analyses. The assay was successfully applied to 310 primary samples collected in Germany from 2007 to 2009.

Characterisation of the CipC-like protein AFUA_5G09330 of the opportunistic human pathogenic mould Aspergillus fumigatus.

Aspergillus fumigatus is currently the major airborne fungal pathogen that menaces immunocompromised individuals. Germination of inhaled conidia is a hallmark of the early infection process, but little is known about the underlying mechanisms. The intention of our ongoing studies is the identification of A. fumigatus proteins that are differentially expressed during germination and may provide insights in the germination process. Using a proteomic approach, we identified AFUA_5G09330 as a major hyphal-specific protein. This result was confirmed using monoclonal antibodies generated in this study. AFUA_5G09330 belongs to a fungal-specific protein family. The eponymous CipC protein of A. nidulans has been shown to be induced by concanamycin A, and transcriptional data from Cryptococcus neoformans demonstrate a strong up-regulation of the expression of a homologous gene during infection. Our data provide evidence that AFUA_5G09330 is a monomeric, cytoplasmic protein. We found no evidence for an overexpression of AFUA_5G09330 induced by concanamycin A or other stress conditions. AFUA_5G09330 is exclusively found in the hyphal morphotype that enables an invasive growth of A. fumigatus during infection. Further studies are required to define the biological function of this hyphae-specific protein and its potential relevance for the pathogenicity of A. fumigatus.

Synthesis of glycine betaine from choline in the moderate halophile Halobacillus halophilus: co-regulation of two divergent, polycistronic operons.

The moderately halophilic bacterium Halobacillus halophilus can synthesize glycine betaine from choline. Oxidation of choline is induced by salinity and repressed by exogenous glycine betaine. The genes encoding the choline dehydrogenase (gbsB) and the glycine betaine aldehyde dehydrogenase (gbsA) were identified and shown to constitute an operon. Divergent to this operon is another operon containing gbsR and gbsU that encode proteins with similarities to a transcriptional regulator and a glycine betaine-binding protein respectively. Synthesis of the four Gbs proteins was strictly dependent on the choline concentration of the medium. Salinity was essential for the production of GbsB and increased the production of GbsA, GbsR and GbsU. Glycine betaine repressed the production of all four Gbs proteins with half maximal inhibition at 0.1 mM glycine betaine.

Role of respiration in the germination process of the pathogenic mold Aspergillus fumigatus.

Inhalation of resting conidia is usually the first step of a systemic infection caused by the opportunistic fungal pathogen Aspergillus fumigatus. In the lung, the inhaled spores encounter an environment that permits germination. However, the relative importance of certain environmental conditions for conidial activation and subsequent hyphae formation has so far not been analyzed in detail. In this study, we studied the role of oxygen during germination. We found that inhibitors of the respiratory chain were nearly as efficient in blocking germination as cycloheximide, an inhibitor of protein synthesis, which is already known to prevent germination of Aspergillus nidulans. We also found that A. fumigatus is unable to grow or germinate under anaerobic conditions, and using the fluorescent mitotracker dye we detected active mitochondria already at the stage of swollen conidia, which indicates that respiration is an early event during germination. In line with these data, we found that significant oxygen consumption was detectable early during germination, whereas no oxygen consumption was measurable in suspensions of resting conidia. In summary, the present study provides evidence that respiration is absolutely required for the germination of A. fumigatus conidia.

A genetic screen identifies mutations in the yeast WAR1 gene, linking transcription factor phosphorylation to weak-acid stress adaptation.

Exposure of the yeast Saccharomyces cerevisiae to weak organic acids such as the food preservatives sorbate, benzoate and propionate leads to the pronounced induction of the plasma membrane ATP-binding cassette (ABC) transporter, Pdr12p. This protein mediates efflux of weak acid anions, which is essential for stress adaptation. Recently, we identified War1p as the dedicated transcriptional regulator required for PDR12 stress induction. Here, we report the results from a genetic screen that led to the isolation of two war1 alleles encoding mutant variants, War1-28p and War1-42p, which are unable to support cell growth in the presence of sorbate. DNA sequencing revealed that War1-28 encodes a truncated form of the transcriptional regulator, and War1-42 carries three clustered mutations near the C-terminal activation domain. Although War1-42 is expressed and properly localized in the nucleus, the War1-42p variant fails to bind the weak-acid-response elements in the PDR12 promoter, as shown by in vivo footprinting. Importantly, in contrast with wild-type War1p, War1-42p is also no longer phosphorylated upon weak-acid challenge, demonstrating that phosphorylation of War1p, its activation and DNA binding are tightly linked processes that are essential for adaptation to weak-acid stress.

Role of rufinamide in the management of Lennox-Gastaut syndrome (childhood epileptic encephalopathy).

Rufinamide, a triazole derivative that is structurally distinct from currently marketed antiepileptic drugs (AEDs), is in development for the adjunctive treatment of Lennox-Gastaut syndrome (LGS) in children and adults. Rufinamide is well absorbed after oral administration, demonstrates low protein binding, and is metabolized by enzymatic hydrolysis without involvement of cytochrome P450 enzymes, conferring a low drug interaction potential. In a randomized, double-blind trial involving 138 adult and pediatric patients with LGS, compared with placebo, rufinamide 45 mg/kg/day resulted in significantly superior reductions in drop attacks (median change -42.5% vs +1.4% with placebo) and total seizures (-32.1% vs -11.7% with placebo), accompanied by significantly higher responder rates. These results are comparable with findings reported for other AEDs in randomized, controlled clinical trials in patients with LGS. Rufinamide produced statistically significant seizure reduction which was maintained during long-term therapy and accompanied by good tolerability. The most frequently reported adverse events from a pooled safety database evaluating short- and long-term therapy were headache (22.9% and 29.5%), dizziness (15.5% and 22.5%) and fatigue (13.6% and 17.7%). Rufinamide therefore presents a favorable efficacy and tolerability profile and is a promising candidate for the adjunctive therapy of LGS.

Global phenotypic analysis and transcriptional profiling defines the weak acid stress response regulon in Saccharomyces cerevisiae.

Weak organic acids such as sorbate are potent fungistatic agents used in food preservation, but their intracellular targets are poorly understood. We thus searched for potential target genes and signaling components in the yeast genome using contemporary genome-wide functional assays as well as DNA microarray profiling. Phenotypic screening of the EUROSCARF collection revealed the existence of numerous sorbate-sensitive strains. Sorbate hypersensitivity was detected in mutants of the shikimate biosynthesis pathway, strains lacking the PDR12 efflux pump or WAR1, a transcription factor mediating stress induction of PDR12. Using DNA microarrays, we also analyzed the genome-wide response to acute sorbate stress, allowing for the identification of more than 100 genes rapidly induced by weak acid stress. Moreover, a novel War1p- and Msn2p/4p-independent regulon that includes HSP30 was identified. Although induction of the majority of sorbate-induced genes required Msn2p/4p, weak acid tolerance was unaffected by a lack of Msn2p/4p. Ectopic expression of PDR12 from the GAL1-10 promoter fully restored sorbate resistance in a strain lacking War1p, demonstrating that PDR12 is the major target of War1p under sorbic acid stress. Interestingly, comparison of microarray data with results from the phenotypic screening revealed that PDR12 remained as the only gene, which is both stress inducible and required for weak acid resistance. Our results suggest that combining functional assays with transcriptome profiling allows for the identification of key components in large datasets such as those generated by global microarray analysis.

Weak organic acid stress inhibits aromatic amino acid uptake by yeast, causing a strong influence of amino acid auxotrophies on the phenotypes of membrane transporter mutants.

The ability of yeasts to grow in the presence of weak organic acid preservatives is an important cause of food spoilage. Many of the determinants of acetate resistance in Saccharomyces cerevisiae differ from the determinants of resistance to the more lipophilic sorbate and benzoate. Interestingly, we show in this study that hypersensitivity to both acetate and sorbate results when the cells have auxotrophic requirements for aromatic amino acids. In tryptophan biosynthetic pathway mutants, this weak acid hypersensitivity is suppressed by supplementing the medium with high levels of tryptophan or, in the case of sorbate sensitivity, by overexpressing the Tat2p high affinity tryptophan permease. Weak acid stress therefore inhibits uptake of aromatic amino acids from the medium. This allows auxotrophic requirements for these amino acids to strongly influence the resistance phenotypes of mutant strains. This property must be taken into consideration when using these phenotypes to attribute functional assignments to genes. We show that the acetate sensitivity phenotype previously ascribed to yeast mutants lacking the Pdr12p and Azr1p plasma membrane transporters is an artefact arising from the use of trp1 mutant strains. These transporters do not confer resistance to high acetate levels and, in prototrophs, their presence is actually detrimental for this resistance.

Moderately lipophilic carboxylate compounds are the selective inducers of the Saccharomyces cerevisiae Pdr12p ATP-binding cassette transporter.

Saccharomyces cerevisiae displays very strong induction of a single ATP-binding cassette (ABC) transporter, Pdr12p, when stressed with certain weak organic acids. This is a plasma membrane pump catalysing active efflux of the organic acid anion from the cell. Pdr12p action probably allows S. cerevisiae to maintain lower intracellular levels of several weak organic acid preservatives than would be expected on the basis of the free equilibration of the acid across the cell membrane. This in turn facilitates growth in the presence of these preservatives and therefore yeast spoilage of food materials. Pdr12p appears to confer resistance to those carboxylic acids that, to a reasonable degree, partition into both the lipid bilayer and aqueous phases. Its gene (PDR12) is strongly induced by sorbate, benzoate and certain other moderately lipophilic carboxylate compounds, but not by organic alcohols or high levels of acetate. PDR12 induction reflects the operation of a previously uncharacterized S. cerevisiae stress response, for which the induction signal is probably a high intracellular pool of the organic acid anion.

War1p, a novel transcription factor controlling weak acid stress response in yeast.

The Saccharomyces cerevisiae ATP-binding cassette (ABC) transporter Pdr12p effluxes weak acids such as sorbate and benzoate, thus mediating stress adaptation. In this study, we identify a novel transcription factor, War1p, as the regulator of this stress adaptation through transcriptional induction of PDR12. Cells lacking War1p are weak acid hypersensitive, since they fail to induce Pdr12p. The nuclear Zn2Cys6 transcriptional regulator War1p forms homodimers and is rapidly phosphorylated upon sorbate stress. The appearance of phosphorylated War1p isoforms coincides with transcriptional activation of PDR12. Promoter deletion analysis identified a novel cis-acting weak acid response element (WARE) in the PDR12 promoter required for PDR12 induction. War1p recognizes and decorates the WARE both in vitro and in vivo, as demonstrated by band shift assays and in vivo footprinting. Importantly, War1p occupies the WARE in the presence and absence of stress, demonstrating constitutive DNA binding in vivo. Our results suggest that weak acid stress triggers phosphorylation and perhaps activation of War1p. In turn, War1p activation is necessary for the induction of PDR12 through a novel signal transduction event that elicits weak organic acid stress adaptation.

Phosphorylation of eukaryotic initiation factor 2 by heme-regulated inhibitor kinase-related protein kinases in Schizosaccharomyces pombe is important for fesistance to environmental stresses.

Protein synthesis is regulated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in response to different environmental stresses. One member of the eIF2alpha kinase family, heme-regulated inhibitor kinase (HRI), is activated under heme-deficient conditions and blocks protein synthesis, principally globin, in mammalian erythroid cells. We identified two HRI-related kinases from Schizosaccharomyces pombe which have full-length homology with mammalian HRI. The two HRI-related kinases, named Hri1p and Hri2p, exhibit autokinase and kinase activity specific for Ser-51 of eIF2alpha, and both activities were inhibited in vitro by hemin, as previously described for mammalian HRI. Overexpression of Hri1p, Hri2p, or the human eIF2alpha kinase, double-stranded-RNA-dependent protein kinase (PKR), impeded growth of S. pombe due to elevated phosphorylation of eIF2alpha. Cells from strains with deletions of the hri1(+) and hri2(+) genes, individually or in combination, exhibited a reduced growth rate when exposed to heat shock or to arsenic compounds. Measurements of in vivo phosphorylation of eIF2alpha suggest that Hri1p and Hri2p differentially phosphorylate eIF2alpha in response to these stress conditions. These results demonstrate that HRI-related enzymes are not unique to vertebrates and suggest that these eIF2alpha kinases are important participants in diverse stress response pathways in some lower eukaryotes.