Surfactant coated aerosol powders and their properties

The hygroscopic growth of aerosols is an important factor effecting particle size. The consequence of the hygroscopic growth of pharrnaceutical aerosols is a change in their deposition characteristics, such that there is an increase in the total amount deposited in the lung. In this study the hygroscopic growth of disodium fluorescein (DF) aerosol powders was investigated by coating the powders with lauric and capric acids. The coating procedure was carried out in dichloromethane and chloroform, which acted as cosolvents for the fatty acids. An assessment of the extent and the nature of the coating was carried out. The qualitative assessment of the coating was achieved by infra-red spectroscopy, electronscanning chemical analysis and scanning electron microscopy. The quantitative analysis was carried out by differential refractometry, ultra-violet spectroscopy and gas liquid chromatography. These powders were generated under conditions approaching those in the lung, of 97 % relative humidity and 37"C. Coated and uncoated DF aerosol powders were introduced into a controlled temperature and relative humidity apparatus, designed and constructed for the investigation of hygroscopic growth in these studies. A vertical spinning disc device was used to generate the powders. Under conditions of controlled temperature and relative humidity mentioned, the growth ratio of disodium fluorescein alone was 1.45 compared with 1.68, for a nominal coating of DF with lauric acid of 0.12 gg-1, 1.0 for a nominal lauric acid coating of 0.2 gg-1, and 1.02 for a nominal capric acid coating of 0.18 gg-1. The range of control of hygroscopic growth of these aerosols has implications for the deposition of these preparations in the respiratory tract. These implications are discussed in the light of the current knowledge of the effects of hygroscopic growth on the deposition of pharmaceutical and environmental aerosols. A series of experiments in which pulmonary ventilation using a simple radioaerosol generator and delivery system are reported showing that particle size determination may be used to aid the design of diagnostic aerosol generators.

Role of contact lenses in relieving ocular allergy

Purpose: To examine the potential barrier and lubricating effects of modern daily disposable contact lenses (DD) against airborne antigens. Methods: Ten patients with skin prick and ocular conjunctival provocation confirmed allergic sensitivity to grass pollen were recruited (average age 27.4±7.7 years). Each had their ocular symptoms (on a 0 none to 5 extreme scale) and appearance of bulbar and limbal conjunctival redness, palpebral conjunctival redness and roughness, and corneal and conjunctival fluorescein staining (CCLRU scale) graded before and five minutes after exposure to 400 grains grass pollen/m3 for 2 minutes in a purpose-designed exposure chamber to simulate the conditions of a ‘very high’ pollen-count day. This was repeated on three occasions separated by >72 hours wearing etafilcon A (sDD), nelfilcon A with enhanced lubricating agents (ELDD) and no contact lenses in random order out of the pollen season. Each sign and symptom was compared to baseline for each condition. The duration of the symptoms was also recorded. www.clinicaltrials.gov NCT01125540 Results: Only symptoms of burning and stinging were significantly reduced in severity by ELDD (Chi-Sq=7.6, p=0.02), but overall symptoms were significantly reduced in duration (F=3.60, p=0.05). Bulbar hyperaemia, corneal and conjunctival staining, and palpebral conjunctival roughness were significantly reduced by DD wear (p<0.01), with limbal and palpebral conjunctival redness further reduced in ELDD (p<0.05). Conclusion: Daily disposable contact lenses offer a barrier to airborne antigen which is enhanced by modern lenses with enhanced lubricating agents.

Design, synthesis and biological evaluation of potential antibacterial oligonucleotides

Purine and pyrimidine triplex-forming oligonucleotides (TFOs), as potential antibacterial agents, were designed to bind by Hoogsteen and reverse Hoogsteen hydrogen bonds in a sequence specific manner in the major groove of genomic DNA at specific polypurine sites within the gyrA gene of E. coli and S. pneumoniae. Sequences were prepared by automated synthesis, with purification and characterisation determined by high performance liquid chromatograpy, capillary electrophoresis and mass spectrometry. Triplex stability was assessed using melting curves where the binding of the third strand to the duplex target, was assessed over a temperature range of 0-80°C, and at pH 6.4 and 7.2. The most successful of the unmodified TFOs (6) showed a Tm value of 26 °C at both pH values with binding via reverse Hoogsteen bonds. Binding to genomic DNA was also demonstrated by spectrofluorimetry, using fluorescein-labelled TFOs, from which dissociation constants were determined. Modifications in the form of 5mC, 5' acridine attachment, phosphorothioation, 2'-0-methylation and phosphoramidation, were made in order to. increase Tm values. Phosphoramidate modification was the most with increased Tm values of 42°C. However, the final purity of these sequences was poor due to their difficult syntheses. FACS (fluorescent activated cell sorting) analysis was used to determine the potential uptake of a fluorescently labelled analogue of 6 via passive, coJd shock mediated, and anionic liposome aided, uptake. This was established at 20°C and 37°C. At both temperatures anionic lipid-mediated uptake produced unrivalled fluorescence, equivalent to 20 and 43% at 20 and 37°C respectively. Antibacterial activity of each oligonucleotide was assessed by viable count anaJysis relying on passive uptake, cold shocking techniques, chlorpromazine-mediated uptake, and, cationic and anionic lipid-aided uptake. All oligonucleotides were assessed for their ability to enhance uptake, which is a major barrier to the effectiveness of these agents. Compound 6 under cold shocking conditions produced the greatest consistent decline in colony forming units per ml. Results for this compound were sometimes variable indicating inconsistent uptake by this particular assay method.

Oxidative injury induced by hypochlorous acid to Ca-ATPase from sarcoplasmic reticulum of skeletal muscle and protective effect of trolox

Hypochlorous acid (HOCl) concentration-dependently decreased ATPase activity and SH groups of pure Ca-ATPase from sarcoplasmic reticulum (SERCA) of rabbit skeletal muscle with IC(50) of 150 micromol/l and 6.6 micromol/l, respectively. This indicates that SH groups were not critical for impairment of Ca-ATPase activity. Pure Ca-ATPase activity was analysed individually with respect to both substrates, Ca(2+) and ATP. Concerning dependence of ATPase activity on HOCl (150 micromol/l) as a function of free Ca(2+) and ATP, V(max) of both dependences decreased significantly, while the affinities to individual substrates were not influenced, with the exception of the regulatory binding site of ATP. On increasing HOCl concentration, fluorescence of fluorescein-5-isothiocyanate (FITC) decreased, indicating binding of HOCl to nucleotide binding site of SERCA. A new fragment appeared at 75 kDa after HOCl oxidation of SR, indicating fragmentation of SERCA. Fragmentation may be associated with protein carbonyl formation. The density of protein carbonyl bands at 75 and 110 kDa increased concentration- and time-dependently. Trolox (250 micromol/l) recovered the Ca-ATPase activity decrease induced by HOCl, probably by changing conformational properties of the Ca-ATPase protein. Trolox inhibited FITC binding to SERCA.

Spontanoeusly resolved macroaneurysm associated with a congenital anomalous retinal artery

Purpose: To report the outcome of retinal artery macroaneurysm associated with a congenital anomalous retinal artery. Methods: The patient was examined with ophthalmoscopy and fluorescein angiography. Results: A 60-year-old woman presented with visual acuity of 20/80 in her right eye. She was found to have an abnormal retinal vessel emerging from the optic disk, passing toward the center of the macula, and looping back toward the disk. The center of the loop had a leaking macroaneurysm, which was treated conservatively. At the 4-month follow-up visit, the visual acuity in the right eye had improved to 20/20 with resolution of most of the macular edema, exudates, and hemorrhage. Conclusion: This case demonstrates that retinal artery macroaneurysm can be successfully managed with a conservative approach, even when associated with a congenital anomalous retinal vessel.

Consensus on recording of gas permeable contact lens fit

Purpose: To develop a new schematic scheme for efficiently recording the key parameters of gas permeable contact lens (GP) fits based on current consensus. Methods: Over 100 established GP fitters and educators met to discuss the parameters proposed in educational material for evaluating GP fit and concluded on the key parameters that should be recorded. The accuracy and variability of evaluating the fluorescein pattern of GP fit was determined by having 35 experienced contact lens practitioners from across the world, grading 5 images of a range of fits and the topographer simulation of the same fits, in random, order using the proposed scheme. The accuracy of the grading was compared to objective image analysis of the fluorescein intensity of the same images. Results: The key information to record to adequately describe the fit of an GP was agreed as: the manufacturer, brand and lens parameters; settling time; comfort on a 5 point scale; centration; movement on blink on a ±2 scale; and the Primary Fluorescein Pattern in the central, mid-peripheral and edge regions of the lens averaged along the horizontal and vertical lens axes, on a ±2 scale. On average 50-60% of practitioners selected the median grade when subjectively rating fluorescein intensity and this was correlated to objective quantification (r= 0.602, p< 0.001). Objective grading suggesting horizontal median fluorescein intensity was generally symmetrical, as was the vertical meridian, but this was not the case for subjective grading. Simulated fluorescein patterns were subjectively and objectively graded as being less intense than real photographs (p< 0.01). Conclusion: GP fit recording can be standardised and simplified to enhance GP practice. © 2013 British Contact Lens Association.

Predicting success with silicone-hydrogel contact lenses in new wearers

Purpose: to evaluate changes in tear metrics and ocular signs induced by six months of silicone-hydrogel contact lens wear and the difference in baseline characteristics between those who successfully continued in contact lens wear compared to those that did not. Methods: Non-invasive Keratograph, Tearscope and fluorescein tear break-up times (TBUTs), tear meniscus height, bulbar and limbal hyperaemia, lid-parallel conjunctival folds (LIPCOF), phenol red thread, fluorescein and lissamine-green staining, and lid wiper epitheliopathy were measured on 60 new contact lens wearers fitted with monthly silicone-hydrogels (average age 36 ± 14 years, 40 females). Symptoms were evaluated by the Ocular Surface Disease Index (OSDI). After six months full time contact lens wear the above metrics were re-measured on those patients still in contact lens wear (n= 33). The initial measurements were also compared between the group still wearing lenses after six months and those who had ceased lens wear (n= 27). Results: There were significant changes in tear meniscus height (p= 0.031), bulbar hyperaemia (p= 0.011), fluorescein TBUT (p= 0.027), corneal (p= 0.007) and conjunctival (p= 0.009) staining, LIPCOF (p= 0.011) and lid wiper epitheliopathy (p= 0.002) after six months of silicone-hydrogel wear. Successful wearers had a higher non-invasive (17.0 ± 8.2. s vs 12.0 ± 5.6. s; p= 0.001) and fluorescein (10.7 ± 6.4. s vs 7.5 ± 4.7. s; p= 0.001) TBUT than drop-outs, although OSDI (cut-off 4.2) was also a strong predictor of success. Conclusion: Silicone-hydrogel lenses induced significant changes in the tear film and ocular surface as well as lid margin staining. Wettability of the ocular surface is the main factor affecting contact lens drop-out. © 2013 British Contact Lens Association.

Characterization of tissue transglutaminase in human osteoblast-like cells

Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular ε(ϒ-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,414C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p <0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.

Application of transglutaminases in the modification of wool textiles

The use of the protein-crosslinking enzymes transglutaminases (EC 2.3.2.13), as biocatalysts in the processing of wool textiles offers a variety of exciting and realistic possibilities, which include reducing the propensity of wool fabric to shrink and maintaining or increasing fabric strength. Guinea pig liver (GPL) transglutaminase or the microbial transglutaminase isolated from Streptoverticilium mobaraense, when applied to wool either alone or following a protease treatment, resulted in an increase in wool yarn and fabric strength (up to a 25% increase compared to a control). This indicates that transglutaminases can remediate the negative effects of proteolytic treatments in terms of loss in fibre strength. Incubation of samples pretreated with different oxidative and reducing agents with both sources of transglutaminases led to significant increases in tensile strength for all samples tested, suggesting that yarn strength lost following chemical treatments can also be recovered. The two different transglutaminases (TGases) could also impart a significant reduction in fabric shrinkage. The incorporation of primary amine transglutaminase substrates into wool fibres, with a view to altering wool functionality, was demonstrated using the incorporation of the fluorescent primary amine fluorescein cadaverine (FC). Incubation of wool with this fluorescent amine and transglutaminase led to high levels of incorporation into the fibres. The treatment of wool textiles with transglutaminases indicates that a number of novel and radically different finishes for wool textiles can be developed.

A collagen IV matrix is required for guinea pig gastric epithelial cell monolayers to provide an optimal model of the stomach surface for biopharmaceutical screening

The surface epithelial cells of the stomach represent a major component of the gastric barrier. A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. Primary cultures of guinea pig gastric mucous epithelial cells were grown on filter inserts (Transwells®) for 3 days. Tight-junction formation, assessed by transepithelial electrical resistance (TEER) and permeability of mannitol and fluorescein, was enhanced when collagen IV rather than collagen I was used to coat the polycarbonate filter. TEER for cells grown on collagen IV was close to that obtained with intact guinea pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [ 3H]glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on plastic culture plates, but no major difference was found between cells grown on collagens I and IV. The proportion of cells, which stained positively for mucin with periodic acid Schiff reagent, was greater than 95% for all culture conditions. Monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide, and were resistant to acidification of the apical medium to pH 3.0 for 30 min. A screen of nonsteroidal anti-inflammatory drugs revealed a novel effect of diclofenac and niflumic acid in reversibly reducing permeability by the paracellular route. In conclusion, the mucous cell preparation grown on collagen IV represents a good model of the gastric surface epithelium suitable for screening procedures. © 2005 The Society for Biomolecular Screening.

The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein

Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.

25th RCOphth Congress, President's Session paper:25 years of progress in medical retina

The quarter century since the foundation of the Royal College of Ophthalmologists has coincided with immense change in the subspecialty of medical retina, which has moved from being the province of a few dedicated enthusiasts to being an integral, core part of ophthalmology in every eye department. In age-related macular degeneration, there has been a move away from targeted, destructive laser therapy, dependent on fluorescein angiography to intravitreal injection therapy of anti-growth factor agents, largely guided by optical coherence tomography. As a result of these changes, ophthalmologists have witnessed a marked improvement in visual outcomes for their patients with wet age-related macular degeneration (AMD), while at the same time developing and enacting entirely novel ways of delivering care. In the field of diabetic retinopathy, this period also saw advances in laser technology and a move away from highly destructive laser photocoagulation treatment to gentler retinal laser treatments. The introduction of intravitreal therapies, both steroids and anti-growth factor agents, has further advanced the treatment of diabetic macular oedema. This era has also seen in the United Kingdom the introduction of a coordinated national diabetic retinopathy screening programme, which offers an increasing hope that the burden of blindness from diabetic eye disease can be lessened. Exciting future advances in retinal imaging, genetics, and pharmacology will allow us to further improve outcomes for our patients and for ophthalmologists specialising in medical retina, the future looks very exciting but increasingly busy.

Anterior eye health recording

Aims: To survey eye care practitioners from around the world regarding their current practice for anterior eye health recording to inform guidelines on best practice. Methods: The on-line survey examined the reported use of: word descriptions, sketching, grading scales or photographs; paper or computerised record cards and whether these were guided by proforma headings; grading scale choice, signs graded, level of precision, regional grading; and how much time eye care practitioners spent on average on anterior eye health recording. Results: Eight hundred and nine eye care practitioners from across the world completed the survey. Word description (p <. 0.001), sketches (p = 0.002) and grading scales (p <. 0.001) were used more for recording the anterior eye health of contact lens patients than other patients, but photography was used similarly (p = 0.132). Of the respondents, 84.5% used a grading scale, 13.5% using two, with the original Efron (51.6%) and CCLRU/Brien-Holden-Vision-Institute (48.5%) being the most popular. The median features graded was 11 (range 1-23), frequency from 91.6% (bulbar hyperaemia) to 19.6% (endothelial blebs), with most practitioners grading to the nearest unit (47.4%) and just 14.7% to one decimal place. The average time taken to report anterior eye health was reported to be 6.8. ±. 5.7. min, with the maximum time available 14.0. ±. 11. min. Conclusions: Developed practice and research evidence allows best practice guidelines for anterior eye health recording to be recommended. It is recommended to: record which grading scale is used; always grade to one decimal place, record what you see live rather than based on how you intend to manage a condition; grade bulbar and limbal hyperaemia, limbal neovascularisation, conjunctival papillary redness and roughness (in white light to assess colouration with fluorescein instilled to aid visualisation of papillae/follicles), blepharitis, meibomian gland dysfunction and sketch staining (both corneal and conjunctival) at every visit. Record other anterior eye features only if they are remarkable, but indicate that the key tissue which have been examined.

Poly(glycerol adipate-co-ω-pentadecalactone) spray-dried microparticles as sustained release carriers for pulmonary delivery

Purpose: The aim of this work was to optimize biodegradable polyester poly(glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL, microparticles as sustained release (SR) carriers for pulmonary drug delivery. Methods: Microparticles were produced by spray drying directly from double emulsion with and without dispersibility enhancers (L-arginine and L-leucine) (0.5-1.5%w/w) using sodium fluorescein (SF) as a model hydrophilic drug. Results: Spray-dried microparticles without dispersibility enhancers exhibited aggregated powders leading to low fine particle fraction (%FPF) (28.79±3.24), fine particle dose (FPD) (14.42±1.57 μg), with a mass median aerodynamic diameter (MMAD) 2.86±0.24 μm. However, L-leucine was significantly superior in enhancing the aerosolization performance ( L-arginine:%FPF 27.61±4.49-26.57±1.85; FPD 12.40±0.99-19.54±0.16 μg and MMAD 2.18±0.35-2. 98±0.25 μm, L-leucine:%FPF 36.90±3.6-43.38±5. 6; FPD 18.66±2.90-21.58±2.46 μg and MMAD 2.55±0.03-3. 68±0.12 μm). Incorporating L-leucine (1.5%w/w) reduced the burst release (24.04±3.87%) of SF compared to unmodified formulations (41.87±2.46%), with both undergoing a square root of time (Higuchi's pattern) dependent release. Comparing the toxicity profiles of PGA-co-PDL with L-leucine (1.5%w/w) (5 mg/ml) and poly(lactide-co-glycolide), (5 mg/ml) spray-dried microparticles in human bronchial epithelial 16HBE14o-cell lines, resulted in cell viability of 85.57±5.44 and 60.66±6.75%, respectively, after 72 h treatment. Conclusion:The above data suggest that PGA-co-PDL may be a useful polymer for preparing SR microparticle carriers, together with dispersibility enhancers, for pulmonary delivery. © Springer Science+Business Media, LLC 2011.

Pigments of sediment cores of the Northwest European Continental Margin

In the context of the European OMEX Programme this investigation focused on gradients in the biomass and activity of the small benthic size spectrum along a transect across the Goban Spur from the outer Celtic Sea into Porcupine Abyssal Plain. The effects of food pulses (seasonal, episodic) on this part of the benthic size spectrum were investigated. Sediments sampled during eight expeditions at different seasons covering a range from 200 m to 4800 m water depth were assayed with biochemical bulk measurements: determinations of chloroplastic pigment equivalents (CPE), the sum of chlorophyll a and its breakdown products, provide information concerning the input of phytodetrital matter to the seafloor; phospholipids were analyzed to estimate the total biomass of small benthic organisms (including bacteria, fungi, flagellata, protozoa and small metazoan meiofauna). A new term 'small size class biomass' (SSCB) is introduced for the biomass of the smallest size classes of sediment-inhabiting organisms; the reduction of fluorescein-di-acetate (FDA) was determined to evaluate the potential activity of ester-cleaving bacterial exoenzymes in the sediment samples. At all stations benthic biomass was predominantly composed of the small size spectrum (90% on the shelf; 97-98% in the bathyal and abyssal parts of the transect). Small size class biomass (integrated over a 10 cm sediment column) ranged from 8 g C/m**2 on the shelf to 2.1 g C/m**2 on the adjacent Porcupine Abyssal Plain, exponentially decreasing with increasing water depth. However, a correlation between water depth and SSCB, macrofauna biomass as well as metazoan meiofauna biomass exhibited a significantly flatter slope for the small size classes in comparison to the larger organisms. CPE values indicated a pronounced seasonal cycle on the shelf and upper slope with twin peaks of phytodetrital deposition in mid spring and late summer. The deeper stations seem to receive a single annual flux maximum in late summer. SSCB and heterotrophic activity are significantly correlated to the amount of sediment-bound pigments. Seasonality in pigment concentrations is clearly followed by SSCB and activity. In contrast to macro- and megafauna which integrate over larger periods (months/years), the small benthic size classes, namely bacteria and foraminifera, proved to be the most reactive potential of the benthic communities to any perturbations on short time scales (days/weeks). The small size classes, therefore, occupy a key role in early diagenetic processes.