Colloidal drug probe : method development and validation for adhesion force measurement using atomic force microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between the drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from a dry powder inhaler (DPI) formulation. Initially model silica probes of approximately 4 μm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-Ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres preattached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

Clinical utility of exhaled nitric oxide

Lower airway inflammation is generally classified as eosinophilic or neutrophilic. In conditions where eosinophilic inflammation predominates such as asthma in children, corticosteroids are usually beneficial. Traditionally, lower airway eosinophilia is measured using cellular count (through bronchoalveolar lavage or induced sputum). Both methods have limited applicability in children. When instruments to measure fractional exhaled nitric oxide (FeNO) became available, it presented an attractive option as it provided a non-invasive method of measuring eosinophilic inflammation suitable for children and adult. Not surprisingly, proposals have been made that FeNO measurement can be clinically used in many scenarios including monitoring the response to anti-inflammatory medications, to verify the adherence to treatment, and to predict upcoming asthma exacerbations. This thesis addresses the utility of FeNO levels in various scenarios, specifically in relation to asthma control and cough, a contentious aspect of the diagnosis of asthma. The thesis consists of a series of systematic reviews (related to the main question) and original studies in children. The over-arching aim of the thesis is to determine if FeNO is a clinically useful tool in the management of asthma and common asthma symptoms. The specific aims of the thesis were, to: 1. Determine if children with asthma have more severe acute respiratory symptoms at presentation with an asthma exacerbation and at days 7, 10 and 14 using validated scales. We also examined if children with asthma were more likely to have a persistent cough on day 14 than children with protracted bronchitis and/or controls. 2. Evaluate the efficacy of tailoring asthma interventions based on sputum analysis in comparison to clinical symptoms (with or without spirometry/peak flow) for asthma related outcomes in children and adults. 3. Evaluate the efficacy of tailoring asthma interventions based on exhaled nitric oxide in comparison to clinical symptoms (with or without spirometry/peak flow) for asthma related outcomes in children and adults. 4. Determine if adjustment of asthma medications based on FeNO levels (compared to management based on clinical symptoms) reduces severe exacerbations in children with asthma. 5. Examine the relationship between FeNO and exercise induced broncho-constriction and cough in children The aims above are addressed in respective chapters and all but one has been published/submitted. A synopsis of the findings are: In study-1 (Aim 1), we found that children with protracted bronchitis had the most severe acute respiratory infection symptoms and higher percentage of respiratory morbidity at day 14 in comparison to children with asthma and healthy controls. The systematic review of study-2 (Aim 2) included 246 randomised adult participants (no children) with 221 completing the trials. In the meta-analysis, a significant reduction in number of participants who had one or more asthma exacerbations occurred when treatment was based on sputum eosinophils in comparison to clinical symptoms. In the systematic review of study-3 (Aim 3), we found no significant difference between the intervention group (treatment adjusted based on FeNO) and control group (treatment adjusted based on clinical symptoms) for the primary outcome of asthma exacerbations or for the other outcomes (clinical symptoms, FeNO level and spirometry). In post-hoc analysis, a significant reduction in mean final daily dose ICS per adult was found in the group where treatment was based on FeNO in comparison to clinical symptoms. In contrast, in the paediatric studies, there was a significant increase in ICS dose in the FeNO strategy arm. Thus, controversy remains of the benefit or otherwise of utilising exhaled nitric oxide (FeNO) in routine clinical practice. FeNO levels are dependent on atopy and none of the 7 published trials have considered atopic status in FeNO levels when medications were adjusted. In study-4 (Aim 4), 64 children with asthma were recruited. Their asthma medications were adjusted according to either FeNO levels or usual clinical care utilising a management hierarchy taking into account atopy. It was concluded that tailoring of asthma medications in accordance to FeNO levels (compared to usual management), taking into account atopy status, reduced the number of children with severe exacerbations. However, a FeNO-based strategy resulted in higher daily ICS doses and had no benefit on asthma control. In study-5 (Aim 5), 33 children with cough and 17 controls were recruited. They were randomised to undertake an exercise challenge on day 1, or dry powder mannitol challenge on day 1 (with alternative challenge being done on day 2). In addition, a 24 hour cough meter, skin prick test, capsaicin cough sensitivity test and cough diary were undertaken. The change in cough frequency post exercise was significantly increased in the children with cough. FeNO decreases post exercise regardless of whether EIB is present or not. Limitations in the studies were addressed in the respective chapters. In summary, the studies from this thesis have provided new information on: • The severity of respiratory symptoms was increased in the early phase of the asthma exacerbation but not in the later recovery phase when compared with controls. • The utility of FeNO in the management of children with asthma. • The relationship of FeNO, cough and EIB in children. • Systematic reviews on the efficacy of tailoring asthma interventions based on eosinophilic inflammatory markers (sputum analysis and FeNO) in comparison to clinical symptoms.

Colloidal drug probe : Method development and validation for adhesion force measurement using Atomic Force Microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between a model drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from dry powder inhaler (DPI) formulations. Model silica probes of approximately 4 lm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres pre-attached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/ hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

Solubility of ibuprofen in aqueous ethanol at low ethanol contents

The solubility of ibuprofen was measured in water (W) - ethanol (E) mixtures from 0 to 50% w/w ethanol at 10, 25 and 40 °C by the dissolution method using UV spectrophotometry to determine the ibuprofen concentrations. The UV calibration for ibuprofen in different water - ethanol mixtures showed Beer - Lambert linearity, however the slopes differed, which indicated the structure of the drug is influenced by the solvent system i.e. the water - ethanol ratio. The ibuprofen solubility in water (zero ethanol) is low (~ 50 ppm) but increases near exponentially with increasing ethanol content. At 40 °C, there is phase separation between 34% and 63% w/w E/(E+W). The solubility data will be used to select precipitation crystallizer conditions to directly produce free flowing ibuprofen particles (<5 m) for developing a dry powder inhaler (DPI) formulation for lung delivery.

Influence of smectite suspension structure on sheet orientation in dry sediments : XRD and AFM applications

The structure-building phenomena within clay aggregates are governed by forces acting between clay particles. Measurements of such forces are important to understand in order to manipulate the aggregate structure for applications such as dewatering of mineral processing tailings. A parallel particle orientation is required when conducting XRD investigation on the oriented samples and conduct force measurements acting between basal planes of clay mineral platelets using at. force microscopy (AFM). To investigate how smectite clay platelets were oriented on silicon wafer substrate when dried from suspension range of methods like SEM, XRD and AFM were employed. From these investigations, we conclude that high clay concns. and larger particle diams. (up to 5 μm) in suspension result in random orientation of platelets in the substrate. The best possible laminar orientation in the clay dry film, represented in the XRD 0 0 1/0 2 0 intensity ratio of 47 was obtained by drying thin layers from 0.02 wt.% clay suspensions of the natural pH. Conducted AFM investigations show that smectite studied in water based electrolytes show very long-range repulsive forces lower in strength than electrostatic forces from double-layer repulsion. It was suggested that these forces may have structural nature. Smectite surface layers rehydrate in water environment forms surface gel with spongy and cellular texture which cushion approaching AFM probe. This structural effect can be measured in distances larger than 1000 nm from substrate surface and when probe penetrate this gel layer, structural linkages are forming between substrate and clay covered probe. These linkages prevent subsequently smooth detachments of AFM probe on way back when retrieval. This effect of tearing new formed structure apart involves larger adhesion-like forces measured in retrieval. It is also suggested that these effect may be enhanced by the nano-clay particles interaction.

Predicting dry eye using non-invasive techniques of tear film surface assessment

PURPOSE. To measure tear film surface quality in healthy and dry eye subjects using three noninvasive techniques of tear film quality assessment and to establish the ability of these noninvasive techniques to predict dry eye. METHODS. Thirty four subjects participated in the study, and were classified as dry eye or normal, based on standard clinical assessments. Three non-invasive techniques were applied for measurement of tear film surface quality: dynamic-area high-speed videokeratoscopy (HSV), wavefront sensing (DWS) and lateral shearing interferometry (LSI). The measurements were performed in both natural blinking conditions (NBC) and in suppressed blinking conditions (SBC). RESULTS. In order to investigate the capability of each method to discriminate dry eye subjects from normal subjects, the receiver operating curve (ROC) was calculated and then the area under the curve (AUC) was extracted. The best result was obtained for the LSI technique (AUC=0.80 in SBC and AUC=0.73 in NBC), which was followed by HSV (AUC=0.72 in SBC and AUC=0.71 in NBC). The best result for DWS was AUC=0.64 obtained for changes in vertical coma in suppressed blinking conditions, while for normal blinking conditions the results were poorer. CONCLUSIONS. Non-invasive techniques of tear film surface assessment can be used for predicting dry eye and this can be achieved in natural blinking as well as suppressed blinking conditions. In this study, LSI showed the best detection performance, closely followed by the dynamic-area HSV. The wavefront sensing technique was less powerful, particularly in natural blinking conditions.

Effects of dry density and grain size distribution on soil-water characteristic curves of sandy soils

The unsaturated soil mechanics is receiving increasing attention from researchers and as well as from practicing engineers. However, the requirement of sophisticated devices to measure unsaturated soil properties and time consumption have made the geotechnical engineers keep away from implication of the unsaturated soil mechanics for solving practical geotechnical problems. The application of the conventional laboratory devices with some modifications to measure unsaturated soil properties can promote the application of unsaturated soil mechanics into engineering practice. Therefore, in the present study, a conventional direct shear device was modified to measure unsaturated shear strength parameters at low suction. Specially, for the analysis of rain-induced slope failures, it is important to measure unsaturated shear strength parameters at low suction where slopes become unstable. The modified device was used to measure unsaturated shear strength of two silty soils at low suction values (0 ~ 50 kPa) that were achieved by following drying path and wetting path of soil-water characteristic curves (SWCCs) of soils. The results revealed that the internal friction angle of soil was not significantly affected by the suction and as well as the drying-wetting SWCCs of soils. The apparent cohesion of soil increased with a decreasing rate as the suction increased. Further, the apparent cohesion obtained from soil in wetting was greater than that obtained from soil in drying. Shear stress-shear displacement curves obtained from soil specimens subjected to the same net normal stress and different suction values showed a higher initial stiffness and a greater peak stress as the suction increased. In addition, it was observed that soil became more dilative with the increase of suction. A soil in wetting exhibited slightly higher peak shear stress and more contractive volume change behaviour than that of in drying at the same net normal stress and the suction.

A Survey of Viruses Affecting Dry Bean and Cowpea in Lebanon

A survey was conducted to identify viruses affecting dry bean (Phaseolus vulgaris) and cowpea (Vigna unguiculata) in Lebanon. Three hundred and thirty four samples exhibiting virus-like symptoms were collected from 13 different locations during the fall growing season of 1984. Samples were stored at 20 deg C until they were tested by the enzyme-linked immunosorbent assay (ELISA) for the presence of blackeye cowpea mosaic virus (B1CMV), bean yellow mosaic virus (BYMV), bean common mosaic virus (BCMV) and cucumber mosaic virus (CMV). In preliminary tests, the extraction buffer 0.1M phosphate + O.1MEDTA, pH 7.4 was found to be far better than the standard extraction buffer and, accordingly, was used for virus extraction for all field samples. Results obtained indicated that around 50% of the bean samples tested were infected with B1CMV. Incidence of BCMV, BYMV and CMV in the samples tested were 4,4 and 1.7%, respectively. B1CMV was detected in 10 locations, whereas, BYMV, BCMV and CMV were found in 1,4 and 4 locations, respectively. Mixed infections such as BCMV, BICMV, BCMV+CMV, BYMV+CMV and BICMV+BCMV+CMV were detected. In 35% of the samples assayed, the causal virus was not identified

Adhesion and dispersion of salmeterol xinofoate from lactose interactive mixtures for inhalation - can surface roughness of carriers determine performance

The objective was to understand the influence of the surface roughness of lactose carriers on the adhesion and dispersion of salmeterol xinafoate (SX) from interactive mixtures. The surface roughness of lactose carriers was determined by confocal microscopy. Particle images and adhesion forces between SX and lactose particles were determined by Atomic Force Microscopy. The dispersion of SX (2.5%) from interactive mixtures with lactose was determined using a twin-stage impinger (TSI) with a Rotahaler® at an airflow rate of 60L/min. SX was analysed using a validated HPLC assay. The RMS Rq of lactose carriers ranged from 0.93-2.84μm, the Fine Particle Fraction (FPF) of SX ranged between 4 and 24 percent and average adhesion force between a SX and lactose particles ranged between 49 and 134 nN. No direct correlation was observed between the RMS Rq of lactose carriers and either the FPF of SX for the interactive mixtures or the adhesion force of a SX on the lactose particles; however, the presence of fine lactose associated with the carrier surface increased the FPF of SX. Dispersion through direct SX detachment from the carrier surface was not consistent with the poor correlations described and was more likely to occur through complex particulate interactions involving fine lactose.