The poor aqueous solubility of many APIs, such as felodipine, are significant dissolution rate limiting factors that often lead to poor oral systemic bioavailability. Solid dispersions have been used as a formulation approach to improve drugs dissolution properties. Most of the reported solid dispersion formulations in the literature are binary mixtures with limited functionalities (with enhancing dissolution being the primary function). The aim of this study is to design, characterise and evaluate complex solid dispersions with intentionally designed microstructures (in the form of phase separations). The potential functionalities of these microstructures were explored by this project. In our view, the complex formulations are more representative of real pharmaceutical products in their final forms. HME-IM is a single processing technique for fabricating formulations with high geometric precision in a rapid, efficient and environmentally friendly way. It was used as the main processing method to produce the solid dispersion based buccal patches in this project. The patches were thoroughly characterised using conventional techniques including DSC, MTDSC, TGA, DVS, ATR-FTIR, PXRD, SEM, EDS, IR imaging, mucoadhesion and in vitro dissolution testing. In order to address the spatial distribution of the phase separations, two non-conventional characterisation methods, thermal analysis by structural characterisation and X-ray microcomputed tomography, were introduced to provide novel insights into the heterogeneity and phase distribution of these formulations. The results revealed that HME-IM patches with 10% drug loading were unsaturated while those with 20-30% w/w drug concentration were saturated or even supersaturated. HME-IM patches containing TPGS were more solubilising to felodipine and more stable compared to Tween 80 containing systems. Thermal analysis by structural characterisation provided rapid detection of heterogeneity and the thermal dissolution of crystalline drug fraction while XμCT provided microscale spatial distribution of different phases. Having shown the advantages of using polymeric blends to formulate solid dispersions that were demonstrated by the felodipine buccal patches, we further explored the use of polymer blends for improving the FDM 3D printability of pharmaceutical solid dosage forms with the potential applications in personalised medicine. This project demonstrated the potential and formulation principles of using HME-IM and FDM 3D printing as formulation methods for production of polymer blend based complex solid dispersions for the purposes of enhanced bioavailability of poorly soluble drugs and providing personalised medicines.

Salbutamol was synthesised in 1967 and is widely known now as a Beta-adrenergic agonist. It is known that p-agonists cause hypertrophy in various mammalian muscles cells, however the mechanisms by which this occurs are not fully understood. Recent studies have suggested that there is great potential for using Beta-agonists, specifically Beta-agonists, in restoring muscle mass and strength in humans. Although, salbutamol is commonly used as a bronchodilator, it has heen identified as having an effect on muscle growth at a dose which is safe for humans. However, like all steroids there are many side effects associated with Beta-agonists. By understanding the mechanisms affected within muscle cells by treatment with salbutamol it will enable not only the understanding of muscle hypertrophy but lead to more specific drugs being designed.

Colloidal Liquid Aphrons (CLAs) have been used in a variety of applications ranging from detergency and solute extraction, to enzyme immobilisation, and have been shown to have very high mass transfer areas due to their size (5-20μm). Also, due to their structure they are very stable while also being naturally buoyant in aqueous solutions, providing natural homogeneity. Over the last few decades their use in the laboratory as an immobilisation support for enzymatic catalysis has been extensive due to their stability, and ability to promote superactivity. Due to these specific characteristics, the possibility of using CLAs as a stable formulation for drug delivery has been particularly attractive. However, in an effort to apply existing research to the area of drug delivery, a new formulation methodology was developed utilizing non-ionic surfactants, nonpolar solvents, and model proteins as active pharmaceutical ingredients with varying molecular weights and isoelectric points (pI). Insights into the chemical forces governing immobilisation showed that hydrophobic interactions were primarily responsible for binding. Adsorption was the main mechanism for immobilisation, with higher affinities being observed with increasing protein concentration and smaller particle size. Superactivity was observed with Lipase and α-chymotrypsin, while aprotinin retained 85% of its inhibitor potency. Evaluation of immobilised enzyme conditions showed that non-ionic CLAs preserved natural pH and temperature optima, while thermodynamic evaluations of activity suggested that the presence of water molecules lead to an active conformation. Characterisation studies on refractive index matched polyaphron systems showed that proteins interacted mainly with the ‘’soapy-shell’’ leading to a hydrated conformation, while calorimetric studies on nonionic surfactant binding proved that surfactant interactions were virtually non-existent. Desorbed enzymes regained their natural conformation illustrating that any small structural effects were reversible upon release. The use of sodium alginate as an additive for enhanced immobilisation proved successful due to induced electrostatic interactions when the pH was lower than the protein pI. Conformational effects upon binding with sodium alginate also proved to be reversible, with an increase in the concentration of the released protein being observed when the pH was >pI. However, the extent of electrostatic interactions on the bioactivity of released proteins was found to be non-denaturing with hydrophilic enzymes, while hydrophobic enzymes were more active upon binding. Finally, insights into proteolytic digestion suggest that electrostatic interactions lead to greater protease vulnerability due to conformational changes induced upon binding.

One of the reasons for the rejection of new drugs during clinical trials is the presence of host cell proteins (HCPs) in the drug formulation. HCPs are immunogenic impurities that can compromise patient safety. Moreover, proteolytic and binding HCPs compromise the integrity, and, hence, the stability and efficacy of a recombinant protein. Therefore, HCPs should be removed from the bioprocess train as soon as possible. Current downstream purification platforms are challenged by HCP-mAb co-elution. A Quality by Design strategy to overcome this problem is to reduce the number of HCPs in the downstream feedstock by tracing their source back to upstream culture and eliminating it. Previous studies have shown that upstream cell culture parameters, e.g. harvest time and culture temperature, significantly affect HCP profiles. However, little is known about how host cells coordinate and regulate their molecular machinery under different cell culture environment that results in different HCP profiles. This study presents experimental results linking cell culture temperature and key process indicators of CHO cell cultures and post-Protein A purification (mAb titre, HCP level and HCP species) by considering the cellular behaviour in terms of cell growth, cell cycle distribution and cell health). This study involved the application of single-use fed-batch bioreactors to culture IgG4 producing GS-CHO cell line, cell health and cell cycle analysis with NucleoCounter, Protein A purification and proteomic analysis with HCP ELISA kits and LC-MS/MS. Cells were more robust under mild hypothermia: over 90% of cells were maintained in a healthy state until the decline phase, and the onset of apoptosis was less evident compared to the results for physiological temperature. IgG4 titre and HCP level at harvest were comparable between the two cases. However, mild hypothermia reduced the HCP variety in HCCF by 36%, including 44% and 27% lower proteases and chaperones, respectively. The differences in HCP variety at harvest resulted in a significantly different HCP profile post-Protein A purification between the two cases. Half of the critically immunogenic HCPs species as determined by the CHOPPI tool were different between the two cases. This study shows that cell culture conditions significantly affect the HCP profile at harvest and that of purified samples.

The transmission of Clostridium difficile infection (CDI) is mediated by spores, which are highly resistant to heat and disinfectants. The healthcare laundry policy, Health Technical Memorandum 01-04 Decontamination of linen for health and social care, provides minimum disinfection conditions and microbiological standards for laundered linen: no bacteria on previously sterile de-sized textiles, > 5 log(10) reduction of a thermotolerant species of bacteria and < 100 cfu with no pathogenic bacteria on sampled linen. Quantification of the survival of spores, from hospital sheets (100% cotton) naturally contaminated with C. difficile spores were laundered in a washer extractor (WE) at a commercial laundry; they failed the microbiological standards. Similar results were achieved in a simulated healthcare WE cycle. The industrial detergent used failed the test for sporicidal activity (BS EN 13704), with a 2.81 log(10) reduction in spores. The method of recovering spores from swatches was important; in the presence of soiling, agitation by vortexing (4.48 log(10) cfu/25cm2) was more effective than stomaching (4.2 log(10) cfu/25cm2, p≤0.05). Spore adherence to cotton occurred over time, with 0% (0 hours) and 51% (24 hours) adherence; adherence decreased to 34% (24 hours) after exosporium removal, suggesting a role in spore adherence to cotton. The possibility cannot be discounted that low-level spore survival on processed linen may be contributing to environmental contamination and asymptomatic carriage.

Substandard and falsified medicines (SF) are a major global public health problem and occur throughout healthcare systems worldwide. With an average occurrence of 10% of all medicines globally, the specific levels range from ~1% of all medicines in high-income countries (HIC) to as much as 30-40% in low-income countries (LIC) and low-middle income countries (LMIC). Current detection methods for SF medicines range from ‘low tech’ approaches, for example, visual appearance, thin layer chromatography (TLC) to those that are technically refined, such as, Raman spectroscopy, Near Infrared (NIR) spectroscopy and Mass Spectrometry (MS). In order to counteract the increasing complexity of anti-counterfeit measures, counterfeiters are finding ever more sophisticated ways to bypass existing screening techniques. Therefore, the aim of this research is to develop a novel rapid screening method to identify SF medicines. The performance of seven different benchtop instruments has been investigated with respect to their potential to deliver rapid assessment of the identity and to quantify the level of Active Pharmaceutical Ingredient’s (API’s) present in tablet samples. The materials used in this research comprised of 29 reference samples and 64 individual group samples from 8 countries, totaling some 867 tablets. Tablets were analysed in both whole and powdered forms. Mass Spectrometry provided the least complex data, followed by Raman and then Attenuated Total Reflectance Fourier Transform Infrared (ATR FTIR). The mass spectrometer was the least reliable instrument, but provided the greatest sensitivity. Successful identification of the API was obtained from ATR FTIR and Raman analysis and from Direct Insertion Probe (DIP) Mass Spectrometry. Quantitative levels of API could be obtained from Ultraviolet (UV) and ATR FTIR measurements, whilst some excipient levels could be determined by Elemental Dispersive X-ray (EDX) spectroscopy. Handheld Raman systems produced some erroneous quantitative information. The majority of samples examined were within ±10% of the stated level, as per the British Pharmacopeia specifications; however, there was some evidence of substandard medication. Substandard medication was suspected in 2 of the 64 pharmaceutical products assessed. At the present state of development, the Raman and ATR FTIR equipment could be used in LMIC’s for the screening of tablet samples. PCA in conjunction with Raman spectroscopy identified an anomalous sample in a set of proprietary preparations from different countries. A combination of the techniques cited confirmed the inclusion of a non-standard excipients into this formulation.

INTRODUCTION: Androgenic alopecia (AA) patients usually have high levels of dihydrotestosterone on their balding scalp area. Currently, dutasteride (DST) is given orally and has systemic adverse effects; diminished sexual desire, increased depression and ejaculation disorder. Topical administration of DST is an appropriate drug-delivery strategy with the potential to reduce systemic side effect, skin irritation and cytotoxicity effects. MATERIALS AND METHOD: Chitosan oligomer (CSO) conjugated with stearic acid (SA) or lauric acid (LA) was synthesised and characterised. Dutasteride-loaded nanostructured lipid carriers (DST-NLCs) were prepared using a melt-dispersion ultrasonication method. DST-NLCs were optimised using a design of experiments approach. DST-NLCs, uncoated and coated with CSO-SA or CSO-LA were characterised for particle size distribution, surface charge and morphology. In vitro release and permeation studies were performed. Cytotoxicity was investigated using human hair follicle dermal papilla cells, and skin irritation was performed using an EpiDermTM RHE model. Cou-6 loaded NLCs were prepared and characterised before proceeding with the cell and skin uptake study. RESULTS: CSO-SA and CSO-LA were successfully synthesised; confirmed using 1H NMR and FTIR. The mean size of DST-NLCs was significantly increased (p < 0.05) when coated with 5% CSO-SA but not with 5% CSO-LA (p > 0.05). The zeta potential changed from negative to positive charge when coating DST-NLCs with CSO-SA or CSO-LA. All formulations were physically stable over six months when stored at 4-8°C. However, DST-NLCs coated with CSO showed aggregation. All formulations exhibited rapid drug release. No dutasteride permeated through pig ear skin after 48 h for all formulations. The cytotoxicity (IC50) for DST nanoparticles, coated and uncoated, was greater than for DST alone (p < 0.05). The in vitro skin irritation study indicated no irritation for all nanoparticle preparations. For the cell and skin uptake studies, all samples showed time-dependent skin and cell uptake. CONCLUSIONS: These stable, low cytotoxic and irritant, positively-charged DST-NLCs with CSO-SA or CSO-LA, represents a promising strategy for topical/ transfollicular delivery of DST.

The rapid development of antimicrobial resistance over the past three decades represents a critical public health threat and urgent challenge. In this context, in order to establish lead compounds or identify novel modes of action one approach is to re-investigate drugs that affect eukaryotic processes for antimicrobial activity, particularly if their drug targets have homologies with bacterial proteins. In this study, regioisomers of LY2183240, a potent inhibitor of anandamide transport and fatty acid amide hydrolase were selected after a small screening study. The 2,5-LY2183240 regioisomer was shown to possess potent antimicrobial activity selective towards certain Gram-positive bacteria, which included Staphylococcus aureus and Bacillus subtilis, but not Enterococcus faecalis or Streptococcus pneumoniae. Conversely, the 1,5-LY2183240-regioisomer had no anti-bacterial activity strongly implicating the position of the carbamoyl on the tetrazole in the structure-activity relationship of the molecule. Investigation of the mechanism of antimicrobial activity suggested that while 2,5- LY2183240 had bacteriostatic activity this was probably not due to inhibition of protein or teichoic acid synthesis. Nevertheless, this activity may be related to the inhibition of bacterial fatty acid synthesis. Supporting this hypothesis, addition of exogenous fatty acids within Tween 80 was able to compromise 2-5-LY2183240 anti-staphylococcal activity. Based on the spectrum of activity and known redundancies at each of the steps in the fatty acid synthesis pathway, the most likely target was deduced to be FabI. However, characterization of a 2,5-LY2183240- resistant mutant revealed no alteration to the deduced amino acid sequence of FabI or relevant changes to FabI protein expression, an observation confirmed by analysis of the fabI promoter region and western blot studies. Since resistance to 2,5- LY2183240 could be mediated through non-target related factors such as drug efflux or drug entry into the cell, FabI could not be ruled out as a potential target. In addition, whole cell protein profiling revealed the 2,5-LY2183240-mutant to have differences in the expressions of several proteins, suggesting resistance may occur through a more global effect. Due to the promiscuous nature of LY2183240, the possibility of having multiple targets in S. aureus that collectively exhibit a bacteriostatic effect cannot be ruled out. This study also showed that the LY2183240 regioisomers are specific inhibitors of class C β-lactamases with optimum inhibitory activity dependent upon the position of the carbonyl on the tetrazole heterocyclic group (Ki values of 1.8 and 2.45 μM for 1,5- and 2,5-LY2183240, respectively). Molecular modelling suggested that LY2183240 regioisomers bind within the catalytic site of class C β-lactamases, interacting with some of the same residues, including Tyr150, Lys315 and Thr316, used by the substrate nitrocefin and β-lactamase inhibitors. The results substantiate the competitive inhibition model determined by enzyme kinetic studies. Furthermore, mass spectrometry data revealed non-covalent interactions between AmpC β- lactamase and LY2183240 regioisomers, which together with the non-reversible inhibitory activity, may indicate that both regioisomers present a high affinity towards this cephalosporinase and dissociate very slowly from the enzyme. LY2183240 may prove useful as a chemical scaffold for the development of novel and highly selective inhibitors of class C β-lactamases.

Background: Diabetes is a threat to peoples’ health and lives around the world. Particularly, there is a large increase in its prevalence in the Middle East, especially type 2 diabetes. Knowledge, beliefs and attitudes are the major contributing factors to medicine misuse and poor glycaemic control. Insulin is the last treatment option that patients with type 2 diabetes might require to control their disease. In addition to traditional injections, newer insulin delivery methods, such as pens and pumps are available, which may impact medicine use. Aim To identify factors that impact the management of type 2 diabetes by examining patients’ experiences about their medicine/device use and lifestyle and exploring the perspectives of healthcare providers (HCPs) to inform service development. Method The study was conducted in hospitals in Kuwait. All patients (n=43) using medical treatment for the management of type 2 diabetes and HCPs (n=10) who were involved in management of those patients were invited to participate. Data were collected primarily through semi-structured interviews. Data analysis was performed using MAXQDA-11. Results Non-adherence to medicines was prevalent in this study. Interviews with patients revealed that poor health awareness, health beliefs and culture influenced health behaviour, in terms of medicine use and lifestyle. Some of the emergent beliefs were about the disease (downplaying its seriousness), medicines (disbelief in their efficacy) and self-beliefs. Using pens and pumps improved patients’ adherence, quality of life and satisfaction. Interviews with HCPs revealed that some barriers affected management of the disease, such as lack of staff, incentives and equipment. Recommendations to inform healthcare provision were identified. Conclusion To improve the management of type 2 diabetes and health outcomes, many issues need to be considered. For example, addressing patients’ beliefs, and the benefits of insulin pens and pumps and expanding their use. Taking into account concerns of HCPs would be valuable to inform service development.

The expertise of the health professionals involved in care delivery influences the overall quality of care provided in the health sector. Empirical evidence demonstrates that when the competencies that are essential for practice are identified, compiled to form a framework, and used alongside standards of practice: it aids expertise development and fosters improvement of professional performance. This research aimed to identify and evaluate foundation and advanced level pharmacy practice competencies. Its objective was to validate the FIP Global Competency Framework (GbCF v1) as a mapping tool for use in the development of country-specific pharmacy practice frameworks in the African region. Its second objective was to evaluate the competencies that are essential for global advanced pharmacy practice. An online cross-sectional survey of pharmacists from fourteen African countries (n=469) demonstrates that 90% of the foundation level competencies contained in the GbCF v1 are relevant to practice. This validates the GbCF v1 as a mapping tool that can be used to develop country-specific frameworks for early career practitioners in the selected countries. A systematic literature review and content analysis of two national frameworks identified 64 competencies and 237 behaviours for advanced pharmacy practice. An expert panel review (n= 14) developed consensus on these competencies via a modified Delphi technique. Based on consensus, 89.5% of the identified behaviours were similar between the two frameworks. A crossover mapping experiment involving advanced practitioners (n=42) from four countries showed within-subject agreement for matching competencies in the frameworks. This agreement was significant for 84% of the competencies (k ≥ 0.25; p ≤ 0.05). Semi structured interviews of advanced practitioners (n=17) indicates the identified competencies were all relevant to practice. General consensus on similarity between the two frameworks was also obtained from the interviews. This demonstrates the existence of a core set of competencies applicable to advanced pharmacy practitioners from different countries. It provides evidence of the feasibility of defining the competencies essential for global advanced pharmacy practice. The research reported in this thesis provides evidence that was previously lacking on the validity of the Global Competency Framework in specific countries in Africa. It also provides preliminary data on the competencies that are required for global advanced pharmacy practice. Overall, these findings provide valuable insights that can be developed through further research to serve as a driver for global policy.