For a drug to be effective it must be in solution so that it can be absorbed by the body at the site of action. Therefore, it must be soluble in an aqueous environment. For practical reasons many drugs are formulated in the solid state; an understanding of how the drug transitions from solid to solution is hence critical. The two key factors to be considered are the drug’s solubility and dissolution rate. In this work a novel method for the analysis of phase transitions in pharmaceutical materials was developed by combining high energy synchrotron X-ray diffraction with differential scanning calorimetry (DSC-XRD). This was used to collect in situ structural and calorimetric data on temperature driven phase transitions. Initial work was carried out to prove the utility of the technique by examining two enantiotropically polymorphic systems, and subsequently it was used to examine phase transitions in materials exhibiting multiple polymorphs. Building on this, the effect of sugar based excipients on the crystallisation behaviour of paracetamol glasses upon heating was examined, with particular emphasis on the stabilisation of metastable polymorphs II and III. DSC-XRD was further employed in the production of new co-crystals between isonicotinamide and three antioxidant compounds. The ability of four different techniques (solvent evaporation, thermal inkjet printing, heating, and milling) to prepare these co-crystals was investigated in detail. Finally, physical vapour deposition, a novel technique in the field of pharmaceuticals, was investigated for the preparation of amorphous materials, with particular emphasis on drugs considered to be poor glass formers. DSC-XRD has been demonstrated to be an incredibly powerful tool for the analysis of phase transitions and co-crystallisation in pharmaceutical materials, and physical vapour deposition has been shown to have great potential for preparing amorphous drugs from poor glass formers.

Diazepam is an allosteric modulator of GABAA receptors which potentiates GABAA receptor activity resulting in enhanced inhibitory synaptic transmission. Diazepam is used to treat anxiety, insomnia and seizures, however, its use is limited due to the development of tolerance. Here I show that prolonged treatment of cortical neurones with diazepam triggers endocytosis and subsequent downregulation of cell-surface GABAA receptors. Using pharmacological reagents, I have demonstrated that diazepam triggers PLC-dependent release of calcium from the endoplasmic reticulum which activates the phosphatase calcineurin resulting in dephosphorylation of the γ2 subunit of GABAA receptors and their endocytosis. This was elucidated using a combination of biochemical and cell biological approaches. In addition, I have developed HEK293 cell lines stably expressing various subtypes of GABAA receptors to investigate further diazepam and isoguvacine-dependent regulation of GABAA receptors. The same calcium-dependent signalling pathway that regulates cell-surface stability of GABAA receptors in neurones was found to operate in HEK293 cells. Subsequently, I focused on a key component of this signalling pathway; PLCδ1. Using biochemical techniques I have demonstrated that PLCδ1 binds directly to the GABAA receptor β3 subunit at two independent sites. This binding was confirmed by coimmunoprecipitation of PLCδ1 and GABAA receptors from cortical neuronal lysates. Interestingly, upon diazepam treatments, PLCδ1 was shown to dissociate from GABAA receptors, thus leading to mobilisation of calcium from the intracellular stores and activation of calcineurin. To assess how changes in cell-surface expression of GABAA receptors affect the stability of GABAergic synapses, I characterised the size and number of post-synaptic GABAA receptor clusters and the number of presynaptic GABA-releasing terminals following chronic diazepam treatment. I observed a reduction in the size and number of post-synaptic GABAA receptor clusters and a reduction in the number of GABA-releasing terminals. These data are consistent with the loss of cell-surface GABAA receptors following long-term treatments of cortical neurones with diazepam. These changes correlated with an increase in the expression of the early apoptosis marker, cleaved-caspase 3, in glutamatergic neurones suggesting indirect cytotoxic effects of diazepam treatments. The loss of inhibitory GABAergic synapses following chronic diazepam treatment may contribute to the well-known development of tolerance to these clinically important therapies for stress- and anxiety- related neurological disorders.

The interplay between cells and biomaterials constitutes a fertile ground to probe specific cellular functions and cues for therapeutic and research purposes. “Smart” materials encompass an extensive library that can lead to the design of dynamic multi-responsive constructs with great importance in the biomedical field. This work aims to describe diverse strategies on the modification of biological interfaces with synthetic polymers to promote the assembly of living cells and the design of multi-responsive healable cell-encapsulating constructs with interest in 3D in vitro modelling, drug delivery, cell-based therapies and tissue engineering. In the first part, cell membrane engineering approaches are introduced to create a responsive platform for the accelerated and simple formation of cellular aggregates/spheroids, and to study polymer-cell interactions by exploring biorthogonal ligand-receptor multivalent interactions under different conditions. Specifically, boronic acid- and succinimide-based copolymers were first synthesised and fully characterised by physicochemical methods, and found to bind covalently to natural moieties present on the membrane of several cell lines, which can regulate the development of cell spheroids and act as self-supporting “cellular glues”. The second part of the project is dedicated to the development of multi-responsive self-healing hydrogel nanocomposites for biomedical applications, where we further expanded the dynamic crosslinking nature of boronate ester bonds. The proposed gels could be prepared almost instantly, exhibited photo- and thermoreversible transient sol-gel type of transition with excellent healing properties, and no toxicity, which allows the system to be used as a versatile biologic delivery matrix. In summary, the results highlight novel and straightforward approaches that may pave the way to implement a biomaterial-cell platform with broad biotechnological applications.

When an active pharmaceutical ingredient (API) is encapsulated within, or attached to, a carrier, drug safety and efficacy can be considerably increased and new therapies are possible. In this work, two different types of low-dimensional carriers have been explored as potential advanced drug delivery systems. These comprise polymer nanofibers, a type of 1D-system, and two 2D systems: layered double hydroxides (LDHs) and hydroxy double salts (HDSs). Three types of nanofibers were initially produced by electrospinning: polyvinylpyrrolidone (PVP)/sodium ibuprofen (SI), PVP/sodium alginate (SA)/SI and poly(ethylene oxide) (PEO)/SA/SI. The fibers were characterized by X-ray diffraction (XRD), nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR), and drug release explored. The PEO/SA/SI fibers showed interesting pulsatile release charactersitics, and thus additional fibers containing PEO/SA and other drugs (sodium valproate, diclofenac, and naproxen) were produced and investigated. The sodium salts of diclofenac, naproxen and valproic acid were also successfully intercalated into known Zn-containing HDS materials by ion-exchange intercalation. The products were characterised by XRD, FTIR, and NMR. The intercalation processes were studied using in situ X-ray diffraction, and drug release studies were undertaken. The release timescale is potentially useful for commercial applications, but the amount of Zn and (in some cases) the presence of Co and Ni in the materials is potentially a concern. Thus, new biocompatible HDSs containing Zn, Mg and Fe were prepared and loaded with drugs. Drug release was investigated, and the new HDSs were formulated into tablets. The latter were subject to the standard pharmacopoeial tests. Finally, LDHs were explored. Phosphonoacetic and sulfoacetic acid were successfully intercalated, and the products characterised by XRD, FTIR, and NMR. The intercalation process was probed using in situ XRD, and interesting intermediate phases observed. Molecular modelling was used to explore these systems, and drug release investigated.

This thesis is an effort to study the concepts of skin barrier function in relation to skin physiology and biochemistry. The study focused on anatomical differences, effects of photoaging, the sensitivity of skin and pigmentation differences. The most photoexposed area of the skin is the face, so the study was designed to evaluate the factors responsible for barrier function in the facial stratum corneum. Firstly, stratum corneum (SC) protein estimated using a colorimetric assay was compared to the non-destructive Squamescan® method. The study found a good correlation between the two methods for forearm and cheek SC protein. The anatomical differences in relation to filaggrin (filaggrin) degrading proteases bleomycin hydrolase (BH) and calpain-1 (C-1) together with the pyrrolidone carboxylic acid (PCA) were analysed from tape strips of cheek, forearm and leg. The results showed the highest activity of filaggrin degrading proteases in the tapes 4-12. Interestingly, the lowest PCA level was quantified from the cheek, in spite of higher protease activity compared to the other two sites. The next study aimed at understanding the variations of these biomarkers in relation to pigmentation and photodamage. The results showed that the photodamage is associated with a significant decrease in SC barrier function. The study also investigated the role of ethnic differences in SC biochemistry and demonstrated that subjects with the highest level of photodamage had increased levels of plasmin activity and reduced cell maturation. Finally, the last study focused on sensitive skin. Biomarkers were measured in samples taken from the cheek. BH and PCA were found to be lowest in sensitive subjects. The lower corneocyte maturity in the sensitive group was well correlated with the lower transglutaminase activity. This thesis highlights the need to improve NMF levels and the activities of late stage filaggrin degrading enzymes together with a proper differentiation of corneocytes in order to improve the SC barrier. In conclusion, the thesis reports new methods of quantification of the filaggrin degrading enzyme activity, plasmin activity and PCA levels from tape strips. New data have also been generated for variation of these biomarkers in different anatomical sites, ethnicities and skin conditions generated which will provide more information about the molecular biochemistry of SC.

No description available.

BACKGROUND: Little is known about Arab women’s (AW) perceptions of medication use in Kuwait. Women with cardiovascular disease (CVD) manage their condition in the context of their lives and responsibilities, so a culturally adapted intervention may have a greater impact on individual behaviour. This study aims to assess adherence to CVD medications among Arab women with cardiovascular disease (AW-CVD), to explore barriers to adherence from the perspectives of both patients and healthcare providers (HCP), to examine possible service developments and to make recommendations to optimise treatment outcomes. METHOD: A mixed methods approach was used, including a cross-sectional survey (N=270) followed by 29 face-to-face and telephone semi-structured interviews with AW-CVD and 17 face-to-face interviews with HCP. Settings: Multiple health centres across Kuwait (N=10). Survey instruments: Patients’ demographics, clinical variables, the 8-item Modified Morisky Adherence Scale (MMAS-8), the Brief Illness Perception Questionnaire, the Beliefs about Medication Questionnaire (Necessity and Concern Scale), social support from family and friends section of the Chronic Illness Resources Survey, measurement of healthcare provisions, beliefs about herbal remedies, and measurement of CVD self-care behaviours. All interviews investigated perceived barriers to medication adherence. The HCP interviews further explored HCPs’ current practice, their suggestions on how to improve current practice and a discussion on the difficulties and opportunities of applying these suggestions. Alongside a review of existing worldwide models of chronic disease management, the collected data helped inform the development of culturally sensitive chronic care services and behaviour change intervention. RESULTS: Ninety-three per cent of AW-CVDs were sub-optimally adherent to medications based on MMAS-8. Several barriers to medication adherence were identified from both AW-CVD and HCP perspectives: patient-related factors, HCP-related factors, disease-related factors, therapy-related factors and social factors. The HCPs tried to support medication adherence by educating the patients, simplifying the treatment regimen and ensuring the availability of medications. However, a number of limitations in their work structure restrict such progress, including time constraints, shortage of work force, lack of training and resources, lack of performance evaluations and poor cooperation among HCPs. The development of a chronic disease management model was proposed to overcome this gap. Such a model would suggest integrated services in which the HCP is provided with the necessary skills and resources to educate patients to become proactive and make informed decisions with the support of their communities. CONCLUSION: This research extends our knowledge of barriers to CVD medication adherence. The findings of the thesis suggest that there is a definite need for reorganization of the current chronic care system in Kuwait. This information was used to develop a chronic disease management model. The challenge now for future studies is to understand the feasibility of the proposed model.

The experience (or mere anticipation) of side effects influences patients’ beliefs about specific treatment and pharmaceuticals in general. In this thesis I postulate that beliefs about medication may also contribute to side effect reporting. Five studies were conducted to test this hypothesis and to explore putative underlying mechanisms. The majority of studies examining the relationship between medication beliefs and side effect reporting are cross-sectional, precluding inferences about the direction of the relationship. I present evidence from a prospective study showing that baseline medication beliefs predict side effect reporting in a large multinational sample of women initiating bone-loss treatment. The claim that non-pharmacological factors like medication beliefs can influence side effect reporting is supported by research on the nocebo effect (side effects to placebo). A sham trial was conducted showing that medication beliefs predict symptom reporting and attribution of symptoms as side effects in healthy volunteers taking Modafinil placebo. Many symptoms that are frequently listed as side effects are also common in patients randomized to placebo and un-medicated healthy volunteers. Using an analogue scenario I demonstrate that people with more negative beliefs about medication are more likely to misattribute a common symptom as a side effect. A related analogue study explored whether pre-existing beliefs about medication act as schemas that bias the way people process information about side effects. I show that individuals with more negative beliefs about pharmaceuticals recall and recognize fewer side effects from a patient leaflet, spend less time reading side effect information and are consequently more likely to attribute an unlisted symptom as a side effect. Finally, I present findings from a feasibility study of interventions to modify unhelpful medication beliefs in order to reduce side effect attribution. Together these findings confirm the importance of medication beliefs in side effect reporting and point to possible intervention strategies.

The use of trio exome next-generation sequencing (NGS), an analysis of the entire complement of coding exons within an affected index patient and unaffected parents, is increasingly being utilised in the analysis of patients with congenital diseases. This thesis describes the use of trio exome sequencing in the analysis of three distinct circumstances. First, the analysis of a cohort of eight trios in which the index patients suffer from startle disease, characterized by dysfunction of inhibitory glycinergic synapses. This revealed a single case of allelic drop-out of a known GLRA1 p.Y307C variant in a novel de novo pattern of inheritance, three cases where known or expected pathogenic variants had been identified in genes known to cause a seizure phenotype (DOLK and DMD), and an association with variants in ATRN, a null mutant of which is associated with a seizure phenotype in Mus musculus. Second, whole exome analysis of three individual cases with rare diseases was performed; a case of hypomyelinating leukodystrophy with a homozygous NKX6-2 p.L54Qfs variant, a case of microcephaly and pontocerebellar hypoplasia with a MED14 de novo p.R1162C variant, and a case of syndromic intellectual disability with hypotonia and a homozygous DRG1 p.Y295* variant. None of these genes had yet been implicated in disease, but all show some functional implication in each of the respective disorders. Third, novel de novo GRIN1 p.M818R and GRIN2B p.M824V variants were identified in further cases with developmental delay and hypotonia, adding to recent publications expanding the phenotype of variants within the NMDA receptor subunits. In each of these cases, trio exome sequencing was requested due to unclear disease aetiology and the resulting data indicates, in the majority of these cases, novel or putative genotype-phenotype correlations.

Hand hygiene is a major and fundamental topic in patient management to avoid health care-associated infections. According to the WHO alcohol-based hand rubs are the preferred means in clinical antisepsis. However, the frequent use of alcohol-based hand rubs can cause adverse events, which negatively impact user compliance, demonstrating a risk for patients. Adverse events are based on protein denaturation and stratum corneum lipid extraction. The aim of this work was to develop in vivo assays for lipid-processing enzymes in the stratum corneum as biomarkers to be routinely used in the evaluation of hand sanitiser formulations. Tape stripping is a well-established method to obtain stratum corneum samples. Fluorogenic substrates were used to develop selective enzymatic assays for phospholipase A1, A2, lipase, β-glucocerebrosidase, and sphingomyelinase activity. Consecutive tapes were removed to understand the activity – depth profiles of the enzymes. In addition, the established non-invasive analytical methods of transepidermal water loss and spectrophotometric intracutaneous analysis were combined with the enzymatic measurements in clinical studies. The characteristic activity – depth profiles of enzymes changed under induced skin barrier alterations by ethanol exposure, indicating compensatory and inflammatory responses. In human subjects, the formulation containing of 0.4% glycerol in addition to 70% ethanol resulted in a reduced transepidermal water loss and reduced enzyme activity. This was based on its hydrating properties of the skin. Niacinamide at 0.3% added an additional benefit on barrier integrity, increasing cohesion between corneocytes and reduced lipase activity. Glycine has hydrating properties and was able to reduce transepidermal water loss added to the 70% ethanol formulation. Glycine increased the activity of β-glucocerebrosidase in healthy skin and hence also offers an interesting new approach in hand sanitisers by potentially altering the Cl- flux in keratinocytes increasing lamellar body secretion and delivering higher amounts of intercellular lipids. The successfully developed assays demonstrate a minimally invasive method to identify the state of the skin barrier integrity in human subjects. This approach allowed the identification of the benefits of glycerol, niacinamide, and glycine in hand sanitisers, regarding the skin barrier integrity. Further excipients and compositions can be routinely studied with these methods in the future to optimise hand sanitiser formulations and consequently improve user compliance. However, the assays are not limited to the evaluation of hand sanitiser but the biomarkers can also be applied to study other formulations or active pharmaceutical ingredients as well as in the understanding, characterization, and monitoring of disease states of the skin.