Many different nanoparticle delivery systems have been reported as potential cancer therapeutics, however, the tumour penetration and uptake characteristics have been determined for very few systems. Animal models are effective for assessing tumour localisation of nanosystems, but difficult to use for studying penetration beyond the vasculature. In this work, defined HCT 116 colorectal cancer spheroids were used to study the effect of nanoparticle size and surface modifications on their penetration and uptake. Incubation of spheroids with Hoechst 33342 resulted in a dye gradient which facilitated discrimination between the populations of cells in the core and at the periphery of spheroids by flow cytometry based on the degree of Hoechst staining. This model was used to compare doxorubicin and Doxil, a range of model polystyrene nanoparticles in different sizes (30 nm, 50 nm, 100 nm) and with different surface chemistry (50 nm unmodified, carboxylated, aminated) and polyethylene glycol modified NPs prepared from a promising new functionalized biodegradable polymer (poly(glycerol-adipate), PGA). Unmodified polystyrene nanoparticles (30 nm/50 nm) were able to penetrate to the core of HCT 116 spheroids more efficiently than larger polystyrene nanoparticles (100 nm). Penetration was also dependent on surface charge. PGA NPs of 100 nm showed similar penetration into spheroids as 50 nm polystyrene nanoparticles, and PEG surface modification significantly improved penetration into the spheroid core. The new spheroid model with Hoechst staining is shown to be a useful model for assessing NPs penetration and demonstrates the importance of controlling physical properties when designing nanomedicine.

The tendency of the majority of active pharmaceutical ingredients (APIs) to exist in different solid forms with keeping their chemical structures is called polymorphism. This phenomenon has gained a lot of interest in the pharmaceutical industry, hoping to avoid producing unexpected transformations of compounds during and after synthesis. The optimal way to avoid that is to subject the API, at the early stage of development, under various conditions in order to obtain an elegant (safe, effective, and stable) drug for the next formulation step. The aim of this thesis was to investigate some factors that affect the appearance of different polymorphs during screening of some APIs. Four model drugs were selected: paracetamol; carbamazepine; mefenamic acid; and flufenamic acid. All have been well-characterised previously in terms of solid-state forms. Piezoelectric, or 2D inkjet printing technique was used as a main technique in fabrication of nanoarrays of APIs onto predefined design on a solid tunable substrates because of its ability to control the delivered quantities of the the printed materials accurately, without any direct contact with the used substrate that may cause a sample cross-contamination. Light optical microscope was used to investigate the behaviour of the printed droplets during and after solvent evaporation and turn to dried spots, and to confirm the crystalline state of some spots by using the polarised light in the same microscope. Raman spectroscopy at low-wavenumber, or phonon region (40-400 cm-1) was used for the first time to identify the resulted polymorphs after the printing process as its ability to probe the alterations that happen in the molecular skeleton inside the crystal lattice , in addition to molecular region (400-1800 cm-1) to analyse the resulting spots. In chapter three, the piezoelectric inkjet printing technique was successfully used for the first time to miniaturise, screen, and study the stability of the APIs at nano quantities in the range of (1-500 ng), about six-ordered magnification less than the reported studies. It was found that the variation in the printed quantities can produce different states and polymorphs. Stability with time was also studied for all the printed samples and it was noticed the variation in time for some printed drugs to convert from solid amorphous to crystalline state. In chapter four, the advantage of the ability of the gold-coated slide to undergo further chemical modifications was exploited to create new substrates. Chemical modification of the gold substrates was carried out by treating them with two types of thiols to form self-assembly monolayers (SAMs) and use them as substrates in polymorph screening of some APIs. The new prepared SAMs were examined by preliminary tests like atomic force microscope (AFM) and water contact angle (WCA) measurements to investigate the texture of the new substrates before using them in printing process. It was found that changing the chemical structure of the substrate can lead to different polymorphs. In chapter five, an attempt to create highly hydrophobic substrates was done to investigate whether it can affect the propensity of APIs for polymorphism. Fluorinated compounds were used in this chapter as they are considered more hydrophobic than the substrates used in the previous part of the work The effect of the fluorinated substrates on appearance of new polymorphs was studied. Two fluorinated compounds were selected for preparation of high-water repellent surfaces and using them as substrates as they have the ability to limit the spreading of the printed droplets of the API, and allow the molecules to be constructed layer by layer and form a condense spot. The new fluorinated substrates were examined before using them in printing, and they exhibited high WCA. Another FLUF polymorph (VI) was investigated in addition to the two reference (I and III) polymorphs used in FLUF polymorphic screening. It was found that the intensity of the Raman peaks of the printed spots of APIs was good and clear to recognise when using fluorinated SAMs as a substrate, while the fluorinated substrate prepared from Flutec LE15 exhibited fluorescence effect due to the interactions between the glass and the drug’s spot spectrum.

As poor solubility continues to be problem for new chemical entities (NCEs) in medicines development the use and interest in solid dispersions as a formulation-based solution has grown. Solid dispersions, where a drug is typically dispersed in a molecular state within an amorphous water-soluble polymer, present a good strategy to significantly enhance the effective drug solubility and hence bioavailability of drugs. The main drawback of this formulation strategy is the inherent instability of the amorphous form. With the right choice of polymer and manufacturing method, sufficient stability can be accomplished. However, finding the right combination of carrier and manufacturing method can be challenging, being labour, time and material costly. Therefore, a knowledge based support tool based upon a statistically significant data set to help with the formulation process would be of great value in the pharmaceutical industry. Here, 60 solid dispersion formulations were produced using ten, poorly soluble, chemically diverse APIs, three commonly used polymers and two manufacturing methods (spray drying and hot-melt extrusion). A long term stability study, up to one year, was performed on all formulations at accelerated conditions. Samples were regularly checked for the onset of crystallisation during the period, using mainly, polarised light microscopy. The stability data showed a large variance in stability between, methods, polymers and APIs. No obvious trends could be observed. Using statistical modelling, the experimental data in combination with calculated and predicted physicochemical properties of the APIs, several multiple linear regression (MLR) models were built. These had a good adjusted R2 and most showed good predictability in leave-one-out cross validations. Additionally, a validation on half of the models (eg. those based on spray-drying models) using an external dataset showed excellent predictability, with the correct ranking of formulations and accurate prediction of stability. In conclusion, this work has provided important insight into the complex correlations between the physical stability of amorphous solid dispersions and factors such as manufacturing method, carrier and properties of the API. Due to the expansive number of formulations studied here, which is far greater than previously published in the literature in a single study, more general conclusions can be drawn about these correlations than has previously been possible. This thesis has shown the potential of using well-founded statistical models in the formulation development of solid dispersion and given more insight into the complexity of these systems and how stability of these is dependent on multiple factors.

Natural products play a pivotal role in the exploration of new cancer therapies of which the plant kingdom is a substantial source. Conofolidine is a novel bisindole alkaloid isolated from Malayan plant Tabernaemontana Corymbosa and belongs to the family of the known vinca alkaloid conophylline. To our knowledge, no published work existed at the time of commencement of this project. Herein, we report for the first time recognition of conofolidine’s exceptional anticancer activity, from a panel of Malayan bisindoles (namely leucophyllidine, bipleiophylline and alstonia macroline-sarpagine bisindoles) that were indiscriminately screened against various human-derived carcinoma cell lines. Preliminary data showed that conofolidine exerted remarkable inhibition of cell proliferation and colony formation of cancer cells (e.g. GI50 = 0.054 and IC50 < 0.1 μM in MCF-7) through either induction of apoptosis or senescence. Apoptosis was confirmed by accumulation of cleaved PARP and activation of caspases 3/7. Alternatively, increased β-galactosidase positive cells accompanied by transformation of cell shape to spindle like with enlarged cell size ascertained senescence induction. G1 cell cycle arrest and S-phase depletion were observed in the majority of tested cell lines. These cell cycle perturbations were confirmed by decreased expression of positive regulators (CDK2, cyclin A2 and c-Myc) and increased expression of CDK inhibitors p21WAF1/CIP1, p27KIP1 and p15INK4b. Conofolidine caused several aberrant mitotic phenotypes exemplified by multi-nucleation, mitotic slippage, changed polarity, membrane blebbing and DNA fragmentation. Compromised DNA integrity was confirmed by increased γ-H2AX foci and/or level indicating DNA double strand breaks. Conofolidine increased ROS production, which partly contributed to DNA damage, apoptosis- and senescence-induction. A proteomic study conducted following exposure of HT-29 cells to conofolidine (72 h; 0.602 μM) corroborated ROS generation by the increased expression of several ROS scavengers e.g. NQO1. Phospho-proteomics analyses revealed significant suppression of p-EGFR, p-Akt, p-ERK and p-STAT signal transduction. Such suppression caused c-Myc destabilisation with consequent eliciting of either apoptotic or senescent phenotypes. The variation in the basal phosphorylation levels of these signalling proteins in the different tested cell lines determined their fates. Additionally conofolidine down-regulated mutant-p53 at transcription, expression and post-translational levels in mutant-p53 (R273H) cell lines which could partly contribute to its suppressive actions on signalling pathways and cell cycle. Proteomic analyses showed decreased expressions of MCM (2-7) including MCM4 through which mutant-p53 (R273H) could drive initiation of DNA replication. Conofolidine’s ability to suppress MCM family (together with ROS production) provides an additional mechanism for conofolidine to induce DNA damage and genomic instability. Taken together, we present conofolidine in this study as potential anticancer candidate and provide mechanistic insight to its molecular targets and pathways, which encourage further future work.

Biological medicinal products (BMPs) are medicinal products extracted from, or synthesised by, a biological system. While BMPs have been proven to be effective in treating chronic and life-threatening diseases, the utilisation of these medicines was associated with creating financial burdens for healthcare systems worldwide. Expiry of patents and marketing exclusivity rights offers an opportunity to develop similar and not identical follow-on copies called ‘biosimilar medicinal products’ (BSPs). The inability to produce an identical copy of the original product is related to the heterogenic molecular nature of BMPs and the complexity of the manufacturing processes involved. The United Kingdom (UK) was frequently reported to have only a limited uptake of BSPs. Factors related to prescribers’ reservations and lack of encouraging national policies were reported to limit BSPs’ utilisation in the UK. In February 2015, the first high-cost monoclonal antibody (mAb), ‘infliximab’ (IFX-BSP), was launched; it was the first BSP to be commissioned locally compared to the previously nationally commissioned BSPs. Since its launch, there have been several encouraging national initiatives led by NHS-England and NICE. These conditions created new market daynamics that might affect uptake rates of BSPs, particularly IFX-BSP. This thesis explores factors affecting BSPs’ utilisation and integration in clinical practice in England. The aim and objectives were actualised following an explanatory sequential mixed methods design. The study’s first component assessed BSPs’ integration levels in local medicines formularies and investigated the prescribing practices of IFX-BSPs by English Acute Trusts. Guided by first component results, the second explanatory component investigated factors that led to the observed utilisation rates of IFX-BSPs. The explanatory study was achieved through 59 in-depth semi-structured interviews with healthcare professionals, who were involved in assessing, commissioning, introducing, managing and prescribing IFX-BSPs. In general, formularies screening showed that the integration levels of targeted BSPs were less than expected from a generic medicines driven market. Data showed variations in the uptake levels between therapeutic and geographical areas within Great Britain; a phenomenon which needs to be further investigated and explained. In England, assessing prescribing practice showed that with time Acute Trusts were becoming more accepting of utilising IFX-BSPs and were beginning to switch their R-BMP stabilised patients over to the BSPs. Interviews suggested that prescribers’ knowledge, experience, common medical practice, professional societies and ethical obligations had influenced prescribers’ acceptance and attitudes toward BSPs, in general, and to the switching of stabilised patients in particular. Data showed BSPs’ utilisation was not only affected by prescribers’ attitudes, but it was also influenced by pull-push dynamics between providing Acute Trusts and commissioning CCGs. The knowledge gained from this research could be used to inform the future implementation of upcoming BSPs. Insights acquired from this research regarding interactions between providers and commissioners might be extrapolated to implementing interventions locally, especially when there is an absence of nationally specific policies or guidelines.

Les nanoparticules constituent une forme médicamenteuse d’avenir dans le domaine pharmaceutique. Capables de véhiculer de nombreux principes actifs, elles peuvent les libérer dans l’organisme après administration in vivo mais aussi les piloter vers des cibles prédéfinies. Ce travail repose sur la préparation, par une technique de double émulsion, de nanoparticules constituées de deux polymères : la poly-e-caprolactone et un polymère polyacrylique et polycationique (Eudragit® RS). La première partie a consisté en la mise au point de nanoparticules encapsulant de l’insuline et capables de la libérer in vitro et in vivo. Ces nanoparticules ont développé une activité hypoglycémiante après administration orale. En effet, l’inconvénient majeur du traitement du diabète par l’insuline reste un mode d’administration contraignant pour le diabétique : la voie injectable. La possibilité d’administrer l’insuline par voie orale constituerait un progrès révolutionnaire dans le domaine de la diabétologie. Les nanoparticules préparées ont donc démontré leur capacité à diminuer le glucose sanguin de rats diabétiques après administration orale. Le mécanisme de passage de l’insuline semble être dû à un contact intime entre le mucus chargé négativement et les nanoparticules de charge opposée créant ainsi un fort gradient local de concentration. La seconde partie du travail a consisté en la mise au point de nanoparticules dites furtives c’est à dire capables d’éviter leur reconnaissance par le système phagocytaire mononucléé après administration intraveineuse. L’objectif était de créer un procédé innovant de furtivité reposant sur la formation d’interactions électrostatiques entre l’héparine, glycosaminoglycane polyanionique, et l’Eudragit® RS, polymère polycationique. Le potentiel de furtivité a été démontré par l’augmentation in vivo de la demi-vie plasmatique de deux principes actifs de natures différentes : le chlorhydrate de propranolol et l’insuline. / Nanoparticles are innovative dosage forms in drug delivery. They can act as prolonged release dosage forms but they also have the potentiality to target specific physiological compartments after in vivo administration. This thesis is based on the preparation of nanoparticles according to a double emulsion process. The nanoparticles are formed of two biocompatible polymers: poly-e-caprolactone and a polyacrylic and polycationic polymer (Eudragit® RS). The first part describes the development of insulin-loaded nanoparticles able to release insulin both in vitro and in vivo. It has been demonstrated that such nanoparticles incorporated high amounts of insulin and were able to display a hypoglycemic activity after oral administration. It is well known that the major drawback of diabetes treatment remains the injectable administration route for insulin. The ability to administer insulin orally would be a tremendous progress in the field of diabetes. Our prepared nanoparticles have demonstrated their ability to reduce blood glucose in diabetic rats after oral administration. It is believed that the positively charged insulin-loaded nanoparticles may interact with the negatively charged mucus and create a high local gradient concentration which would favor the intestine permeation. The second part of the thesis was devoted to the development of stealth nanoparticles able to avoid the recognition by the mononuclear phagocytosis system after intravenous administration. The objective was to create a new system based on the formation of electrostatic interactions between heparin, a polyanionic glycosaminoglycane, and Eudragit® RS, polycationic polymer. The stealth potential has been demonstrated by increasing the in vivo plasma half-life of two drugs namely propranolol hydrochloride and insulin.

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We address the issue of shuffling loads in Automated Storage/Retrieval Systems (AS/RS) in this paper. The objective is to pre-sort the loads into any specified locations in order to minimize the response time of retrievals. 1D, 2D and 3D AS/RS racks have been designed in order to achieve the shuffling efficiently. The shuffling algorithms are described in detail. The response time of retrieval, the lower and upper bounds of energy consumption are also derived. Results of the analysis and numerical experiments show that the shuffling algorithms are quite efficient. / Singapore-MIT Alliance (SMA)

Cerebral ischaemia, traumatic injury to the brain, inflammatory neurological disorders and HIV infections are amongst the most prevalent causes of neurodegeneration. Neuroprotective strategies are usually to limit the progressive secondary injury that generally occurs, thus limiting overall tissue damage. Neuroprotective strategies are usually to limit the progressive secondary injury that generally occurs, thus limiting overall tissue damage. Sodium channel blockers have been often used for this matter as they prevent the cascade of events culminating in free radical generation and eventually neuronal apoptosis. Newer compounds, such as antiperoxidants and free radical scavengers, show encouraging experimental results, but their clinical use is still very limited. Phenytoin being a popular drug in the treatment of epilepsy has also been used as a neuroprotectant during certain neurological emergencies and in pharmacological prophylaxis of post-traumatic epilepsy. Furthermore this agent functions by prolonging inactivation of voltage gated sodium channels. In these sets of experiment the neuroprotective properties of phenytoin were examined. The histological study revealed that phenytoin confers protection to the CA1 and CA3 regions of the hippocampus under the insult of QUIN. Cells maintain their characteristic shape and minimal tissue necrosis occurs in the presence of this agent. The in vitro effect of this antiepileptic drug on free radicals generation shows that phenytoin does not reduce or prevent the formation of these reactive species. Lipid peroxidation was induced using QUIN and iron (II), two known neurotoxins. The study reveals that only lipid peroxidation induced using iron (II) is reduced by phenytoin. These experiments were carried out in whole rat brain homogenate. These studies show that phenytoin possesses poor free radical scavenging properties. However, the dose-related reduction of iron-induced lipid peroxidation allows for speculation that phenytoin interacts with iron in order to reduce neuronal damage. Metal binding studies were performed using UV, IR and electrochemical analysis to examine the interaction of phenytoin with iron (II) and iron (III). Phenytoin, when added to iron (II) in solution, first oxidises the latter to iron (III) and maintains it in that form. A shift in the peak was observed in the UV spectrum when iron was added to phenytoin. Moreover, electrochemical studies indicate that the interaction between the metal and the ligand is very weak. The IR analysis it shows that phenytoin may be coordinating with iron through the Nitrogen atom on the phenytoin molecule. These studies show that phenytoin maintains iron in its oxidised form, which is a good property to possess as a neuroprotectants. Pineal organ culture showed that phenytoin does not increase melatonin production but slightly and non-significantly reduces the levels of this pineal hormone. However there is a significant rise in precursor NAS levels. As melatonin is known to possess antioxidant and free radical scavenging properties, this could mean that this drug can cause the CNS to become more susceptible to attacks by reactive oxygen species.

Osteoarthritis (OA) is a complex, multifactorial, and slowly progressive disease where there is currently no effective medical treatment. Research in understanding the mechanisms of OA has been advanced by preclinical studies in rodent models of OA. Recent evidence highlights the role of different classes of lipids in OA pathogenesis. Therefore, the main aim of this thesis was to apply both targeted and untargeted (global) lipidomics mass spectrometry based an alytical methods, in conjunction with univariate and multivariate statistical analysis, in various tissues from three established rodent models of OA; meniscal transection (MNX), monosodium iodoacetate (MIA), and destabilization of the medial meniscus (DMM). The overall goal was to identify statistically differentiated lipids between controls versus OA rodents that may reflect changes in the pathophysiology of OA and associated pain. In addition, a global lipidomics workflow was developed by me, following the latest trends used within the wider metabolomics community, ensuring robustness and reproducibility in the identification of putative metabolite/lipid biomarkers for diseases. Experiments in this thesis using a targeted oxylipin liquid chromatography tandem mass spectrometry (LC-MS/MS) method showed that statistical significant changes in the levels of certain oxylipins were observed. More specifically, 11,12-DHET (mean concentration: 0.26 pmol/g in control, 0.54 pmol/g in MNX; p < 0.01), 14,15-DHET (0.46 pmol/g in control, 0.75 pmol/g in MNX; p < 0.05) and 8-HETE (5.46 pmol/g in control, 7.40 pmol/g in MNX; p < 0.05) were statistically increased in the MNX compared to control (sham) rats in ventral spinal cord in the MNX rat model of OA. These findings are supported by literature since these three lipids exhibit pro-inflammatory properties and thus are expected to increase in the OA group where inflammation is the main feature of OA. Regarding the MIA rat model levels of other oxylipins in synovial fluid were differentially expressed in the MIA compared to saline (control) rats. Arachidonic acid (AA), (272.3 pmol/g in control, 435.3 pmol/g in MIA; p < 0.05) was increased in the MIA-treated compared to saline-treated rats, while 9-HODE (4.42 pmol/g in control; 1.21 pmol/g in MIA; p < 0.05) was statistically decreased in the MIA compared to saline rats. Since AA has been reported to be released from membrane phospholipids in OA, the observation that AA is statistically increased in synovial fluid in MIA- compared to saline-treated rats bears strong significance. In addition, maps of oxylipins metabolism were generated to visualize the pathways underlying the changes of lipid concentrations in plasma between control and OA rats for both MNX and MIA rat models. Therefore, applying a targeted oxylipin LC-MS/MS method in different tissues of MNX and MIA rat models of OA is a successful approach and informative about changes in pathophysiology of OA, underlying significant alterations in oxylipins concentrations. Although the global lipidomics approach was able to measure different classes of lipids that might account for differences in plasma between MNX/MIA and sham/saline-treated rats, this approach exhibited weak MVA (multivariate analysis) models. In contrast to MNX and MIA rat models, the global LC-MS lipidomics profile in plasma from a DMM mouse model of OA exhibited excellent MVA models with good prediction scores. Twenty-six statistically significant lipids were identified, using the lipidomics workflow that I have developed, and when four of these lipids were used to build Receiver Operative Curves (ROC) the model produced high prediction (84%) power in separating sham from DMM mice. The identity of these four lipids was classified as being fatty acids (FAs), sterols, sphingolipids, and diacylglycerols (DAG). In addition, MS/MS experiments were performed to confirm the identity of significant lipids. Thus, it was shown herein that applying a global lipidomics LC-MS approach in plasma from the mouse DMM model, using only a small number of mice (15 in total), can be informative about significant changes in the “lipidome” in OA and can be used as a robust means of predicting OA in mice based on their global lipidomics profile. Lastly, correlation statistical analysis was applied between levels of lipids in the various tissues, pain behaviour, and histopathology parameters in the three rodent models of OA. Although many oxylipins/lipids levels were found to be statistically correlated with the aforementioned parameters, the most striking finding is that 9-HODE and AA were both found to be positively correlated with Weight Bearing (WB), a parameter of pain behaviour, in plasma and synovial fluid in the MIA rat model of OA. Since plasma reflects systemic inflammation and synovial fluid reflects local (inflammation) 9-HODE (p < 0.01 in plasma; p < 0.05 in synovial fluid) and AA (p < 0.01 in plasma and synovial fluid) are oxylipins that potentially depict systemic and local changes in WB differences, and subsequently in OA related pain. This finding is supported by literature since both AA and 9-HODE are both agonists of a pain receptor (i.e. transient receptor potential vanilloid 1, TRPV-1). Thus, it was proved in this thesis that correlation analysis can be used as an additional and complementary statistical tool in an effort to determine the role of lipids in OA pathogenesis in rodent models of OA. In conclusion, applying both targeted oxylipin LC-MS/MS and global lipidomics LC-MS analytical methods capable of measuring either oxylipins or the whole “lipidome” in vivo, have provided novel findings to support the involvement of these lipids in OA and associated pain.