This thesis makes an original contribution to the field of formulation development by providing new experimental data and insights into the effect of processing and formulation conditions on the quality of lyophilised biopharmaceuticals. The quality attributes of lyophilised products include: a quick reconstitution time, product elegance and protein stability, which are known to be affected by processing and formulation parameters. However, choice of formulation excipients or processing conditions often relies on previous experience rather than mechanistic insight. The motivation of this thesis was therefore to provide a greater understanding of how process variables andexcipient choice affect these quality attributes. Bovine serum albumin (BSA) and immunoglobulin G (IgG) were used as model proteins to investigate formulation conditions, which included the excipient, the lyophilisation cooling profile and duration of the annealing step. BSA was also used as a model protein to explore the effects of sucrose and arginine as lyoprotectants. Unique to this study was the investigation of arginine salts as lyoprotectants, wherein the counterions were dicarboxylic acids with increasing chain length. Two key results regarding quality attributes were observed. Firstly, characterisation of the lyophilised structure established that there was an optimal annealing time, beyond which there was an increase in primary drying time, batch heterogeneity and variable moisture content. Secondly, a relationship was found between decreasing dicarboxylic acid chain length and improved protein stability. To explain these findings, two mechanisms are proposed that account for ice crystal growth during annealing and the observed changes in protein stability at the molecular level. Significantly, this research provides insights for future formulation development studies.

Global pharmacy education and pharmacy practice continue to face remarkable changes. Many countries are undergoing major transformations in the field of pharmacy education. In developing countries, there is an increasing trend towards adopting the PharmD degree. Thai pharmacy education has transitioned from having two entry-level degrees – a 5-year BPharm (with three main tracks: pharmaceutical care, pharmaceutical sciences, social and administrative pharmacy) and a traditional 6-year PharmD (pharmaceutical care) programme – to a single national 6-year PharmD programme or ‘an all-PharmD programme’ (including industry pharmacy and pharmaceutical care tracks). Similar to other countries that have also been transitioning to ‘an all-PharmD programme’, there was limited evidence for the merit of the transition in Thailand. Although opinions and questions put forth on social media networks triggered debates about the need for the transition, there is a lack of an evidence-based and in-depth investigation about the reasons for this transition. This thesis explores the experiences and perceptions held by stakeholders concerning the transition towards an all-PharmD programme in Thailand. The study used a mixed methods approach through a quantitative analysis of surveys (Phase 1 and Phase 2) and interviews with stakeholders who were involved in pharmacy education (Phase 3). Findings from the three phases and other resources were triangulated and validated (by comparing and confirming them) to provide a better picture of the transition of pharmacy education in Thailand. Phase 1: A survey of the status of Thai pharmacy education. This study aimed to explore the status of pharmacy education in Thailand by using a questionnaire survey. The surveys were distributed to the deans of all 19 faculties of pharmacy in Thailand. The response rate was 84% (n = 16). Characteristics of the Faculties of Pharmacy, the teaching staff, types of PharmD programme, the number of training sites and quality assurance mechanisms were reported. The results showed that the Thai PharmD curriculum includes industrial pharmacy and clinical pharmacy tracks that differentiate it from the traditional US PharmD programme, which only focuses on patient care. There was a shortage of academic staff in the pharmaceutical care area and some faculties needed to better prepare for their training sites. Phase 2: A survey of the pharmacists’ perceptions towards the suitability of the PharmD graduates employed in hospital and community pharmacy settings and the competencies difference between the BPharm and the PharmD graduates. This study aimed to explore Thai pharmacists’ perceptions regarding the suitability of the PharmD graduates employed in hospital and community pharmacy settings, as well as the competency differences between the BPharm and PharmD graduates. A cross-sectional survey questionnaire was distributed to 180 hospital pharmacists and 200 community pharmacists during two conferences. The response rate was 55.6% among hospital pharmacists and 20% in the community pharmacists group. The findings highlighted that the PharmD graduates were suited for large hospital settings as they were well coordinated with the health care team. However, there were concerns regarding the suitability of the PharmD graduates for primary care settings, because of their lack of training in health promotion. Half of the respondents perceived PharmD graduates as having higher competencies in clinical activities and being more prepared to work than BPharm graduates. However, the other half of the respondents perceived the competencies of both pharmacy qualifications as being similar; PharmD graduates provide non-clinical activities similar to BPharm graduates, due to numerous barriers (e.g., high workload in dispensing services and the shortage of pharmacists) preventing PharmD pharmacists from providing direct pharmaceutical care services. Phase 3: Thai stakeholder’s perceptions of the introduction of the PharmD programme: a qualitative study. This qualitative study aimed to understand the experiences and perceptions of stakeholders, regarding the transition to an all-PharmD programme in Thailand. Semi-structured interviews were conducted with 130 stakeholders (e.g., policy makers, educators, health care providers, patients, students, and parents). The data were audio recorded, transcribed verbatim and analysed using an inductive thematic analysis. Three main themes were derived from the findings: 1) influences on the transition (e.g., the US-Thai consortium for the development of pharmacy education); 2) perceived benefits (e.g., improved pharmacy competencies from generalist to specialists); and 3) concerns (e.g., the higher cost of a longer period of study, and insufficient preceptors and training sites). This PhD study carries important implications for both universities and policy makers. Faculties of pharmacy should consider a long-term plan to develop sufficient qualified academic staff and preceptors. Policy makers should prepare a strategic plan for the future workforce supply and requirements, increase the flexibility of the PharmD curriculum during its transition stage, and prepare a supportive and enabling system for PharmD graduates to provide advanced practice at their full potential. Close coordination between faculties, the Pharmacy Council of Thailand and pharmacy professional organisations is needed to ensure that pharmacy education provides the necessary competencies for graduates to offer a high level of needed pharmacy services. Further research focussing on the outcome, impact and efficiency of the PharmD programme is also needed. To conclude, in this thesis, the issues surrounding the transition to an all-PharmD programme in Thailand were carefully investigated. This study reflects the influences and the requirements of the transition that it was initiated, in order to meet the need for higher levels of competency for the nation’s pharmacists and is influenced by many factors. The stakeholders perceived benefits from the transition. They thought that the PharmD graduates will have higher competencies and be ready to work as pharmacists compared to graduates from the previous pharmacy curriculum. The findings also addressed the following issues concerning curriculum change: the higher costs of a longer period of study, the mismatch between the pharmacy graduates’ competency and the job market’s needs and the shortage of qualified preceptors.

Lung cancer is considered a major health concern and is responsible for most cancer-related deaths. Nasopharyngeal carcinoma (NPC) is another type of cancer that is predominantly in China and has a low survival rate, which makes it a serious health issue. There is currently no cure for lung cancer and NPC, so it was decided to investigate derivatives of the highly bioactive natural product, cardamonin, for a potential drug candidate. 19 analogues of cardamonin were synthesised and tested against A549 (lung) and HK1 (NPC) cell lines. The techniques employed in synthesising the analogues were one-step reactions which included alkylation, acylation, reduction, condensation, cyclisation and complexation reactions. The analogues were fully characterised. MTS assay showed that several derivatives, such as the allyl derivative of cardamonin (2) and cardamonin’s Cu (II) complex (19), had more potent cytotoxic activities than cardamonin. Furthermore, the active analogues have generally demonstrated lower toxicity towards normal MRC5 cells. Structure-activity relationship (SAR) analysis showed the importance of the ketone and alkene groups for bioactivity, while substituting cardamonin’s phenolic groups with more polar moieties resulted in activity enhancement. As part of the SAR study and further exploration of chemical space, the effect of metal coordination on cytotoxicity was also investigated, but it was only possible to successfully obtain the Cu (II) complex of cardamonin (19), and the metal ion enhanced bioactivity. 19 was the most potent analogue possessing IC50 values of 13.2 μM and 0.7 μM against A549 and HK1 cells, corresponding to a 5- and 32-fold increase in activity, respectively. It was also able to inhibit the migration of A549 and HK1 cells. Mode of action studies revealed that 19 induced DNA damage in both cell lines resulting in G2/M-phase arrest, which further led to apoptosis via the activation of caspase-9 and caspase-3/7. Moreover, qPCR analysis showed that 19 inhibited the expression of the mammalian target of rapamycin (mTOR) by >50% in A549 and HK1 cells which indicated that it exerted its anticancer activity, at least in part, via inhibition of the mTOR signalling pathway. So molecular docking of cardamonin and 19 to mTOR was performed and the study showed that the higher activity of 19 might be due to formation of further hydrogen bond interactions with the receptor resulting in a higher binding free energy of -9.8 kcal/mol. Therefore, all these assays have further proven the high bioactivity of 19. However, further in vivo and animal model studies would have to be conducted in order to confirm the potential of 19 as an anticancer agent.

The blood-brain barrier (BBB) is the most extensive and restrictive barrier to brain delivery for therapeutic agents. A low proportion of low molecular-weight agents can cross into the CNS. This decreases further as the molecular weight increases, meaning therapeutic antibodies, oligonucleotides and other supramolecular entities effectively cannot reach therapeutic levels within the CNS. Targeting ligands against receptors thought to undergo transcytosis across the brain microvascular endothelial cells (BMECs), can boost CNS delivery of therapeutics. Understanding these mechanisms, in an in-vitro setting, has proved challenging, due to the constraints of cell culture systems and the difficulty to replicate the in-vivo environment. With even the most extensively studied targeting receptor, transferrin receptor, not producing clear evidence to suggest the occurrence of transcytosis. To understand in-vitro trafficking of brain targeting ligands a pulse-chase assay, in combination with sub-cellular localisation microscopy was developed and compared to the current permeability-based assay method. The characterisation was done by comparison of transferrin receptor ligands; native holo-transferrin, the 8D3 antibody and a low-affinity variant; with the non-specific uptake probe, dextran. The method could distinguish between the two endocytosis methods, with concentration-dependent efflux efficiency observed with the targeted probes. The combination of techniques was then applied to the novel targeting ligand, Rabies-Virus Glycoprotein (RVG) peptide, to assess its suitability as a brain delivery. Studies were performed to confirm the target receptor of the RVG peptide, including competitive uptake, siRNA knockdown methods. The RVG peptide demonstrated desirable delivery characteristics, and the target receptor was confirmed as the α7 nicotinic acetylcholine receptor. Finally, attempts were made to develop a total internal reflection fluorescence (TIRF) microscopy assay for the assessment of ligand arrival at the basolateral membrane of BMECs. Initial work for this was performed with the transferrin receptor and transferrin, using both labelled ligand and photoswitchable receptor constructs. In summary, the pulse-chase assay provides a complementary technique to permeability assays for the assessment of brain targeting ligand trafficking in BMEC cell-lines in-vitro.

The potential applications of transition metals-based nanoparticles are expanding in the biomedical field. Oxides of iron are the matter of investigation in this study where various preparations of ultrafine superparamagnetic iron oxide nanoparticles (USPIONs) were fabricated using flow injection technology with spinning disc reactor. Basically, two types of preparation parameters were examined; first, instrument-related (physical) parameters and, second, chemistry-related parameters. USPIONs fabricated by this instrument showed fine-tuning size adjustment. Subsequent surface modification of these nanoparticles produced hydrophobic, hydrophilic and neutral amino acids modified surface, whereby aminoacid ‘monomers’, rather than polymeric materials were used. Transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements, thermogravimetric analysis (TGA) and fourier-transform infrared-attenuated total reflection spectroscopy (FTIR-ATR) were employed to characterise the coated nanoparticles. The data show that ultrafine, 4-9 nm sized coated nanoparticles show good dispersion upon TEM imaging. Measurement of number of monomers molecules effectively associated with USPIONs suggest formation of multilayer of amino acid adsorbed on nanoparticles (NPs). Prediction of NPs- amino acid association mechanism by FTIR-ATR study reveals presence of either monodentate or bidentate molecular adsorption on the surface of USPIONs. In the second stage of the project, interactions of differently modified USPIONs with epithelial cell layer (model of intestinal epithelium) are now investigated. An intestinal adenocarcinoma cell line (Caco-2) is used for performing the in vitro studies. The toxicity of three types of USPIONs (Asp-, His-, and Phe-USPIONs) reveals that these particles have potential toxic effect on biological system. Relatively long term exposure to these particles (24 hours) with high concentration 250 μg/ml and more was found to enhance apoptotic mode of cell death. Cell-NPs interaction study displayed presence of different forms of cellular interaction which are supposed to be related to USPIONs surface chemistry. While some of Phe-USPIONs are found internalised and accumulated inside some cells, Asp-USPIONs exhibit different interaction mode where the cell membrane of most cells is covered with thin layer of NPs without significant cell penetration. This gives an indication that metal oxide NPs (USPIONs) that are associated by their surface with small molecules could render these NPs with aggravated toxicity and cell-NPs interaction and hence long term effect.

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.