Understanding how drug molecules interact with our body and what effects they may induce as a result is of fundamental concern to the pharmaceutical industry. Crucially we want to use this knowledge to our advantage during the drug discovery process in order to manipulate a drug’s efficacy in vivo - therefore the development of new technologies, able to effectively screen numerous desirable drug-membrane interactions, is of key importance. The first half of this thesis details the development of a vesicle leakage assay, as a means to assess the effect of cationic amphiphilic drugs (CADs) on model lipid membranes. Having demonstrated the reproducibility of the assay, the assay was transferred into a microfluidic format where water-in-oil droplet systems act as individual experimental vessels. As such, it has been demonstrated that the use of fluorescence lifetime techniques can provide a way in which to translate this assay into a high-throughput format. The second part of the thesis is concerned with using droplet interface bilayers (DIBs) as a means to probe the effect of exogenous species upon a single lipid bilayer, as opposed to bulk vesicle populations. Several advantages exist for using such a system, including for example the ease with which one is able to control the composition of the aqueous compartments on either side of the bilayer and to form asymmetric bilayers. An assay, involving the use of pH gradients, is detailed, where proof-of-concept experiments illustrate that pH sensitive dyes could be used to report the extent to which lipid bilayers are perturbed by drug molecules for example. Furthermore, a novel automated approach has been developed, offering advantages over the manual manipulation of lipid-containing droplets for DIB formation, where a microfluidic approach is used for their generation in high-throughput. Consequently, this approach enables the formation of multiple DIBs, where the composition may be differed and the droplet dimensions controlled, enabling the formation of DIB networks that can be arranged in either two- or three-dimensions.

The transport of compounds around the body has been a topic of interest for many years, and the advent of non-invasive biological imaging in living tissue has made huge advances in the characterisation and localisation of cellular receptors for use in drug targeting. However, there remains a significant paucity of knowledge regarding how the majority of drug molecules are transported about the body, when they often exhibit negligible aqueous solubility and the body expresses no trans-membrane pumps or chaperone proteins that recognise them and facilitate their movement. This leads to large attrition rates in drug discovery programmes, as compounds with high binding constants or inhibitive activity in vitro fail to perform in vivo, due to poor bioavailability or non-specific sequestration away from the tissue of interest. In this study, the interactions between a number of drug and lipid molecules were investigated and the effects upon both the lipids’ chemical and bulk membrane structures were analysed. This revealed some of the mechanistic causes of the previously observed hydrolytic activity a number of common drug compounds exhibit toward lipid membranes and identified parameters affecting the observed rates of reaction. The findings also suggest approaches by which this behaviour might be predicted, or even tuned to deliver optimum pharmacological characteristics.

Mitragyna speciosa Korth (Kratom), a herb of the Rubiaceae family is indigenous in southeast Asia mainly in Malaysia and Thailand. It is used as an opium substitute and has been increasingly abused by drug addicts in Malaysia. Recently, the potent analgesic effect of plant extract and its dominant alkaloid mitragynine (MIT) were confirmed in vivo and in vitro. MIT acted primarily on μ- and δ-opioid receptors, suggesting that MIT or similar compounds could be promising alternatives for future pain management treatments. However the potential cytotoxicity of this plant is unknown. Therefore, the cytotoxicity of methanol-chloroform extract (MSE) and MIT on human cell lines (HepG2, HEK 293, MCL-5, cHol and SH-SY5Y cells) has been examined. MSE appeared to exhibit dose-dependant inhibition of cell proliferation in all cell lines examined, at concentration > 100 μg/ml with substantial cell death at 1000 μg/ml. SH-SY5Y was the most sensitive cell line examined. MIT showed a similar response. Clonogenicity assay was performed to assess the longer-term effects of MSE and MIT. The colony forming ability of HEK 293 and SH-SY5Y cells was inhibited in a dose-dependant manner. Involvement of metabolism in cytotoxicity was further assessed by clonogenicity assay using rat liver S9 (induced by Arochlor 1254); toxicity increased 10-fold in both cell lines. To determine if cytotoxicity was accompanied by DNA damage, the Mouse lymphoma tk gene mutation assay was used. The results were negative for both MSE and MIT. Studies on the involvement of metabolism in cytotoxicity of MSE and MIT were performed using MCL-5 and it appeared that CYP 2E1 is involved in activation of cytotoxicity. Studies with opioid antagonists were performed using SH-SY5Y cells treated with MSE and MIT. Naloxone (μ and δ receptor antagonists), naltrindole (δ receptor antagonist) and cyprodime hydrobromide (μ receptor antagonist) confirmed that MSE cytotoxicity was associated with μ and δ receptor while MIT mainly acted on μ receptor. Studies on mechanism of MSE and MIT cytotoxicity showed that cell death observed at high dose was preceded by cell cycle arrest, however MSE cell arrest was independent of p53 and p21 while MIT showed opposite result. Studies have been undertaken to examine the nature of this cell death. Morphological examinations showed that cell death induced by MSE was cell type dependant, in which SH-SY5Y cells appeared to die via apoptosis-like cell death while HEK 293 and MCL-5 cells predominantly via necrosis. Biochemical assessments confirmed that MSE induced cell death independent of p53 or caspases pathway while MIT cell death appeared to be associated with p53 and caspases pathway. The involvement of reactive oxygen species (ROS) generation in MSE and MIT mediating cell death was performed using SH-SY5Y cells. The results appeared negative for both MSE and MIT treated cells. Collectively, the findings of these studies suggest that MSE and its dominant alkaloid MIT produced cytotoxicity effects at high dose. Thus, the consumption of Mitragyna speciosa Korth leaves may pose harmful effects to users if taken at high dose and the evidence for involvement of CYP 2E1 in increasing the MSE cytotoxicity suggests that caution may be required if the leaves are to be taken with CYP 2E1 inducers.

P2X7 receptors exhibit a mainly non-neuronal localisation on immune and glial cells and primarily function as non-selective cation channels. After prolonged or repeated exposure to agonist, functional and cellular changes occur: the formation of a large diameter pore, cell lysis and the release of mature, biologically active interleukin-1β (IL-1β) a potent inflammatory cytokine. It is this repertoire of functions, along with its localisation that underlies the hypothesis for its involvement in pain processing. The biophysics and pharmacology of rat P2X7 receptors were investigated using stable cell lines. Increases in the current amplitude were shown to be dependent upon the agonist concentration and current deactivation was agonist application number and voltage dependent. These results increased our understanding of the receptor, but have also had implications for the design of protocols to investigate antagonist potency and efficacy. GSK31418A was identified as a potent, reversible and voltage-independent antagonist of rat and human P2X7 receptors. GSK314181A was >10000 fold selective over P2X4 receptors and >1000 fold selective over P2X2/3 receptors. GSK314181A produced a significant reversal of FCA-induced hypersensitivity when profiled in vivo, providing further validation of the role of P2X7 receptors in inflammatory pain. Although the influence of glia cells on neuronal activity in the CNS is now well documented, the role of peripheral glia, Schwann cells and satellite cells of sensory ganglia, is less well established. Non-neuronal cells in DRG cultures were shown to express P2X7 receptors by pharmacological, biophysical and immunofluorescence techniques. Native P2X7 receptors expressed on these cells were shown to have many of the properties of recombinant P2X7 receptors, in regards to the response to agonist activation and pharmacology. Finally, I have shown that Lamotrigine is an effective inhibitor of recombinant rat and human P2X7 receptors and native P2X7 receptors expressed in DRG. The potent inhibition of human P2X7 by Lamotrigine was replicated with the chemical analogue and neuroprotective agent Sipatrigine. However, little effect was recorded for a P2X7 antagonist in two models of epileptiform activity studies.

Strong opioids, morphine and oxycodone, are used in the management of moderate to severe cancer pain, but there is wide inter-individual variation in patients' response to these drugs. Here we identify the clinical and pharmacogenetic factors influencing individuals' response and build a model that predicts a favourable clinical outcome to these pain relieving strategies. Two clinical studies were conducted. Firstly, 298 subjects were recruited to a prospective, observational case-control study; data were obtained on 205 morphine responders and 93 morphine non-responders, also known as "switchers‟. Secondly, a randomised control trial was established to compare the clinical response rates to morphine and oxycodone used as first line strong opioids for cancer pain; analysis was carried out on 100 subjects randomised to receive trial medications "A‟ and "B‟ respectively. Clinical, haematological, biochemical, metabolite and genotype data were collected on all subjects; metabolite samples were processed using high performance liquid chromatography and genotyping of single nucleotide polymorphisms (SNP) in candidate genes was performed using sequence specific primer assays whilst other data were obtained by genome-wide SNP analysis. Clinical factors associated with response to strong opioids included age, plasma morphine and metabolite concentrations, concomitant medications and the presence of visceral pain. Genetic variation in the gene encoding the glutamate receptor subunit 2A was associated with response to morphine and a number of polymorphisms were associated with secondary clinical phenotypes. There was no difference in the number of subjects responding to trial medications but dose conversion ratios between trial medications were asymmetric suggestive of cross-tolerance. These findings support a clinical and pharmacogenetic basis of response to strong opioids but the factors influencing response to pain relieving strategies need further definition.

The permeability of the neuronal membrane to different ions determines both resting membrane potential (RMP) and input conductance. These parameters determine the cells response to synaptic input. In this thesis I have examined how the molecular properties of potassium and chloride ion channels can influence neuronal excitability in ways that have not previously been considered. For example, two‐pore domain potassium (K2P) channels open at rest to generate a persistent potassium ion efflux. In addition to its accepted role in setting the RMP, I have tested the hypothesis that this conductance is sufficient to repolarise the membrane during an action potential (AP) in the absence of voltage‐dependent potassium channels (Kv). We tested this prediction using heterologous expression of TASK3 or TREK1 K2P channels combined with conductance injection to simulate the presence of a voltage‐gated sodium conductance. These experiments demonstrated that K2P channels are sufficient to support APs during short and prolonged depolarising current pulses. The membranes permeability to chloride ions can also be affected by extrasynaptic GABAA receptors containing the delta subunit (δ‐GABAARs) that produce a tonic conductance due to their high apparent affinity for GABA. The anaesthetics Propofol and THIP are both believed to alter neuronal excitability by enhancing this persistent chloride flux. We have examined how this anaesthetic action is affected by the steady‐state ambient GABA concentrations that are believed to exist in vivo. Surprisingly, the anaesthetic enhancement of δ‐GABAARs is lost at low ambient GABA concentrations. Therefore, I would suggest that the anaesthetic potency of these drugs is affected by the resting ambient GABA concentration in a manner that has not previously been appreciated. In the current Thesis I have examined the molecular and pharmacological properties of two very different ion channel families that both generate a leak conductance, and I will present models that link the behaviour of these ion channels to their ability to modulate neuronal excitability.

The development of acquired resistance to chemotherapeutic drugs is often a limiting factor in the treatment of cancer. In this study, I investigated alterations in MAP kinase signalling and metabolism in a doxorubicin/epirubicin-resistant breast cancer cell line. Doxorubicin, also known as adriamycin, is one of the most important drugs in the treatment of breast cancer. The MAP kinases ERK, JNK, and p38 have all been linked to drug response and tumorigenesis, as well as apoptosis, in different cell types. I found that ERK activity was downregulated in the resistant cell line, while transcripts of the ERK phosphatases DUSP5 and DUSP6 were enriched. The resistant cells also contain less FOXO3a, a broadly pro-apoptotic transcription factor that regulates many aspects of cellular activity. I also used NMR-based metabonomics to generate a metabolic profile of the parental and doxorubicin-resistant cell lines. Many metabolic changes are seen during tumorigenesis, with further changes seen after the development of drug resistance. An increase in glycolysis is the best known, but alterations in choline metabolism and glutamine usage are also commonly seen in cancer. My results confirmed an increase in glycolysis in the resistant cells, as well as altered glutamine metabolism, and also provided novel findings for future work. The reduction in intracellular glutamine in the resistant cells was correlated with a loss of expression of the metabolic enzyme glutamine synthetase. My results show that the doxorubicin-sensitive parental cell line expresses glutamine synthetase, which was required for maximal proliferation rate. Conversely, the lack of glutamine synthetase in the doxorubicin-resistant cells caused them to be dependent on the provision of extracellular glutamine for growth. This may have implications for the treatment of drug resistant breast cancers.

Glucocorticoids (GCs) have been used for decades in the treatment of chronic inflammatory and autoimmune diseases, thanks to their powerful anti‐inflammatory properties. However, long term treatment can lead to deleterious side effects, and some patients also experience resistance to their therapeutic effects. GCs act through the glucocorticoid receptor (GR) to regulate transcription both positively and negatively. Negative regulation of transcription involves a process known as transrepression, in which ligand‐activated GR impairs transcriptional activation by nuclear factor κB (NF‐κB) and other transcription factors. It is widely believed that transrepression accounts for most of the anti‐inflammatory effects of GCs, whereas the activation of transcription (transactivation) is responsible for most side effects of GCs. Based on this principle, several pharmaceutical companies are trying to identify selective GR modulators (SGRMs) that preferentially induce transrepression rather than transactivation. Such compounds are predicted to retain the anti‐inflammatory properties of classical GCs but cause fewer side effects. There are several problems with a dogma that equates anti‐inflammatory effects of GR with transcriptional repression. One is that GCs have long been known to destabilise many pro-inflammatory mRNAs, and this property is not explained by the transrepression model. Another issue is that GCs induce the expression of many factors with powerful anti-inflammatory effects. One of these is dual specificity phosphatase 1 (DUSP1), an enzyme that dephosphorylates and inactivates mitogen‐activated protein kinases. Studies of the Dusp1‐/‐ mouse have underlined the importance of the phosphatase in the antiinflammatory response to GCs. In this work, I investigated the role of DUSP1 in (1) the post‐transcriptional regulation of pro‐inflammatory mRNA stability by GCs and (2) the anti‐inflammatory actions of SGRMs. 1‐ The classical dexamethasone (dex) was shown to upregulate DUSP1 in mouse macrophages, and to inhibit the expression of cyclooxygenase 2 (COX‐2) in a manner that was partially dependent on DUSP1. Dex destabilised COX‐2 and interleukin 1α mRNAs, and this post‐transcriptional effect appeared to require DUSP1. 2‐ Two SGRMs were characterised and shown to preferentially mediate transrepression rather than transactivation. However, they were capable of inducing the expression of DUSP1 in several different cellular systems, and their capacity to inhibit the expression of COX‐2 was correlated with DUSP1 induction. Finally, several of the anti‐inflammatory effects of the SGRMs were found significantly impaired in mouse macrophages lacking DUSP1.

Dual specificity phosphatase 1 (DUSP1) limits the expression of pro-inflammatory gene products by dephosphorylating and inactivating mitogen-activated protein kinases (MAPKs), in particular p38 MAPK and cJun N-terminal kinase. In many mouse and human cell types, glucocorticoids (GCs) increase the expression of DUSP1. The resulting inhibition of intracellular signalling pathways is thought to contribute to the powerful anti-inflammatory effects of GCs. The purpose of this project was to define mechanisms by which GCs control expression of mouse and human DUSP1 genes. Orthologous genes of distantly related species often respond to GCs via regulatory elements that are evolutionarily conserved in sequence and position. Therefore interspecies sequence comparison was used as one tool to identify GC-responsive regions (GRRs) of mouse and human DUSP1 genes. Functional assays of GRRs were carried out by transient and stable transfection of mouse and human cells. Interactions of these cis-acting elements with transcription factors (including the GC receptor itself, GR) were assessed by in vitro DNA-binding assays and chromatin immunoprecipitation. A number of conserved, putative GRRs were identified up to 29 kb upstream of the DUSP1 transcription start site (TSS). Surprisingly, some of these were found to be differently utilised by mouse and human DUSP1 genes, in spite of their sequence similarity. Strongly GC-responsive elements were found 4.6 and 1.3 kb upstream of the human DUSP1 TSS. The corresponding regions of the mouse DUSP1 gene were unresponsive or very weakly responsive to GC. Instead the mouse gene appeared to be regulated via an element 29 kb upstream of the TSS, the corresponding human element being unresponsive. GC responsiveness correlated with the ability to recruit GR in vivo, but could not be explained on the basis of differences in the sequences of GR binding sites. These observations suggest that recruitment of GR to DUSP1 loci and transcriptional activation in response to GC are critically dependent on “accessory” transcription factors. GC responses of mammalian DUSP1 genes appear to have evolved via gain and/or loss of binding sites for accessory factors, whilst maintaining overall output. GC regulation of DUSP1 genes involves atypical GR binding sites and, unusually, does not appear to require dimerisation of GR.

The overall aim of this PhD was to improve our understanding, including the clinical potential, of neonatal Nav1.5 (nNav1.5) expression in human metastatic breast cancer. Mainly, the strongly metastatic MDA-MB-231 cells were used throughout the studies. The specific aims were threefold, as follows: 1) To test the effects of several types of voltage-gated sodium channel (VGSC) blocker on nNav1.5 mRNA and protein expression and metastatic cell behaviours (MCBs); (2) to determine the effects of hypoxia on the drug treatments and MCBs; and (3) to elucidate a possible association of carbonic anhydrase-9 (CA9) and nNav1.5 expression/activity. There are three main Results chapters. Results-1 demonstrates the effects of the drugs on MCBs of MDA-MB-231 cells under normal oxygen level (normoxia). Two classes of blocker were used: a) Local anaesthetics (lidocaine and procaine) and (b) blockers of persistent current (INaP) (ranolazine and riluzole). In addition, a specific VGSC blocker, tetrodotoxin (TTX), was incorporated as a control. At concentrations not affecting the cells‘ viability or proliferative activity, 24 h treatment with all the drugs tested significantly reduced MCBs (lateral motility, transverse migration and Matrigel invasion). Concurrently, the treatments reduced nNav1.5 mRNA and protein levels. TTX had a similar effect. Overall, this chapter showed (i) that MCBs in MDA-MB-231 cells were enhanced by VGSC activity and (ii) that INaP played a significant role in the enhancement. Results-2 shows that viability and proliferative activity of MDA-MB-231 cells were not affected by hypoxia (mostly 2 % oxygen applied for 24 h). However, hypoxia increased the cells‘ invasiveness and this was accompanied by upregulation of HIF-1α (protein), nNav1.5 (mRNA) and CA9 (both mRNA and protein). Treatment for 24 h with INaP blockers; ranolazine and riluzole under hypoxia reduced lateral motility, transverse migration and Matrigel invasion. At concentrations not affecting cell viability and proliferation, the effects of ranolazine and riluzole in suppressing MCBs were generally greater under hypoxia compared to normoxia. It was concluded (i) that hypoxia enhanced VGSC-mediated MCBs and (ii) that the enhancements were likely to be increase in INaP amplitude induced by hypoxia. Results-3 examined a possible functional link between hypoxia (HIF-1α and CA9) and VGSCs. Acetazolamide was used as a general inhibitor of CAs and siRNA was used to silence specifically CA9. Under hypoxia, treatment with acetazolamide for 24 h had no effect on invasion, and treatment with TTX was without effect on CA9 expression. In contrast, silencing CA9 using siRNA (siCA9) reduced CA9 and nNav1.5 expression and Matrigel invasion was also significantly inhibited. It was concluded (i) that CA9 played a role in cellular invasion and (ii) that nNav1.5 was down-stream to CA9 in the control of MCBs. The Thesis is concluded with a General Discussion and Conclusion chapter integrating the findings and highlighting their clinical potential.