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Pancreatic cancer has been considered as the most lethal cancer type among adults for many decades. Especially with the failure to improve survival rates for the last 40 years, despite all the progress in targeting therapies. With the lack of therapeutic options, attention has been directed towards innovative platforms in a bid to overcome the resistant and aggressive nature of the disease. Micro-RNAs are endogenous small, non-coding RNAs considered to be post-translational regulators for the expression of targeted genes. The utilization of miRNAs as therapeutic agents has showed promising rapid progress in the last 10 years but are limited because of the lack of appropriate delivery systems that will ensure reach of the pharmacological targets. On the other hand, the bottleneck with using gemcitabine, the first line chemotherapeutic agent, is the rapidly developing resistance coupled with low bioavailability. The aim of this project is to elucidate the potential role of miR-143, miR-145 and gemcitabine triphosphate in the treatment of pancreatic cancer when delivered in fusogenic peptide-based nanoparticles. To date, results have shown that the fully characterised nanoparticles can transfect pancreatic cell lines with efficient delivery of the cargo intracellularly, as detected by FACS and qRT-PCR analysis. The restoration of miR-143 and miR-145 demonstrated disruption in BxPC-3 functionality while no response was observed with MIA-PaCa2. On the other hand, PANC-1 had variable responsiveness to both miRNAs which highlights the significant role of cellular variation and molecular signature on the effectiveness of miR-143/145 as a therapeutic option. In contrast, administering gemcitabine triphosphate via the developed nanoparticles resulted in consistent efficacy among the cell lines suggesting a common mechanism of evading gemcitabine resistance and intraindividual variability. Furthermore, RALA/GTP nanoparticles demonstrated potentiation of the cytotoxic effect of the chemotherapeutic agent by 8- to 24-fold compared to the parental drug.

Nucleoside analogues are molecules that share structural similarities with nucleosides and interfere with DNA replication. Many nucleoside analogues such as gemcitabine, a cytidine analogue, are an established part of cancer therapy. When integrated into the DNA, gemcitabine functions as a chain terminator and causes replication stalling, which leads to cell death. Other effects of Gemcitabine include dNTP pool depletion and inhibition of enzymes involved in DNA replication. Despite its established clinical use, the cellular mechanisms behind gemcitabine resistance and removal are not well understood. Previous research has shown that MRE11 is involved in the gemcitabine removal and thus contributes to resistance of cancer cells against gemcitabine. MRE11 is a core enzyme part of the MRN complex. It is frequently mutated in certain types of colorectal cancer. The MRN complex is involved in DNA double strand break detection and repair as well as other pathways such as topoisomerase removal. Mirin, a small molecule inhibitor, has been shown to be an inhibitor of the MRE11 exonuclease function. The research conducted in the context of this thesis shows that mirin exposure sensitises DT40 chicken B lymphocytes and human MRC5 lung fibroblasts to gemcitabine treatment. Furthermore, DT40 MRE11H129N/- cells innately deficient in MRE11 nuclease function showed sensitivity to gemcitabine as well, but no further sensitisation after mirin treatment. These results suggested that MRE11 exonuclease function might remove gemcitabine from the DNA. Analysis of the gemcitabine content of the DNA of gemcitabine-treated human and chicken cells via mass spectrometry showed that both mirin-treated and DT40 MRE11H129N/- cells have increased levels of genomic gemcitabine compared to control cells. Combined, these findings show that mirin specifically inhibits MRE11 exonuclease activity, and that MRE11 exonuclease activity contributes to the removal of gemcitabine from the DNA. Additional experiments with a library of S. pombe strains with mutations in genes involved in DNA damage detection and repair, cell cycle, and genome maintenance identified a number of mutants sensitive to treatment with hydroxyurea, gemcitabine, fludarabine, or cytarabine; homologous repair mutants and cell cycle checkpoint mutants displayed high sensitivity specifically. These findings show that DNA repair mechanism and knowledge of the genetic composition of cells such as mutations in cancer cells can be utilised to increase the efficiency of drug treatment, by personalising cancer treatment and tailoring treatment to individual patients.

Drugs enter cells via proteinaceous membrane transporters which are normally used for the transportation of nutrients and metabolites. Drug efficacy is thereby mainly determined by the distribution and kinetics of influx and efflux transporters for which they are substrates. The idea that the transporter-mediated disposition of drugs can be altered by the addition of a second small molecule, that of itself has no inhibitory pharmacological effect, but that influences the expression of transporters for the primary drug, was studied. This strategy is here referred to as the 'binary weapon' approach. The experimental system tested the ability of such a molecule to increase the toxicity of the nucleoside analogue gemcitabine to pancreatic cancer cells. An initial phenotypic screen of a 500-member polar drug (fragment) library, using the MTT assay to estimate cell viability, yielded three candidate fragments which showed efficacy in enhancing the toxicity of gemcitabine by at least 10%. Cheminformatics analyses were used to screen the rest of the 2000 members of this library suite, and the structures of 20 fragments had a Tanimoto similarity (with MACCS encoding) >0.7 to the reference fragments. Wet-lab screening experiments were performed, and indeed each fragment was itself active (the cheminformatics thus providing for a massive enrichment). The top six representatives from the whole library, showing activity at the lowest concentration studied (3 uM), were considered to be the final active fragments. These fell into three clusters whose members bore reasonable structural similarities to each other (two were in fact isomers), lending strength to the self-consistency of both the predicted and experimental strategies used. Moreover, none of the six active fragments seemed to have any such effects on the non-cancerous pancreatic cell line hPDE, the human embryonic kidney cell line HEK293, or the neuroblastoma cell line SH-SY5Y, implying that there is, or can be, at least some degree of specificity in the 'binary weapon' approach. Given the experimental evidence that some of the Maybridge fragments were increasing gemcitabine's toxicity towards Panc1 cells, it was intriguing to understand the underlying mechanism of action. ENT1-3 influx and ABCC2, 3, 4, 5 and 10 efflux transporters displayed measurable transcripts in Panc1 cells, along with a ribonucleoside reductase RRM1 known to affect gemcitabine toxicity. Very strikingly, the addition of gemcitabine alone increased the expression of the transcript for ABCC2 by more than 12-fold, and that of RRM1 by more than fourfold, and each of the fragment 'hits' served to reverse this. However, MK571, an inhibitor of ABCC2, was without significant effect, possibly implying that the effect of RRM1 is the more significant contributor to the phenotypic changes noted. It proved difficult to find inhibitors of RRM1. It seems, therefore, that while the effects measured here were mediated more by efflux than influx transporters, and potentially by other means, the binary weapon idea is hereby fully confirmed: it is indeed possible to find molecules that manipulate the expression of transporters involved in the bioactivity of a pharmaceutical drug. This opens an entirely new area of chemical genomics-based, transport-mediated drug targeting. Moreover, internal experimental controls to ensure that unwanted off-target effects were kept to a minimum showed that many of the library fragments were indeed stimulating cell growth. To investigate this observation, phenotypic screening of the Maybridge fragment library, again using the MTT assay to estimate cell viability, and cheminformatics analyses to determine the Tanimoto similarity between fragments, yielded five molecules which enhanced cell growth by at least 10%, displaying potential uses in the biotechnology industry where the growth rate stimulation of mammalian cells is usually sought after. The efficacy of these fragments was further tested in the presence of gemcitabine. Only one of these fragments proved to be bimodal, enhancing Panc1 cell growth rate in the absence of gemcitabine by at least 10%, however, also showing efficacy in enhancing the drug's toxicity by the same percentage when treatment was administered in the presence of gemcitabine.

Pancreatic cancer is a deadly disease with a five-year survival rate of less than 5%. Gemcitabine is the standard of care in the treatment of pancreatic cancer, however, limitations in effectiveness are widespread and need drastic improvement. This project aimed to develop a novel drug combination capable of enacting cytotoxic effect, eliminating the protein levels of mucin glycoproteins while having minimal reductions on human nucleoside transporters in pancreatic cancer cells. Consequently, I investigated the growth inhibitory, cell viability, mucin depleting and transporter depleting effects of bromelain, NAC, and gemcitabine as single agents or in combination against a panel of pancreatic cancer cell lines in vitro. I observed that bromelain and NAC exert cytotoxic effect as single agents, though combination treatments significantly increase potency. The addition of bromelain and BromAc to gemcitabine results in significant inhibition of cell proliferation, survival, and viability. The levels of hENT1/2, hCNT1/3 and MRP5 protein showed no significant reductions following treatment with bromelain, NAC, Gem/bromelain, and Gem/NAC, while BromAc treatments displayed significant reductions in hCNT1/3 protein levels. All transporters increased in protein levels following gemcitabine treatment with significant reductions observed in hENT2 and, hCNT1/3. Bromelain and NAC treatments displayed no significant changes in the levels of mucin, while BromAc displayed significant reductions in MUC1/4/5AC/13/16. All mucins displayed an increase in protein levels following Gem treatment and decreases following treatment with Gem/bromelain and Gem/NAC, apart from MUC13 which displayed increases. The Gem/BromAc treatment resulted in significant decreases in protein levels for all examined mucins. Correlations were identified between the transporters hENT2, hCNT1/3 and mucins MUC1/4/13/16. In addition, all examined mucins, hCNT1, and hCNT3 were seen to correlate with cell proliferation and viability though insufficient data was obtained to accurately predict synergy. To summarize, the addition of bromelain and NAC to gemcitabine results in a significant increase in potency and decrease in toxic dose. This is due in part to the dissolution of mucin glycoproteins encompassing pancreatic cancer cells, thus exposing the human nucleoside transporters required for the uptake of gemcitabine.

Replication stress can be produced by various exogenous or endogenous factors that perturb the movement of replication forks. To overcome the stress, the cells provoke the DNA replication checkpoint to mobilize several pathways to protect the forks, maintain genome integrity, and promote cell survival. Defects in the replication checkpoint cause forks collapse, leading to chromosomal DNA damage or cell death. Although the replication checkpoint is crucial for genome integrity in all eukaryotes, the underlying mechanisms remains to be fully understood. To investigate the mechanisms of DRC, hydroxyurea (HU), an established inhibitor of ribonucleotide reductase (RNR), has been widely used in laboratories as an inducer of the replication stress. It depletes dNTP pools, slows down the movement of DNA polymerase at the forks, and thus activates the replication checkpoint pathway in yeasts and in mammalian cells. Unfortunately, HU also targets other cellular components, which may complicate the studies, leading to ambiguous description of the checkpoint mechanisms. The purpose of this study is to find an RNR inhibitor that produces the replication stress more specifically than HU in the fission yeast Schizosaccharomyces pombe. We examined three RNR inhibitors guanazole, triapine, and gemcitabine under several experimental conditions that are commonly used in the laboratories for checkpoint studies. We found that among the three drugs, guanazole and triapine produce the replication stress more specifically than HU under the chronic drug exposure conditions such as the spot assay. Under acute drug treatment conditions, however, guanazole and triapine cause other cellular stresses more significantly than HU. Therefore, using guanazole and triapine in chronic drug exposure conditions and HU in acute treatment can produce a specific replication stress under various experimental conditions and thus benefits the checkpoint studies in S. pombe and possibly the research in other model systems.

Platinum- and gold-based compounds have emerged as promising anticancer agents. Pt(IV) prodrugs are more stable than their Pt(II) counterparts and, with the flexibility provided by the two additional axial ligands, are able to be structurally fine-tuned. These features make Pt(IV) compounds a great platform for overcoming systemic toxicity and drug resistance issues characteristic of Pt(II) compounds and for preparing multi-action anticancer drugs. Gold(I) bis-N-heterocyclic carbenes (Au(I) bis-NHCs) are able to localize specifically in mitochondria and inhibit the activity of thioredoxin reductase (TrxR); this renders them both good mitochondria-targeting agents and potent antitumor agents. However, there has been no record in the literature of coupling these two powerful anticancer compounds. Here, we report the synthesis of a conjugate comprising an Au(I) bis-NHC, oxaliplatin, and gemcitabine. A synergistic effect between the gold and the platinum component was inferred from preliminary results in the cell proliferation experiments. Further investigations will be needed to elucidate and characterize the putative synergism and other biological properties.

Cancer is a deadly malignant disease that is costly to treat and still impossible to cure. Cancer treatments often come with side effects that impact the overall health of patients. Breast cancer is the most common type of cancer in the United State and bladder cancer mortality and recurrence rates are still high among cancer patients. Thus, safe and effective regimens are needed to combat these diseases. In this study, the safe and efficacious combination of gemcitabine, cisplatin, and romidepsin (Gem plus Rom+Cis) to treat two types of cancer: bladder and breast cancer, was formulated. In vitro data from this study revealed that the combination of Gem plus Rom+Cis synergistically reduced cell vitality, clonogenic survival, invasion, and anchorage independent growth of bladder and breast cancer cell lines. The tolerable drug doses and administration schedules of Gem plus Rom+Cis for mice model were determined. Athymic nude mice (nu/nu) treated with the triple combination showed no significant weight loss or sign of toxicity at the end of studies. Gem plus Rom+Cis efficaciously controls the development of tumors in CDX models. Results showed that breast and bladder CDXs treated with Gem plus Rom+Cis had significantly smaller tumor sizes comparing to double combination of Gem plus Cis or Rom+Cis or untreated control. Data from urothelial carcinoma studies revealed that the apoptosis of bladder cancer cells was found to be enhanced through the ERK-Nox-1 pathway via reactive oxygen species (ROS) induction. Furthermore, drug resistance was reduced due to the suppression of glutathione (GSH) and possibly by reducing factors that associated with epithelial to mesenchymal transition (EMT). The data also suggested that the combination reduced invasiveness and anchorage independence in cancer cell lines as a consequence of suppressing the expression levels of proteins that are related to metastasis and EMT process. In conclusion, this triple combination was revealed to be more effective in controlling tumor development than double or mono anti-cancer therapy. Thus this combination should be considered as an advanced regimen for cancer treatment that hopefully could be translated into a clinical trial to reduce cancer-related mortality and improved quality of life for cancer patients.

Gemcitabine (2’-2’-difluoro-deoxycytidine) is an intravenously administered nucleoside analogue used in the treatment of various solid tumor cancers. While oral gemcitabine administration would offer a more patient-friendly, less complex, and less expensive alternative to intravenous administration, the clinical utility of oral gemcitabine administration is hindered by a low oral bioavailability of approximately 10%. This low oral bioavailability was previously believed to be the result of gemcitabine’s low intestinal permeability and oral absorption, followed by significant presystemic metabolism by the enzyme cytidine deaminase (CDA). In one study, we sought to define the mechanisms of gemcitabine intestinal permeability, the potential for saturation of intestinal uptake, and the transporter(s) responsible for mediating the oral absorption of drug using in situ single-pass intestinal perfusions in mice. Concentration-dependent studies were performed for gemcitabine over 0.5 to 2000 µM, along with studies of 5 µM gemcitabine in a sodium-containing buffer +/- thymidine (which can inhibit concentrative (i.e., CNT1 and CNT3) and equilibrative (i.e., ENT1 and ENT2) nucleoside transporters) or dilazep (which can inhibit ENT1 and ENT2), or in a sodium-free buffer (which can inhibit CNT1 and CNT3). Our findings demonstrated that gemcitabine was, in fact, a high-permeability drug in the intestine at low concentrations, that jejunal uptake of gemcitabine was saturable and mediated almost exclusively by nucleoside transporters, and that jejunal flux was mediated by both high-affinity, low-capacity (Km = 27.4 µM, Vmax = 3.6 pmol/cm2/s) and low-affinity, high-capacity (Km = 700 µM, Vmax = 35.9 pmol/cm2/s) transport systems. Thus, CNT(s) and ENT(s) at the apical membrane allow for gemcitabine uptake from the lumen to enterocyte, whereas ENT(s) at the basolateral membrane allow for gemcitabine efflux from the enterocyte to portal venous blood. These results further show that systemic exposure following oral gemcitabine administration is limited by extensive CDA-mediated presystemic metabolism and potentially by saturation of nucleoside transporter-mediated intestinal uptake following oral administration of large doses (i.e., doses which generate saturating gemcitabine concentrations in the intestinal lumen). A subsequent study evaluated the in vivo performance of a peptide transporter 1 (PEPT1)-targeted amino acid ester prodrug of gemcitabine, 5’-L-valyl-gemcitabine (V-Gem), developed to increase gemcitabine’s oral bioavailability. V-Gem was previously shown to be a substrate of the intestinally expressed PEPT1 and stable against CDA-mediated metabolism. However, earlier studies did not evaluate the in vivo oral performance of V-Gem as compared to parent drug. Therefore, we evaluated the pharmacokinetics and in vivo oral absorption of gemcitabine and V-Gem following intravenous and oral administrations in mice. These studies revealed that V-Gem undergoes rapid systemic elimination (half-life < 1 min) and has a low oral bioavailability (< 1%). Most importantly, the systemic exposure of gemcitabine was not different following oral administration of equimolar doses of gemcitabine (gemcitabine bioavailability of 18.3%) and V-Gem (gemcitabine bioavailability of 16.7%). Single-pass intestinal perfusions with portal blood sampling in mice revealed that V-Gem undergoes extensive activation (i.e., conversion to gemcitabine) in intestinal epithelial cells and that gemcitabine undergoes first-pass metabolism in intestinal epithelial cells. Thus, formulation of gemcitabine as the prodrug V-Gem does not increase systemic gemcitabine exposure following oral dosing, due, in part, to the metabolic instability of V-Gem in intestinal epithelial cells. These findings suggest that future development of gemcitabine prodrugs for oral administration should focus on prodrugs with high intestinal permeability, high presystemic stability, and complete in vivo conversion to gemcitabine.

Due to chemoresistance and a high propensity to formlungmetastasis, survival ratesin pediatric osteosarcoma (OS) are poor. With the aim to improve anticancer activity in pediatric OS, a multidrug nanomedicine was designed using the alkyl-lysophospholipid edelfosine (EF) co-assembled with squalenoyl–gemcitabine (SQ–Gem) to form nanoassemblies (NAs) of 50 nm. SQ–Gem/EF NAs modified the total Gem pool exposure in the blood stream in comparison with SQ–Gem NAs, which correlated with a better tolerability and a lower toxicity profile after multiple intravenous administrations in mice. For in vivo preclinical assessment in an orthotopic OS tumor model, P1.15 OS cells were intratibially injected in athymic nude mice. SQ–Gem/EF NAs considerably decreased the primary tumor growth kinetics and reduced the number of lung metastases. Our findings support the candidature of this anticancer nanomedicine as a potential pediatric OS therapy

The strategy to improve the lipophilicity of gemcitabine by conjugation with a lipid (e.g. docosahexaenoic acid (DHA) or stearoyl acid) allows to prevent the deamination of gemcitabine by cytidine deaminase, main cause of gemcitabine degradation in vivo. In addition, this strategy may confer gemcitabine a strong antitumor activity compared to parental drug. 4-(N)-docosahexaenoyl 2´, 2´-difluorodeoxycytidine (DHA-dFdC) is a novel compound with strong in vitro and in vivo antitumor activity in several cancer models (e.g. pancreatic, breast, leukemia, and lung cancer). However, the toxicity of DHA-dFdC has not been tested in an animal model. In this dissertation, a preclinical short-term toxicity study to evaluate the tolerability of DHA-dFdC was performed in healthy DBA/2 mice. DHA-dFdC showed a dose-dependent toxicity, affecting mainly the spleen. The repeat-dose maximum tolerated dose (RD-MTD) of DHA-dFdC was 50 mg/kg. DHA-dFdC exhibits a strong efficacy at or below its RD-MTD in mouse models of pancreatic cancer or leukemia. Unfortunately, DHA-dFdC has two main issues, chemical instability and poor solubility in water. In this dissertation, a solid lipid nanoparticle (SLN) formulation of DHA-dFdC was developed to improve its chemical stability and water solubility. The SLN formulation improves the apparent water solubility of DHA-dFdC by 2.6-fold, as compared to a previously developed DHA-dFdC in Tween 80-ethanol-water solution. The SLN formulation as a lyophilized powder also improves the chemical stability of DHA-dFdC. In addition, DHA-dFdC-SLNs showed stronger antitumor activity than DHA-dFdC in Tween-ethanol-water solution in a mouse model when given intravenously. Finally, this dissertation demonstrates in a mouse model that the absolute oral bioavailability of DHA-dFdC in the DHA-dFdC-SLN formulation is 68%. It is concluded that the SLN formulation of the DHA-dFdC (i.e. DHA-dFdC-SLNs) improves the chemical stability of DHA-dFdC and increases its apparent water solubility, and the DHA-dFdC-SLNs can be administered intravenously or orally

Nanodiamonds (NDs) are promising candidates for drug delivery due to their small size, biocompatibility and stable fluorescence. Their large surface area and ease of functionalization allow the surface to be functionalized with various bioactive compounds. However, the strong tendency to form aggregates in aqueous environment limits their bio-applications. In this Thesis, NDs were coated with synthetic hydrophilic polymers to improve both colloidal stability and to create sites accommodating for anticancer drugs. Several issues exist with the direct administration of anticancer drugs, amongst these are the severe systemic side effects and metabolization before reaching the diseased sites. Incorporating the drugs into nanoparticles presents an elegant method to protect drugs from metabolism and minimize side effects. A gemcitabine prodrug monomer with a pH or glutathione (GSH) responsive linkage was incorporated into a polymer by RAFT polymerization. The polymer was then grafted onto the surface of NDs by copper free click chemistry or supramolecular chemistry. Melittin was encapsulated onto the surface functionalized with polymer containing carboxylic acids via electrostatic interactions. When the surface of NDs was functionalized with ?-cyclodextrin conjugated polyacrylic acid, the positively charged doxorubicin and paclitaxel-loaded polymer with adamantane end group were loaded onto the surface simultaneously. After internalization into cancer cells, the nanoparticles loaded with drugs can release their payloads responsive to the intracellular microenvironment, such as a decrease in pH or an increase in GSH concentration. Additionally, these drugs are mostly retained in the nanoparticles before uptaken by cells, allowing for the on-demand delivery of the encapsulated drugs. Cleavable linkages were employed for gemcitabine and paclitaxel whilst melittin and doxorubicin were loaded electrostatically. Both these methods released the respective drugs in their original form, hence preserving the therapeutic efficacy of these drugs.

The functional heterogeneity of cancer phenotypes and responses to therapeutics is a huge obstacle to clinical cures and an intense area of study. A relatively new hypothesis posits the existence of a subpopulation of tumor cells, termed cancer stem cells, which are primarily responsible for tumor propagation and resistance to therapy. Identification of such cells may be important to develop targeted therapies for more effective cancer treatment. This dissertation focuses on the validation and functional study of cancer stem cells associated with human prostate and pancreatic cancers. By leveraging the fact that surgical castration leads to enrichment of stem/progenitor cells in the prostate, we idenitifed CD166 as a novel marker for prostate stem cells. We show that CD166 can further enrich and refine the established prostate stem/progenitor cell and cancer stem cell population in both murine and human systems. While genetic deletion of CD166 would not inhibit the development of the prostate gland or the formation of prostate cancer in our genetically engineered model systems, this protein may serve as an attractive marker for identifying and targeting prostate cancer stem cells. Further attempts were made at identifying stem/progenitor populations for prostate and pancreatic cancer through various discovery and targeted approaches. We found that Lgr5, a marker of stem cells in the gut and skin, is not expressed in prostate and pancreatic epithelial tissue, but only in prostatic stroma. Novel surface markers such as CD138 were found to be highly upregulated in pancreatic tumors, but specificity for cancer stem cells was lacking. Lastly, we studied the functional role of cancer stem cells in gemcitabine therapy resistance in a human pancreatic cancer model. We found that the PI3K and ubiquitin-mediated proteosomal degradation pathways can be used to stratify gemcitabine treatment response in patient tumors. Inhibition of Skp2 expression and therapeutic treatment with PI3K inhibitors proved to be effective sensitiziers to gemcitabine treatment. Importantly, cancer stem cell content and function did not mediate gemcitabine relapse. As such, our studies reveal the cancer stem cell paradigm may be more complicated and cancer context-dependent, and must be rigorously functionally proven prior to clinical adoption.

Cancer is a collection of over one hundred different types of diseases and is responsible for the leading cause of death in the United States. More strikingly, cancer mortality rates have remained relatively unchanged for the past several decades, indicating significant clinical demand for improved cancer therapy. Gemcitabine, known clinically as Gemzar®, is used to treat a variety of human cancers, however, clinical efficacy is modest due to it’s brief blood circulation, rapid clearance, manifestation of tumor-drug resistance, and lack of drug specificity. This thesis sought to develop a solid lipid nanoparticle-based platform to passively and actively target a gemcitabine lipophilic derivative, 4-(N)-stearoyl gemcitabine, into tumor cells over-expressing epidermal growth factor receptor (EGFR) after intravenous injection. Considering gemcitabine is hydrophilic and the core of the nanoparticle is solid (hydrophobic), we lipophilized gemcitabine by conjugating a stearoyl group to its N-terminus to form 4-(N)-stearoyl gemcitabine. Second, we incorporated stearoyl gemcitabine into lecithin-based nanoparticles. The nanoparticle formulation was prepared from lecithin/glyceryl monostearate-in-water emulsions. Third, we grafted the gemcitabine nanoparticles with polyethylene glycol chains with reactive end groups that are capable of conjugating with a targeting moiety on the surface to actively target tumors that over-express EGFR. Taken together, the overall objective of the research presented in this thesis is to develop, characterize, and evaluate the anti-tumor performance in vitro as well as in mice against both human and mouse tumor models.

The first part of this dissertation describe the characterization of the DNA binding activity of high mobility group A (HMGA) protein and provide an analysis of the use of a DNA aptamer as a potential cancer therapeutic treatment in human pancreatic cancer cell lines. The second part describes the application of NMR-based metabonomics in mammalian cell line studies. Chapter 1 provides a background on the current knowledge of HMGA and its role in cancer, as well as an introduction to NMR-based metabonomics and its use in cell line studies. Chapter 2 presents the characterization of HMGA/DNA complex formation using electro mobile shift away and isothermal calorimetry. Based on this study, a HMGA-targeted, DNA aptamer inhibitor was designed, and its effect on cancer cell growth was examined in human pancreatic cancer cell lines. This HMGA inhibitor study, discussed in Chapter 3, also examined the effect of the co-treatment using the DNA aptamer together with the chemotherapy reagent, gemcitabine, which showed significant decrease in pancreatic cancer cell viability. Chapters 4, 5, and 6 describe cell line metabonomics studies using NMR spectroscopy. Cell line metabonomics are useful for the identification of potential biomarkers for a certain disease. Chapter 4 describes a comparative study of metabolic profiles of three human pancreatic cancer cell lines and a normal human pancreatic ductal epithelial cell line, H6C7. This study identified not only metabolites unique to the cancer cells compared to normal cells, but it also identified metabolic differences between cancer cell lines. Furthermore, the use of NMR-based metabonomics in cell line studies enabled identification of metabolic pathways that can be targeted by drug treatment, which can ultimately be used to monitor the effect of cancer drug therapy. Chapters 5 and 6 demonstrate the capability of NMR-based metabonomics to study the effect of drug treatments in two different systems: a burn-injury model and a breast cancer model. In both systems, several metabolites were identified as potential markers for assessing these drug treatments, and it is anticipated that this research will provide a framework for further analysis of these drugs as potential therapeutic agents. Chapter 7 summarizes the presented research and discusses future work.

Multiple anti-cancer drugs are often combined to increase their effectiveness. This study investigates the combination between an epidermal growth factor receptor (EGFR) inhibitor and gemcitabine, a commonly used chemotherapy agent that causes cell death by incorporation into DNA. Clinical trials combining EGFR inhibitors with gemcitabine-based chemotherapy in non-small cell lung cancer (NSCLC) have not produced a survival advantage. This may be caused by antagonism between the two drugs or mutations that promote such, possibly RAS mutation. Furthermore, ERK, a critical growth regulator downstream of RAS, may play a role. Therefore the aim of this study is to explore the cytotoxic interaction between gemcitabine and an EGFR inhibitor as well as the mechanisms underlying this interaction. The impact of KRAS mutation will also be investigated. Two NSCLC cell lines (A549 and H322) with divergent KRAS mutation status were treated with gemcitabine and the EGFR inhibitor AG1478. Cytotoxic interaction was determined with median effect analysis. Cell cycle alteration was assessed using flow cytometry while EGFR downstream signalling was assessed using immunoblotting and ELISA. ERK was inhibited using U0126 to explore the effect on cytotoxic interaction. The effect of KRAS was explored using siRNA-mediated knockdown in A549. Cytotoxic interaction was concentration and schedule dependent with antagonism in AG1478 pre-treatment and high concentration AG1478. G1 arrest, being observed with both high concentration AG1478 and high concentration gemcitabine, was inconsistently associated with antagonism. On the other hand, ERK phosphorylation was increased by gemcitabine and its suppression by AG1478 was related to antagonism particularly in H322. Antagonism caused by inhibition of ERK was further confirmed following U0126 treatment. Greater antagonism was observed in the KRAS mutant cell line and KRAS knockdown by siRNA resulted in increased sensitivity to AG1478 as well as combination treatment. In conclusion, ERK appears to be an important determinant of cytotoxic interaction with its phosphorylation favouring synergism. However, the impact of other EGFR downstream pathways warrants further investigation. Furthermore, KRAS mutation confers resistance to AG1478 and combination treatment. Both ERK and KRAS may potentially be important biomarkers for EGFR inhibitor combination therapy.

Gemcitabine is a nucleoside analog used as a radiosensitizer for the treatment of locally advanced cervical carcinoma. Yet, despite its efficacy when administered concomitantly with radiation, gemcitabine therapy is not without side effects. The utility of delivering gemcitabine directly to the cervix was explored through the use of a novel drug delivery device, CerviPrep(TM). Local administration to the cervix led to clinically relevant concentrations of gemcitabine in cervical tissue and plasma, while no gemcitabine was detected in the systemic circulation and no side effects were reported. Our data suggest that targeting gemcitabine delivery to the cervix can limit systemic exposure and toxicity while achieving cytotoxic concentrations of drug at the target site. Despite its widespread use in cervical carcinoma, little is known about the disposition of gemcitabine in this tissue. As a nucleoside analog, gemcitabine is a substrate for the equilibrative nucleoside transporters (hENT), and patient response to gemcitabine therapy has been associated with the expression of these proteins. A characterization of hENT1 and hENT2 in both malignant and normal cervical tissue was undertaken, and while no effect of malignancy was observed on hENT1 protein expression, hENT2 protein was nearly three-fold higher in malignant cervical tissue when compared to normal tissue. Expression of hENT mRNA was highly variable and not associated with malignancy. We also examined the effect of dFdU on gemcitabine disposition, as this relatively inactive metabolite is present at much higher concentrations in the plasma than gemcitabine following intravenous administration of the parent compound. We report a novel interaction between dFdU and gemcitabine whereby dFdU competes with gemcitabine for transport via hENT1 and hENT2. The presence of dFdU appears to enhance the retention of gemcitabine intracellularly leading to an increase in the amount of active gemcitabine triphosphate. As more gemcitabine is phosphorylated in the presence of dFdU, a "metabolic sink" is created, further increasing gemcitabine uptake into the cell via hENT1 and hENT2. These data suggest that both transport and intracellular metabolism are equally important components of gemcitabine disposition and cytotoxic potential, and that the presence of dFdU increases intracellular exposure to this nucleoside analog.

We performed pharmacometric modeling of irinotecan, PQIP and gemcitabine. Irinotecan is a DNA topoisomerase-I inhibitor used in colorectal and brain tumors. One of our aims was to show that that cranial radiation, delivered during irinotecan administration, can enhance uptake in to cerebral-spinal fluid (CSF), and also to characterize the pharmacokinetics of irinotecan and its metabolites SN38 and APC in plasma and CSF in rats. The concentration-time data in plasma and CSF were analyzed by nonlinear mixed-effects modeling using FOCE-I method in NONMEM. The analysis showed that cranial radiation delivered during irinotecan administration enhances uptake in to CSF. The second aim of dissertation was to quantitatively evaluate the drug administration sequence effects of insulin-like growth factor type-1 receptor inhibitors (IGF-1R) with gemcitabine in vitro . Gemcitabine is a nucleoside analogue approved for a variety of solid tumors, while PQIP is an investigational IGF-1R inhibitor. In vitro experiments were conducted in 24-well tissue culture plates using MCF-7, MDA-MB-231 and Hs-578T human breast cancer cell lines. Viable cells were counted every day following the day of drug exposure. The effect of sequence on cell-kill was analyzed by bayesian techniques using WinBUGS. We found that exposure with gemcitabine first, followed by PQIP (GP sequence) was either superior or equivalent to the reverse sequence (PG sequence) with these drugs. The third aim of dissertation was to perform a population pharmacokinetic analysis of gemcitabine and its metabolites dFdU and dFdCTP in patients with solid tumors and to determine patient specific covariates associated with their disposition. Concentration data were obtained in 40 patients with solid tumors. The concentration-time data were analyzed by nonlinear mixed-effects modeling using first-order conditional estimation method with interaction (FOCE-I) in NONMEM. We found that Body surface area (BSA) and age were significant covariates for the disposition of gemcitabine, while creatinine clearance (CL CR ) and BSA were found to be significant covariates for the disposition of dFdU. We also found that patients who received gemcitabine at a rate less that 25 mg/m 2 /min had higher formation of dFdCTP than those who received gemcitabine at a rate greater than 25 mg/m 2 /min.

Gemcitabine (GEM) is a nucleoside analogue, which is commonly used to treat solid tumours. The activity of GEM is correlated with intracellular accumulation of its active metabolite, gemcitabine-triphosphate (GEM-TP). This thesis aims to explore the effect on GEM accumulation of: the genetics of the tumour and the host; the rate of GEM administration; and the interactions between them. The relationship of single nucleotide polymorphisms (SNPs) in relevant genes to GEM- pharmacokinetics were examined in a group of patients receiving 30 min (standard) and Fixed Dose Rate (FDR) infusion of GEM. Multivariate analysis showed an interaction between CDA c.79A>C SNP, dose rate and week of GEM administration. Furthermore, the CDA c.79A>C SNP was also associated with increased toxicity and dose delay. This data suggests that the benefit of FDR infusion is restricted to the variant genotype of CDA c.79. The increase in accumulation of GEM-TP between the first and second week of GEM administration has been termed (auto-induction of GEM accumulation). A cell line model was established in which the level of GEM-TP measured by HPLC after the second round of GEM exposure was 10-times higher than after the first. This increase was not associated with a significant increase in dCK gene expression, as measured by quantitative-realtime PCR. Induction of GEM-TP accumulation was not selective to GEM but seen with both cytarabine and cisplatin. This suggests that the mechanism is either due to post translational modification of DCK or alteration of GEM transport as a non-specific response to inhibition of DNA synthesis. The effect of DNA mismatch repair on antiproliferative activity of GEM and platinum drugs was demonstrated using paired cell lines deficient (HCT116) and proficient (HCT116+Ch3) in mismatch repair and by comparison of the effect of cisplatin with oxaliplatin. There was no evidence to support an effect of DNA mismatch repair on synergy/antagonism between GEM and cisplatin. This work provides a model for the optimising the pharmacology of GEM through a comprehensive approach considering: pharmacogenetic variation in both the tumour and host; and by understanding the mechanisms of regulation of GEM accumulation.

BACKGROUND: To determine the dose-limiting toxicity (DLT), maximum tolerated dose, recommended dose (RD) and preliminary evidence of activity of escalating doses of irinotecan (CPT-11) fixed-dose-rate infusional gemcitabine (FDR-GMB) and bevacizumab in pretreated metastatic colorectal cancer (mCRC) patients. Pharmacogenomic analysis was performed to investigate the association between VEGF single-nucleotide polymorphisms and clinical outcome. PATIENTS AND METHODS: A total of 89 mCRC patients were recruited in a two-step study design; 28 were included in the dose-finding study and 59 in the pharmacogenomic analysis. The FDR-GMB of 1000 mg m⁻², bevacizumab 5 mg kg⁻¹ and CPT-11 doses ranging from 100 to 160 mg m⁻² were explored. The VEGF protein serum levels were quantified by EIA. Allelic discrimination was performed to genotype polymorphisms in the VEGF gene. RESULTS: CPT-11 RD was 150 mg m⁻². Diarrhoea and neutropenia were the DLT. After a median follow-up of 42 months, the median time to progression (TTP) and overall survival were 5.2 and 19.9 months, respectively. VEGF levels were significantly correlated with VEGF-2578AA and VEGF-460CC genotypes, and a trend was observed with VEGF+405GG genotype. The presence of any of these genotypes correlated with a longer median TTP (8.8 vs 4.5 months, P=0.04). CONCLUSION: The triplet combination tested in this study is effective and well tolerated. A possible predictive role for VEGF gene polymorphisms and baseline VEGF circulating levels is suggested.

Due to the inherent chemoresistance of some cancer types, for example renal cell carcinomas (RCC), treatment approaches using cytotoxic drugs ultimately fail. This necessitates the search for alternative therapy. Immunotherapy agents like Interferon and Interleukin-2 (IL-2) have activity in some patients but are not curative. Thus, strategies to improve immunotherapy (IT) are needed. One approach is to use chemotherapy (CTX) to enhance IT. This could come about by exploiting immuno-modulatory effects of specific cancer drugs. Alternatively, CTX could be used to “sensitize” tumor cells to lytic effects of immune cells. The current investigation tested the hypothesis that tumor cells exposed to CTX will exhibit increased sensitivity to lysis by lymphokine activated killer (LAK) cells and investigated possible candidate genes involved in the molecular mechanisms responsible for the observed effect.

Gemcitabine (dFdCyd) is a nucleoside analog that has demonstrated synergistic cell killing with ionizing radiation (termed radiosensitization) in a variety of solid tumor cells. The diphosphate derivative, dFdCDP, inhibits ribonucleotide reductase, which primarily produces a decrease in dATP levels in solid tumor cells, resulting in decreased DNA synthesis. The triphosphate derivative, dFdCTP, competes with dCTP for incorporation into DNA, which correlates with cytotoxicity, and inhibits DNA synthesis. Although the potent radiosensitizing ability of dFdCyd has been well documented, the mechanism has not been elucidated. This dissertation examines the roles of p53, mismatch repair (MMR), and mismatches in DNA on the mechanism of dFdCyd radiosensitization. Radiosensitization with dFdCyd has correlated with dATP depletion and S-phase accumulation, without apparent alterations in DNA double strand breaks or their repair. The role of p53 and whether its expression would prevent S-phase accumulation due to a G1 block was examined in matched MCF-7 breast cancer cells with either wild-type or mutant p53. Both cell lines exhibited depleted dATP pools, accumulated in S-phase prior to irradiation, and were radiosensitized. Results demonstrated that radiosensitization was not prevented by p53. To account for the strong correlation between dATP depletion and radiosensitization by dFdCyd, we hypothesized that DNA mismatches from the dATP pool imbalance lead to radiosensitization. By comparing cells that differed in their ability to repair DNA mismatches, we demonstrated that HCT116 MMR-deficient cells were better radiosensitized by dFdCyd than the corresponding HCT116 MMR-proficient cells. Using an episomal supF gene as a marker in both MMR-deficient and proficient cells, we determined that dFdCyd and hydroxyurea produced increased nucleotide changes only at radiosensitizing concentrations. We hypothesize that the dFdCyd-induced imbalance in dNTP pools leads to errors in DNA replication, which are normally corrected by MMR. The data suggest that when the MMR capacity is exceeded, the nucleotide misincorporations caused by depleted dATP pools are the lesions responsible for dFdCyd radiosensitization.

2',2'-Difluoro-2' -deoxycytidine (dFdCyd) is a nucleoside analog that has shown clinical activity against a variety of solid tumor types. There are two pathways through which dFdCyd can exert cytotoxicity: (1) the diphosphate derivative, dFdCDP, inhibits ribonucleotide reductase resulting primarily in the depletion of dATP in solid tumor cells; and (2) the triphosphate derivative, dFdCTP, can be incorporated into DNA and interfere with DNA replication. In addition to its inherent cytotoxicity, when combined with ionizing radiation, dFdCyd produces a synergistic effect in cell killing. This dissertation examines the roles of dATP depletion and cell cycle distribution in the mechanism of dFdCyd mediated radiosensitization. In order to study the mechanism of dFdCyd mediated radiosensitization, U251 human glioblastoma cells, which express a mutant p53 and were readily radiosensitized, were compared to D54 human glioblastoma cells, which express wild-type p53 and could not be radiosensitized. Previous studies correlated radiosensitization by dFdCyd with its ability to deplete dATP. The lack of radiosensitization of D54 cells was not due to a lack of dATP depletion. dATP depletion alone was not sufficient to induce radiosensitization; however, dATP depletion did parallel DNA synthesis inhibition. dATP depletion and the subsequent inhibition of DNA synthesis did not contribute significantly to cytotoxicity. Other studies have demonstrated that S-phase cells are more susceptible to radiosensitization by dFdCyd. Before examining the role of the cell cycle in radiosensitization by dFdCyd, an extensive review of dual parameter flow cytometry measuring BrdUrd incorporation and DNA content was performed. Both acid/heat and DNase denaturation of DNA were equally effective; however, indirect labeling of BrdUrd was more sensitive than direct labeling. Following dFdCyd treatment, U251 cells readily accumulated in S-phase, whereas D54 cells did not. However, treatment of D54 cells with hydroxyurea resulted in S-phase accumulation without subsequent radiosensitization. Removal of p53 controlled cell cycle checkpoints by the introduction of HPV-16 E6 into D54 cells promoted S-phase accumulation following dFdCyd treatment and increased the incidence radiosensitization. These studies suggest that dATP depletion and S-phase accumulation are not sufficient for, but may induce the critical DNA lesion of radiosensitization by dFdCyd.