The topic addressed by the 2011 WHO‘s World Health Day was antibacterial resistance. This action served to highlight the serious problem bacterial resistance now presents to healthcare professionals globally. This investigation focussed on the action of four cationic antimicrobial agents against E. coli, S. aureus and P. aeruginosa. Triclosan is widely used clinically and as an ingredient of personal care products that has come under renewed scrutiny by the FDA last year. Colistin is used clinically as salvage therapy against multi-drug resistant bacteria, particularly P. aeruginosa and A. baumanii. NP101 and NP108 are novel antimicrobial peptides, considered as a class with huge future potential in the treatment of not only bacterial but also viral and fungal infections in humans. The action of triclosan on all three test bacteria, including P. aeruginosa, historically considered resistant to triclosan, revealed concentration dependent bacteriostatic/bactericidal effects; reduction in bacterial growth; inhibition of second-phase logarithmic growth in S. aureus; induction of minimal K+ loss in all bacterial species; non-septation and aggregation in all test species. Colistin use was abandoned clinically between 1970-2000 and is not used clinically against Gram-positive species. Colistin exposure induced a common response from test bacteria including the Gram-positive S. aureus; concentration dependent retardation in onset of bacterial growth, without reduction in final bacterial density; significant K+ loss from S. aureus and E. coli but not P. aeruginosa; the development of resistance to the inhibitory colistin concentration by P. aeruginosa and S. aureus but not E. coli; production of small colony variants by P. aeruginosa; formation of spherical aggregates; membrane blebbing and inhibition of normal bacterial septation. Similar responses to NP101 and NP108 were observed to the test bacteria; an all or nothing effect on bacterial growth; prevention of second phase logarithmic growth in S. aureus; induction of significant K+ loss in all bacteria; size and shape alterations; extrusion of intracellular material, membrane blebbing; inhibition of bacterial septation and interruption of binary fission. Each of these peptides was incorporated into lyophilised wafers and tested for in vitro topical antibacterial efficacy and shown to have antibacterial activity against all test species.

Flavonoids have shown much promise for the chemoprevention of PCa but their poor bioavailability is thought to hinder their chemopreventive efficacy in vivo. However, methoxylation of the flavonoid scaffold could improve bioavailability and efficacy. 3’,4’,5’-trimethoxyflavonol (TMFol) was identified as the most potent growth-inhibitory agent against the PCa cell lines tested. TMFol was 5-15 times more growth inhibitory than fisetin and quercetin, two widely studied flavonols. TMFol caused a G2/M arrest in androgen-dependent cells (LNCaP and TRAMP C2) whereas S phase arrest in the androgen-independent cells (PC-3). TMFol induced more apoptosis in the androgen-dependent cells than the androgen-independent one TMFol inhibited the expression and activity of the AR and also repressed the mRNA levels of both AR and PSA. TMFol also modulated a number of key apoptotic proteins. In vitro data suggests that TMFol may modulate similar proteins to that of quercetin and fisetin, however, exerting its activity at much lower concentrations. Pharmacokinetic data revealed that TMFol levels achievable in the prostate tissue were higher than the concentrations required in vitro to exert pharmacological activity. Nude mice were administered either control diet or diet supplemented with 0.2% w/w TMFol one week prior to cell inoculation. TRAMP C2 cells were implanted into the right flanks of the mice and tumours, once established, were measured twice a week. TMFol significantly reduced the size and weight of the tumour versus control (p<0.05). Equimolar concentrations of quercetin and fisetin in the same model failed to exert efficacy. The expression of p27, bax and survivin which were significantly altered in vitro were also significantly changed following TMFol intervention. These results further our understanding of the in vitro and in vivo pharmacology and cancer preventative activity of TMFol and provide evidence that TMFol may be investigated in preference to quercetin or fisetin for the management of PCa.

Resveratrol (trans-3,5,4′-trihydroxystilbene), a naturally occurring polyphenol present in grapes and red wine has undergone investigation as a potential cancer chemopreventive agent. Resveratrol is rapidly and extensively metabolised to its major phase II metabolites, including resveratrol sulfates and glucuronides. It is not yet known whether resveratrol metabolites contribute to the beneficial effects attributed to resveratrol, or whether resveratrol regeneration can occur from the metabolites. Resveratrol and metabolite pharmacokinetics were investigated in the plasma of healthy human volunteers receiving 0.5, 1.0, 2.5 and 5.0 g resveratrol daily. Concentrations were also quantified in malignant and non-malignant colon tissue removed from colorectal cancer patients, who received 0.5 or 1.0 g resveratrol daily. A mixture of resveratrol monosulfates and individual monoglucuronide isomers were synthesised. Resveratrol monosulfates were administered to mice to determine their pharmacokinetics. The effects of the metabolites on proliferation in HCA-7, HT-29 and HCEC colon cell lines were assessed. In healthy volunteers, resveratrol metabolites were shown to be the major species recovered from plasma. Average volunteer plasma AUClast for resveratrol-4′-O-glucuronide and resveratrol-3-O-sulfate were approximately 36- and 81-fold greater respectively than for resveratrol, following 0.5 g resveratrol dosing. In colon tissues from cancer patients resveratrol generally predominated, with resveratrol sulfate glucuronide being the most prominent metabolite. In mice administered monosulfates, resveratrol formation was found to occur, with measurable concentrations in plasma, mucosa, liver, lung and pancreas. The conversion of resveratrol sulfates to resveratrol was also observed in cells in vitro. Uptake of the metabolites and intracellular resveratrol correlated with effects on proliferation. Whilst resveratrol monoglucuronides had a limited inhibitory effect on cell proliferation, monosulfates caused a more pronounced reduction, with the greatest effect in HT-29 followed by HCA-7 cells, and little or no effect in HCEC cells. Further investigations will improve our understanding of the role of resveratrol metabolites in chemoprevention.

Silybinin and its crude form silymarin exhibit cancer chemopreventive efficacy in rodents including models of colorectal carcinogenesis. Silymarin is used clinically as a hepato-protectant against alcohol- and drug-related damage. Silybinin is a strong antioxidant and modulates the insulin-like growth factor (IGF) system in mice in vivo by increasing circulating levels of IGF binding protein-3 (IGFBP-3). In this thesis, the hypothesis that oral consumption of silybinin affords pharmacologically active levels in blood, liver and colorectum was tested. Twelve patients with colorectal carcinoma and twelve with colorectal liver metastases received silybinin phosphatidylcholine (silipide) at varying doses for 7 days. Blood, normal and malignant colorectal or liver tissue were obtained before and after silybinin ingestion. A HPLC-UV method was developed and validated prior to quantifying levels of silybinin in plasma and tissue samples. Plasma metabolites were identified by liquid chromatography-mass spectrometry. Levels of IGFBP-3 and IGF-1 in serum and of the oxidative DNA damage pyrimidopurinone adduct of deoxyguanosine (M1dG) in leucocytes were determined as potential pharmacodynamic markers of silybinin efficacy. Repeated administration of silipide was safe and well tolerated. Silybinin levels recovered from plasma between 1-4 h post final silipide dose were 0.3-4.0 μM. Silybinin monoglucuronide, silybinin di-glucuronide, silybinin mono-sulphate and silybinin glucuronide sulphate were identified as metabolites in the plasma. Silybinin concentrations in liver and colorectal tissues obtained by resection 3-6 h post last silipide dose were 0.3-2.5 and 20-141 nmolesg-1, respectively. Intervention with silipide did not affect blood levels of IGF-1, IGFBP-3 or M1dG. In conclusion, silipide ingestion at safe doses can achieve detectable levels of agent in plasma and liver, and silybinin concentrations reached in the colorectal tract are of the level which has been shown to elicit pharmacological effects in cells in vitro. The results support the further development of silybinin as a potential human colorectal cancer chemopreventive agent.

Resveratrol, found in grape skins and red wine, has potential chemopreventive activity in vitro at concentrations ≥5μM. This project explored the tolerability, pharmacokinetics and pharmacodynamics of resveratrol in two clinical phase 1 studies. In vivo data were supported by in vitro studies designed to mimic the daily dosing protocol, using concentrations observed clinically. Forty healthy volunteers received 29 oral daily doses of 0.5, 1.0, 2.5 and 5.0g resveratrol and 20 colorectal cancer patients 8 oral daily doses of 0.5 and 1.0g prior to surgical resection. The pharmacodynamics of resveratrol were assessed by measuring changes in plasma levels of proteins involved in the IGF system, oxidative DNA damage in whole blood and colorectal tissue (M1dG), effects on inflammatory pathways in plasma (PGE2) and colorectal tissue (COX-2), as well as colorectal tissue proliferation (Ki-67). No serious adverse events were reported. In volunteers, mean peak plasma levels of resveratrol across the dose groups ranged from 44.7-954ng/mL (0.20-4.20μM), and for the main metabolite, resveratrol-3-sulfate, were 4-13 fold-higher. Despite low systemic bioavailability, resveratrol concentrations associated with potential chemopreventive efficacy were observed in colorectal tumour tissue with a mean of 44.0nmol/g detected in patients receiving the 1.0g dose (range 0.30-195nmol/g). Post dosing, IGF-1 levels were reduced by 8% (P=0.03) in volunteers and by 33% (P<0.001) in colorectal cancer patients. In tissue, a reduction in cell proliferation of 5.5% (P=0.05) was observed, whilst there was an increase in COX-2 staining (P=0.004). Apart from the 2.5g dose in volunteers, where a significant increase was observed in blood M1dG (21.6%, P=0.02), resveratrol did not significantly affect plasma PGE2 or markers of DNA damage in either study. In cultured colon cancer cells, daily exposure to resveratrol was associated with increased antiproliferative activity compared to an equivalent single dose, supporting the indication that chronic administration may cause pharmacodynamic changes in humans. The work presented here suggests resveratrol has potential as a cancer chemopreventive agent and controlled clinical trials are now warranted.

Prostate cancer is an ideal candidate for chemoprevention. Tea drinking is a possible explanation for the rarity of prostate cancer among Chinese men. Tea (Camellia sinensis) contains flavonoid polyphenols called catechins, believed to be responsible for this anti-carcinogenesis. In black tea these catechins are oxidised into theaflavins. Catechins and theaflavins both inhibit human prostate cancer cell proliferation in vitro. Catechins inhibit prostate cancer in the TRAMP mouse animal model of the disease. To determine if tea polyphenols warrant investigation in large trials, evidence is required from animal models and biomarkers of cancer prevention identified in small human studies. In the work presented here, prostate carcinogenesis was inhibited by orally administered tea polyphenols in the TRAMP mouse. Following 26 weeks of polyphenol administration median prostate masses were 0.54g, 0.28g and 1.01g for the theaflavin, catechin and control group respectively. This is the first in vivo evidence of prostate cancer chemoprevention by black tea theaflavins and adds to the previously published evidence for the same effect by green tea catechins. In the catechin group, this chemoprevention was associated with a significant reduction in the concentration of oxidative DNA adduct malondialdehydedeoxyguanosine (M1G) in prostate tissue. M1G, a marker of oxidative DNA damage, was therefore proposed as a putative biomarker of prostate cancer chemoprevention. A human trial was then performed involving 18 men randomised to receive four weeks of catechins, theaflavins or no polyphenol prior to transurethral resection of prostate. A significant reduction in M1G was detected in the DNA from prostate tissue of men who had received catechins. Tea polyphenols and particularly catechins may therefore represent prostate cancer prevention agents suitable for study in a larger human intervention trial however, this finding should be first be tested in further better designed biomarker studies using this result to inform decisions on study population size.

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