No description available.

No description available.

Pharmaceutically useful natural products such as the antibiotic erythromycin are often glycosylated. In many cases the glycosylation of natural products are often critical to their bioactivity. Therefore, advances in our understanding of the mechanism of glycosylation by glycosyltransferases are important. These glycosyltransferases could potentially be harnessed to allow rapid access to series of novel unnatural products of medical significance. The work described in this thesis focuses on the bi- and tri- functional glycosyltransferase; gra-14, involved in the formation of the two C-glycoside links in the granaticins and also of the O-glycoside link found in granaticin B and dihydrogranaticin B. A single C-glycoside link between a sugar moiety and its aglycone core is a common feature, in natural products isolated to date only the granaticin's exhibit two C-glycosidic bonds to a single sugar moiety. The glycosyltransferase was cloned from S. violaceoruber Tii22 and a revision of the sequence discovered which was supported by the recently deposited granaticin biosynthetic gene sequence from S. vietnamensis GIMV4.0001, after which overexpression and purification of the protein enabled the production of pure and homogenous recombinant Gra-14. The corresponding protein product was not capable of generating glycosylated products in vitro with variant substrates. The putative natural sugar donor TDP-4-keto-2,6-dideoxy-D-glucose was then reconstituted in both E. coli and S. lividans TK24, unfortunately it did not result in glycosylated derivates of the aglycones. These studies showed that the glycosyltransferase has stringent substrate specificity. Attempts at crystallisation were unsuccessful as the crystals of the glycosyltransferase obtained were not diffraction quality and hampered any attempts for structural and mechanistic studies.

No description available.

Phage display, a powerful polypeptide display technology, affords the rapid identification of peptides and proteins that interact with a target of interest The aims of the project were the phage display identification of peptides that interact with a druggable target in a brain disorder (glioblastoma multiforme) and the identification of peptides that serve as targeting vectors for brain delivery. Validation studies were undertaken to qualify the use of a cyclic 7-mer peptide phage library against targets including streptavidin and paracetamol chosen as examples of a large complex and small simple molecule, respectively. With the aim of identifying peptide phages that bind to the luminal surface of brain micro vasculature, a primary in-vitro porcine model of the blood-brain barrier (BBB) comprising primary brain capillary endothelial cells was established and characterised. An in-vivo phage display was undertaken in the rat with the aim of identifying peptide sequences that mediated translocation across the BBB into brain grey matter. A 7-mer cyclic peptide was identified with sequence AC-SYTSSTM-CGGGS that enhanced the uptake of phages into brain grey matter by 4-fold compared to control wild-type phages. This peptide may serve as a novel targeting vector for the delivery of a therapeutic cargo to the brain. Caveolin-1 was identified as a potential new therapeutic target in in-vitro models of grade IV astrocytomas (glioblastoma multiforme), with siRNA knockdown of caveolin-1 associated with reduced glioma cell proliferation and invasiveness. With the caveolin-1 scaffolding domain (aa 81-101 in the caveolin-1 protein) as a target, an in-vitro peptide phage selection was undertaken and identified a series of peptides that bind the scaffolding domain with high affinity. These peptides will serve as a template for the development of low molecular weight peptidomimetics that inhibit caveolin-1 function. In conclusion, the studies in this thesis have demonstrated the utility of phage display in experimental therapeutics of brain disorders.

This thesis investigates the potential of some commonly used pharmaceutical excipients to alter drug pharmacokinetics. In breath test studies, 3.2ml/kg DMSO prolonged the half-life of 14C-aminopyrine, -erythromycin and -NDMA, indicating in vivo inhibition of metabolism by CYP3A (erythromycin), CYP2E1 (NDMA) and the variety of enzymes that metabolise aminopyrine (CYP 2C11, 2C12, 2B1 and 2B2). However, no effects were apparent at doses typically used in pre-clinical formulations. Aminopyrine and erythromycin breath tests were not affected by propylene glycol (PG) or Solutol HS15, however PG did significantly increase 14C02 exhalation half- life in the NDMA breath test, suggesting a specific effect on metabolism by CYP2E1. While two 2ml/kg doses of DMSO increased plasma ai-AGP levels, none of the excipients tested consistently affected plasma ai-AGP at doses commonly used in pre-clinical formulations, suggesting that they are unlikely to increase protein binding by this mechanism. Transport studies in MDCK-MDR1 cells demonstrated an inhibitory effect of 0.1% Tween 80 and Solutol HS15 on P-Gp. In vivo, Tween inhibited the biliary elimination of 99mTc-MIBI but not 99mTc-HIDA, indicating inhibition of P-Gp, while Solutol inhibited the biliary elimination of both radiopharmaceuticals, suggesting inhibition of MRP and possibly P-Gp. Pre-treatment with 4ml/kg DMSO substantially impaired the renal elimination of 99mTc-DTPA. In contrast, 20% 1.8ml/kg DMSO significantly increased 99mTc-DTPA uptake into the kidneys, suggesting an increase in GFR. Both Tween and Solutol delayed 99mTc-DTPA elimination from the kidneys in some rats, without affecting GFR. However, Solutol did not significantly affect the pharmacokinetics of OAT or OCT substrates, suggesting it did not affect active renal secretion by these transporters. These results demonstrate that excipients can influence drug pharmacokinetics in vivo, after a single acute dose at levels commonly used in pre clinical studies.

In conclusion, the data from the present study showed the value of providing a clinical pharmacy service in reducing the number of drug-induced ADRs. This led to a significant improvement in patient care and cost savings to the health service.

<italic>Visceral leishmaniasis</italic> is the second largest parasitic killer in the world (after malaria) with 59,000 deaths annually. The parasite resides in macrophages, within a compartment called the parasitophorous vacuole, but many anti-leishmanial drugs are toxic and poorly effective due to the low concentrations attained in this compartment. Therefore, the aim of this study was to establish the basis for design of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates that would target the parasitophorous vacuole, with potential to deliver improved treatments for leishmaniasis. Mannose was selected as a ligand for macrophage targeting and amphotericin B as a model drug. First, in vitro macrophage models (RAW 264.7 and THP-1 cells) were established that displayed mannose receptor expression and uptake of mannosylated macromolecular ligands. A library of HPMA copolymer-mannose conjugates containing Oregon Green (OG) were then synthesised to define the optimal mannose loading needed for targeting. To define the fate of internalised conjugates a density gradient subcellular fractionation method was developed for THP-1 cells. Finally HPMA copolymer-amphotericin B mannose conjugates were synthesised and their cell uptake, intracellular fate and preliminary haemolytic and cytoxicity profiles established. HPMA copolymer-OG-Man conjugates with a mannose loading 4 mol % showed significantly higher uptake by THP-1 cells. The subcellular fractionation and confocal fluorescence microscopy confirmed time-dependent trafficking of such conjugates to late endosomes/lysosomes. Release of free OG (as a drug model) from the biodegradable polymer-OG linker was seen. HPMA copolymer-amphotericin B-mannose conjugates were taken up 5-fold faster than the control and accumulated in the late endosome/lysosomal compartment. HPMA copolymer-amphotericin B-mannose conjugates displayed reduced haemolysis and cytotoxicity against the THP-1 cells. This study has established methods to investigate the intracellular trafficking of polymer-drug conjugates, and has demonstrated the potential of mannose-targeted HPMA copolymer conjugates for effective targeting of anti-leishmanial drugs to macrophages.

Despite the ability to modulate SHH signalling, EGF pathway inhibitors targeting pAKT and pERK may not be useful for treating basal cell carcinoma. Alternative SHH signalling inhibitors such as those that target the formation of the primary cilium may be highly effective.

The development of phosphoramidates as a new pronucleotide approach has shown to lead to a significant boost in activity for a series of antiviral nucleoside analogues. Given the importance of nucleoside analogues in anticancer therapy and the need of more potent drugs against what is considered the major cause of death in the developed world, the aim of the present thesis was to investigate phosphoramidates as new potential anticancer prodrugs. A consistent part of the work presented is related to the further SAR investigation of Thymectacin, a BVdU phosphoramidate prodrug that has recently entered clinical trials against colon cancer. Herein, the use of naphthyl as a new phosphate masking group is reported for the first time. The antileukaemic drug Cladribine was a new target for the application of our approach and two series of related phosphoramidates represent the second major contribution to this thesis. Synthesis and cytostatic evaluation were also carried out for novel phosphoramidate derivatives of a different anticancer agent Zebularine, a natural nucleoside Adenosine and an inactive nucleoside analogue IsoCladribine. Through the combination of computational and NMR studies, the absolute stereochemistry of the phosphorus center of separated phosphoramidate diastereoisomers was suggested for the first time. Eventually, a preliminary molecular modelling study was performed on the human Hintl enzyme to investigate its possible role in the activation of phosphoramidates.