Poly (butyl 2-cyanoacrylate) nanoparticles have been prepared with a range of particle sizes by varying the nature and concentration of stabiliser added to the polymerisation medium. Particle size analysis was performed by photon correlation spectroscopy. The range of diameters produced using dextran stabilisers was found to be approximately 100 to 800nm. This could be extended to 3ym using j3 -cyclodextrin and to 20nm using polysorbate 20. The results infer that the nanoparticles are sterically stabilised. The molecular weight of the cyanoacrylate polymer formed during nanoparticle production was found to be dependent on the type of stabiliser used together with the polymerisation pH and monomer concentration. The bulk of the polymer had a relatively low molecular weight (<2000) which indicates that nanoparticles are formed by an aggregative mechanism. Dextran was found to copolymerise with the monomer to give an interfacial layer of the polysaccharide attached by covalent linkages. By using dextrans bearing charged functional groups it was possible to alter the electrophoretic behaviour of the resulting nanoparticles. Partial oxidation of the surface dextran introduced aldehyde groups which were capable of covalently binding a simple amine, aniline, thereby enhancing the uptake and decreasing the release rate of this compound. This technique may be applicable to the covalent coupling of antibodies or cytotoxic agents to the nanoparticle surface. Nanoparticles were radiolabelled with a technetium-99m-dextran complex and the biodistribution of this colloid determined in rabbits by gamma scintigraphy following intravenous injection. Most of the nanoparticle suspension (approximately 50%) was cleared by the liver and spleen. Coating the nanoparticles with non-ionic surfactants (poloxamer 338 or Tetronic 908) failed to alter significantly this distribution pattern.

No description available.

Most of the biological effects of cannabis are due to the activation of specific cannabinoid receptors. To date, two such receptors have been discovered and are found predominantly in the central nervous system (the CB1 receptor) or the immune system (the CB2 receptor). Endogenous cannabinoid receptor ligands, the endocannabinoids, have also been isolated and the mechanisms of their synthesis and degradation postulated. By modulating the activation of cannabinoid receptors and endocannabinoid metabolism, synthetic cannabimimetic compounds have enormous therapeutic potential for the treatment of such diverse symptoms and diseases as pain, inflammation, cancer, hypertension, schizophrenia and multiple sclerosis. This thesis describes the design, synthesis and subsequent biological evaluation of three classes of novel, potentially cannabimimetic drugs, namely aryl ethanolamides, phenylphosphinic acids and alkylphosphinic acids. In order to assess cannabimimetic activity, the ability of these compounds to bind to the cannabinoid receptors and to inhibit endocannabinoid uptake and enzymatic hydrolysis was examined. Affinity for the CB1 receptor was assessed using radioligand binding assays in rat brain membranes. Although none of the compounds proved to be high-affinity CB1 receptor ligands, two aryl ethanolamide compounds exhibited some affinity for this receptor, suggesting that this general class of compound may have cannabimimetic potential. In order to ascertain whether the test compounds had affinity for the CB2 receptor, a radioligand binding assay was developed using porcine spleen membranes. To date, only the human, murine and rat CB2 receptors have been cloned and there has been no detailed examination of the cannabinoid binding profile of the porcine CB2 receptor. The Kd of the radiolabelled cannabinoid [3H]-CP-55,940 was determined in porcine spleen membranes and the Bmax subsequently calculated. The Ki values of a number of cannabinoid receptor ligands were then determined. These values were shown to be similar to the corresponding values obtained using cloned CB2 receptors. However, when the test compounds were assessed in this assay system, no affinity for the CB2 receptor was observed. To determine the effect, if any, of the test compounds on the endocannabinoid uptake system, accumulation of the radiolabelled endocannabinoid [3H]-anandamide into N18TG2 mouse neuroblastoma cells was examined. [3H]-Anandamide accumulation had previously been reported in this cell line but, until now, this mechanism had not been characterized. This accumulation was shown to be time-, temperature- and concentration-dependent and was inhibited by AM404 and bromocresol green, known inhibitors of the endocannabinoid carrier system. [3H]-Anandamide accumulation exhibited a Km value similar to those previously described for rat astrocytes and neurones and the time taken to achieve half maximal rate was shown to be considerably greater than in these rat cells. None of the test compounds significantly inhibited [3H]-anandamide uptake by N18TG2 cells although one phenylphosphinic acid compound, with structural similarities to AM404, appeared to be inhibitory at high concentrations. The final biological target examined was fatty acid amide hydrolase (FAAH), the enzyme that catalyses the hydrolysis of endocannabinoids. For FAAH studies, a novel, inexpensive and rapid spectrophotometric assay was developed as an alternative to the traditional radiochemical- and chromatography-based assays. Using this novel assay system, the Km and Vmax values of rat liver FAAH were determined and shown to be similar to those published in the literature. Known FAAH inhibitors were shown to inhibit FAAH in a concentration-dependent manner with IC50 values comparable to previously published data. In addition, this assay was used to demonstrate differences in FAAH activity between soluble and insoluble membrane preparations from rat liver and brain, possibly indicating the presence of, as yet, unknown FAAH enzymes. Attempts were also made to adapt this assay for use on a microtiter plate, where it was possible to detect FAAH inhibitors. Therefore, this spectrophotometric assay may prove to be of use in the high-throughput screening of chemical libraries for drugs that cause cannabimimetic effects via FAAH inhibition. None of the test compounds synthesized inhibited FAAH activity and this, combined with their lack of biological activity at the other targets tested, showed that they exerted no cannabimimetic effects.

No description available.

The DNA damaging activity of RSU 1069 and seven of its analogues (RSU 1131, RSU 1164, RSU 1150, RB 7040, RSU 1172, RSU 1137 and RSU 1170) plus misonidazole and CB 1954 were investigated using the SOS-Chromotest The SOS-Chromotest is a genotoxicity assay that monitors the induction of the SOS response, which is induced in response to DNA damage. The strains used were PQ37, which possesses a uvrA mutation and is deficient in UvrA excinuclease activity, and PQ35, which is uvr and UvrABC excinuclease conpetent. These strains were exposed to the compounds being investigated under both oxic and hypoxic conditions. The results showed that RSU 1069 and some of its analogues were more active than misonidazole under both oxic and hypoxic conditions. This increase was due to their aziridine side-chains. With the exception of RSU 1137 and RSU 1170 all of the compounds showed altered SOS induction activities between oxic and hypoxic conditions. This alteration was shown to correlate with increased reduction of their nitro-groups under hypoxia. There was a difference in the hypoxic activities of RSU 1069 and some of its analogues between the uvrA-strain and the uvr -strain. With the uvrA-strain RSU 1069 showed decrease activity under hypoxia compared to oxia, whereas, the converse applied with the uvr -strain. This was interpreted to mean that RSU 1069 caused some damage that required an active UvrABC excinuclease to produce an SOS response. It has been proposed that this damage takes the form of DNA crosslinks. RSU 1137 showed insignificant SOS induction and this was demonstrated to be due to its nitro-group not being reduced. It was suggested that the ring opened aziridine side chain of RSU 1137 in some way inhibited its bioreduction. The order of activity of the drugs for SOS induction activities did not correlate with that for their toxicities. This indicated that DNA lesions other than, or in addition to, those responsible for cytotoxicity induced the SOS response. The DNA damaging activity and mutagenicity of RSU 1069 was also investigated using Ml3 phage rfDNA. Radiation reduced RSU 1069 was shown to produce some relatively long lived product that was more active towards DNA than unreduced RSU 1069, as judged by phage survival. Unreduced RSU 1069 was shown to be non-mutagenic, producing mutation rates under 1.5 times background level. The effect of strict hypoxic conditions upon the SOS response was investigated using the SOS-Chromotest with the uvrA tester strain. The results showed that the SOS response was induced under strictly anaerobic conditions in E. coli but that the response was altered compared to that obtained aerobically. The nature of the alteration was not determined as six different compounds, with five different modes of action, were used as SOS-inducers and all showed different types of response under hypoxic conditions.

No description available.

These studies have characterized the peptide transport systems of Candida albicans, with a view to the rational design of peptide antifungal agents exploiting the 'smugglin' concept. In initial studies, a series of polyoxin complexes (peptide-nucleoside antibiotics) and individual components, were isolated from a batch of agricultural fungicide (Polyoxin Z). Isolated fractions were toxic to a particulate chitin synthetase preparation from Candida albicans. Different strains of Candida albicans exhibited varied sensitivities to a series of peptide analogues. From a sensitive strain, B2630, spontaneous mutants were selected for resistance to each analogue; certain mutants showed cross-resistance to other analogues and associated defects in peptide transport. A bacilysin-resistant mutant was cross-resistant to the other analogues and to m- fluorophenylalanylalanylalanine a but retained sensitivity to m- fluorophenylalanylalanylalanine. This mutant showed defective dipeptide transport but normal oligopeptide transport, and was unable to utilize Ala-Ala as a sole nitrogen source. Thus, Candida albicans has distinguishable mechanisms for dipeptide and oligopeptide transport which can be exploited for uptake of peptide-drug adducts. Peptide transport was shown to be stimulated by the presence of peptides (peptone) in the growth medium. On transferring cells from minimal to peptone medium, this stimulatory effect was shown to be rapid, independent of protein synthesis and to override ammonia regulation of peptide transport. The reduction of transport activity on transferring cells from peptone to minimal medium was also rapid. It was speculated that regulation of peptide transport is achieved by a rapid, reversible activation of preformed transport components, or a mechanism of exocytotic insertion and endocytic retrieval of preformed transporters. The effect of protein-modification reagents on transport activity was also examined. Dipeptide transport was specifically inhibited by N-ethyl-5-phenylisoxazolium-3'-sulphonate (Woodwards Reagent K), offerring potential for the specific labelling of the component(s) of this system. Peptide transport was shown not to be sensitive to osmotic shock though a series of uncharacterized polypeptides was released by the shock treatment.

The overall aim of the project was to investigate the role of the cell membrane as a barrier and/or target for drug action and relate this to the development of strategies for overcoming multiple drug resistance (MDR). The effects of doxorubicin on various bacterial strains expressing different levels of anionic phospholipid were compared. Giowth of wild-type Echerichia coli (E. coli) strain MRE600 was severely affected up to 9 hours following doxorubicin treatment (15uM), but resistance occurred after 9 hours. E. coli strain FIDL1 1 was resistant to doxorubicin (1 O0piM) over 9 hours, however, increasing the anionic lipid content showed little difference in sensitivity. The mouse mammary tumour cell line (EMT6-S) and MDR sub-line (EMT6-R) were characterised with regard to growth kinetics, susceptibility to doxorubicin and membrane lipid composition. The log phase doubling times (h) were found to be 21.8 (EMT6-S)and 25.0 (EMT6-R) and the IC 50 values for doxorubicin to be 2.2 x 10-8 M and 1.8 x 10-6 M for EMT6-S and EMT6-R cells, respectively. No difference was observed between the phospholipid profiles of the two cell lines and total fatty acid composition was similar, however, the level of linoleic acid appeared to be higher in the resistant cells. The photocytotoxicity of the cationic dyes methylene blue (MB), toluidine blue (TBO) and Victoria blue BO (VBBO) against the EMT6 cell lines was compared to the cyotoxic effect of doxorubicin and cis-platinurn. The cytotoxic effect of VBBO was enhanced 10-fold by illumination (7.2 J cm2) in both EMT6-S and EMT6-R cells. In order to overcome resistance, however, the EMT6-R cells required a 10-fold greater level of the dye than the parental cells to reach an IC50 value. By contrast, doxorubicin required almost a 100-fold increase in concentration to overcome this resistance. Pre-treatment of EMT6-S and EMT6-R cells with low concentrations of VBBO resulted in a 2-fold increase in doxorubicin toxicity in both cell lines. Pre-treatment with MB and TBO resulted in a 1.4-fold and 2-fold increase in doxorubicin toxicity, respectively, in the sensitive cells, increasing to 2-fold and 3-fold, respectively in the resistant cells. Glutathione (GSH) depletion of EMT6-S and EMT6-R cells did not enhance the photocytotoxicity of VBBO, suggesting that the primary site of action of VBBO is at an intracellular site not protected by GSH or that the mechanism of action is not via the in situ generation of singlet oxygen. Addition of the chemosensitizer, verapamil (7gM), increased the efficacy of doxorubicin by 2-fold in EMT6-S cells and by 18-fold in EMT6-R cells. By contrast, the presence of verapamil did not increase the cytotoxicity of YBBO in either cell line. A series of compounds, PVB, MVB and MOVB, based on the skeleton of VBBO was examined. VBBO was found to be the most effective photosensitizer. The rate of uptake for VBBO, MVB and PVB appeared to be very similar, whereas that of MOVB was slower. The uptake/dose trend was also similar four all four drugs tested and conelated to the levels of lipophilicity of the agents. Confocal microscopy studies showed all the photosensitizers to be distributed widely throughout the cytoplasm, with considerable accumulation of VBBO and PVB in the perinuclear region. Time course studies showed the intracellular distribution of VBBO in both cell lines to be similar, although uptake of the drug appeared slower in the resistant cell line. VBBO was clearly localised throughout the cytoplasm, in a punctate pattern, which may be consistent with the widespread distribution of mitochondria. No interaction with the plasma membrane was evident. By contrast, doxorubicin was found to localise mainly in the nucleus of the sensitive cell line, whereas no nuclear involvement was seen in the resistant cells. The drug was also effluxed more rapidly from EMT6-R cells than EMT6-S cells. Time course studies with EMT6-S cells showed that the drug clearly interacts with both the plasma membrane and the nucleus. These results indicate that the main modes of action for the two drugs differ markedly, suggesting interaction with both the membrane and the nucleus in the case of doxorubicin, but possibly mitochondrial involvement for VBBO.

No description available.

No description available.