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Title: Effects of the counter-ion on the photodynamic potential of a novel tetrapentyl-phenothiazinium salt
Author: Woods, Robert Matthew
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2008
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Abstract:
Photodynamic therapy (PDT) is a promising alternative to conventional therapies for the treatment of tumours and other non-malignant lesions. This technique combines a photosensitive drug (photosensitiser) with light and oxygen to elicit a cytotoxic effect via the generation of reactive oxygen species. The endeavour to develop novel photosensitisers, with improved characteristics over currently available photosensitisers, led to development of the phenothiazinium salts. The most widely known phenothiazinium salt methylene blue is known to possess intrinsic photodynamic efficacy. However, methylene blue also el icits a number of undesirable characteristics such as inducing DNA strand breaks and a lack of significant in vivo or clinical efficacy. Analogues of methylene blue have been developed in an attempt to develop a better photosensitiser with an enhanced efficacy whilst decreasing any undesirable properties. 3,7-bis( dipentylamino)phenothiazin-5-ium (tetrapentyl-phenothiazinium saIt, PPA90S) is a methylene blue analogue where the methyl groups have been substituted for pentyl chains. This new analogue shows potential for development in POT. PPA90S exhibits a net positive charge and exists as a salt with an anionic counter-ion. The influence of this anionic counter-ion on the behaviour of the PPA90S was investigated in this study. Five different PPA90S salts were investigated, two with the inorganic counter-ions iodide and bromide and three organic counter-ions, namely glycolate, gluconate and lactate. It was shown that the different counter-ions were unable to significantly influence many physiochemica l properties, with the exception of the aqueous solubility of the molecule. The PPA905 salts with organic counter-ions were significantly more water soluble with PPA905 lactate showing the greatest water solubility. Some marginal variation was observed in the toxicity of each PPA905 salt to RIF-l cell s, however all of the PPA90S salts were significantly more phototoxic (approximately 100 fold) than methylene blue with LD50 values below 1 μM. Importantly, each PPA90S salt was significantly less cytotoxic in the absence of light, highlighting their potential as photosensitisers. This high degree of phototoxicity was not accompanied with DNA damage such as that seen with methylene blue, reducing any potential genotoxicity. Considering the in vitro data PPA905 lactate was selected as the most suitable PPA90S salt for further development both in vitro and in vivo. Analysis of PPA905 lactate in vitro highlighted discrepancies between different cytotoxicity assays, but PPA90S lactate was shown to be highly phototoxic in all systems used. Furthermore, the high phototoxicity of PPA90S lactate was shown in a variety of cell lines including multi-drug resistant A2780-adr cell. Comparison of the phototoxicity to the wild-type A2780 cells confirmed that PPA905 lactate was not a substrate for the broad spectrum efflux protein P-glycoprotein. Studies investigating the uptake of PPA90S lactate into RIF-1 cells suggested that intracellular uptake proceeds by simple diffusion and endocytosis. It was clear that PPA905 lactate could also exhibit an inhibition of cell prolife ration in the absence of light. PPA905 lactate showed excellent metabolic stability in vitro, however some inhibition of the cytochrome P450 isoforms CYPIA2 and CYP2C19 was observed. Evaluation of PPA905 lactate efficacy in vivo showed that short drug-light intervals (1-6 hours) gave the greatest anti-tumour effect. Under these experimental conditions the maximum tumour necrosis achieved was 85% from a single PPA905 lactate and light dose. Some normal t issue damage was observed in organs located in close proximity to the tumour, however no skin photosensitisation was shown. The pharmacokinetics of PPA905 lactate following an intravenous dose suggested a rapid distribution into many t issues. Highly vascularised tissues or those high in reticulo-endothelial components showed the greatest accumulation, higher than that shown in tumour t issue. In summary, PPA905 lactate showed great potential as a photosensitiser, being non-toxic to mice at the doses used and no anti-tumour effects were observed in the absence of light. The optimisation of treatment protocols are now required for the progressive development of PPA90S lactate.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.599559  DOI: Not available
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