Studies into the Production of Protoporphyrin IX from 5-Aminolaevulinic Acid and its Derivatives

Aminolaevulinic acid-photodynamic therapy (ALA-PDT) has become widely used for the treatment of a range of premalignant, malignant and nonmalignant conditions, particularly in dermatology. Exogenous administration ofALA induces accumulation of endogenous porphyrins, most notably protoporphyrin IX. These porphyrins can be photoactivated by a specific wavelength of light, which, in the presence of molecular oxygen, results in the generation of reactive oxygen species and thus localised cell death. I ,I 11 Despite promising results, ALA-PDT has proved to have a number of limitations, most of which appear to be related to the limited penetration of ALA through biological barriers. A potential solution to this problem was to increase the lipophilicity ofALA by esterifying its carboxylic acid group. It was hoped that the resulting ALA-esters would induce a more favourable porphyrin accumulation than their parent compound. This generally appears to be the case, and a number of ALA-esters are currently being used in the clinic. Since ALA and ALA-esters are precursors to photosensitive porphyrins, it is important to understand how they lead to the accumulation of porphyrin, and the chemical and biochemical properties of ALA and ALA-esters influencing such accumulation. This thesis provides an insight into this novel area of research. It is widely assumed that once inside cells ALA-esters are hydrolysed by nonspecific cellular esterases to ALA which can then be converted into PpIX, however, this has never been proven. It therefore remains possible that unhydrolysed ALA-esters may enter the haem biosynthetic pathway or perhaps influence PpIX accumulation in some other way. Since ALA-esters are analogues of ALA, they are most likely to exert an effect via ALA dehydratase (ALAD). Measurement of the activity of ALAD in the presence of l ALA-esters suggested that whilst ALA-esters are not inhibitors of, or substrates for, ALAD, they can be combined with ALA by ALAD in vitro to form mono-esterified-PBG. It is unlikely that subsequent esterified intermediates would be formed, thus esterases appear to be vital for ALA-ester-induced porphyrin accumulation. The potential formation of PBG-mono-ester in vivo and its subsequent effects on the haem biosynthetic pathway, however, require further investigation. Various chemical and biochemical properties ofALA and ALA-esters, including purity, stability, cellular ALA-ester hydrolysis, cellular uptake and subsequent intracellular porphyrin accumulation can influence the efficacy of ALA- and ALA-ester-PDT. These properties were investigated in vitro. Whilst it was confirmed that ALA and ALA-esters were relatively stable under the experimental conditions in which they were used throughout this thesis, it was highlighted that ALA can be unstable at physiological pHs and ALA-esters can be partially hydrolysed in a range of preparations. pH was identified as an important factor influencing both ALA degradation and ALA-ester-hydrolysis. Clinical and biological formulations should therefore be carefully considered when planning experimental and clinical procedures, and interpreting the resulting data. It was also suggested here that the majority of ALA-esters are hydrolysed at a similar rate, possibly indicating the action of nonspecific esterases, and that it is the uptake of ALA and ALA-esters, rather than the haem biosynthetic pathway, that is rate limiting with respect to ALA- and ALA-ester-induced porphyrin accumulation. The majority of ALA-ester-PDT research to date has focussed on their use in dermatology, however, it is also important to investigate whether ALA-esters can improve the efficacy of other ALA-based applications. The use of ALA-esters in photodynamic antimicrobial chemotherapy (PACT) was therefore examined. Both m-ALA and h-ALA were less effective than ALA against the gram negative bacterium E. coli. This appeared to be due to reduced porphyrin accumulation which was probably a result of reduced uptake and/or hydrolysis. Thus, ALA-esters are unlikely to be useful for the photoinactivation of gram negative bacteria. /7 / /1 1!!iIiIII( IIIIiI! IIII The furthemlent of PDT and its related applications such as PACT not only relies on the development of novel photosensitisers and research into new conditions for which they may be useful, but also on the detailed investigation of existing protocols. The data presented in this thesis highlight the importance of studying the mechanisms of ALAand ALA-ester-induced porphyrin formation, and the chemical and biochemical properties of ALA and ALA-esters in order to optimise ALA- and ALA-ester-based applications.EThOS - Electronic Theses Online ServiceGBUnited Kingdo