Onderzoek van het mechanisme van de toename van de schade door ALA-PTD met behulp van een langdurig licht fractioneringsschema
01 / 2002 - 01 / 2006
Website Nederlandse Kankerbestrijding
INTRODUCTION: Photodynamic therapy (PDT) using protoporphyrin IX (PpIX), endogenously generated by 5-aminolevulinic acid (ALA), has attracted widespread attention. To date it has primarily been used for the treatment of non-melanoma skin lesions, such as actinic keratoses (AK) and basal cell carcinoma (BCC). It has recently received approval in the United States for the treatment of AK of the face or scalp. PDT following systemic administration of ALA is also under investigation for treatment of dysplasia in Barrett's oesophagus. Topical ALA-PDT is only effective for superficial lesions, and often requires multiple treatments. A number of methods have been investigated to increase the effectiveness of therapy, but have achieved limited success. Our extensive clinical and pre-clinical investigations, performed within our group, in a previous project funded by the Dutch Cancer Society have shown that subtly changing how the illumination is performed can critically affect the efficacy of ALA-PDT. An attempt to combine multiple treatments into a single treatment day has revealed an unexpected success. If the illumination is split into 2 equal fractions separated by a dark interval of > 1 h, the damage after PDT is significantly greater than if the same total fluence is delivered in a single fraction. The increase in effectiveness is even greater when the first fraction is decreased (i.e. 5 J cm-2 + 95 J cm-2 is much more effective than 50 J cm-2 + 50 J cm-2). This increase in efficacy seems to hold for every model system and every clinical condition we have investigated. Photodynamic efficacy has been shown to be dependent on a wide range of interrelated treatment parameters, such as the concentration of sensitiser, the fluence delivered, the local fluence rate and concentration of oxygen. In addition to this, in a two-fold illumination scheme, the early response to the first illumination is expected to play a critical role in enhancing the efficacy of the subsequent illuminations. At present, these phenomena occurring in the first few hours after ALA PDT are largely unknown. The overall purpose of the present project is to further develop clinical application of the long-term fractionation scheme. For an optimum exploitation of the advantages of long-term fractionation we must explore the mechanism behind the increase in effectiveness of ALA-PDT. Clarifying the mechanism will show the direction in which further improvements may be sought. Specifically, we will investigate the response of the treated volume to ALA-PDT and test various mechanistic hypotheses in different animal models. However, since many of the effects that we expect to encounter are largely determined by the host we will at every stage cross-validate our results using data from clinical ALA-PDT. Therefore there will be a significant clinical component to this project in which patients will be treated using a long-term fractionation scheme and from which biopsies will be taken. This does not include a large clinical trial, but a number of small clinical protocols focussed on specific phenomena. PLAN OF INVESTIGATION: We intend to determine the time course of localisation of PpIX fluorescence, infiltrating cells, and apoptotic cells in biopsies acquired during ALA-PDT of basal cell carcinoma. These data will be compared to the response of tissues to ALA-PDT in (a) tumours in the rat skin fold observation chamber and (b) tumours transplanted on the rat thigh after systemic ALA administration, and (c) UVB induced skin tumours in the hairless mouse after topical ALA application. In model (a) we will determine if cells and tissues that accumulate PpIX after ALA administration re-synthesise PpIX after illumination. Fluorescence images will be acquired during a complete two-fold illumination for a range of first fraction fluences. We will investigate the differences between the first and second illumination by monitoring changes in blood flow and PpIX photobleaching during illumination for a range of first and second fraction fluences. In (b) we will investigate the role of neutrophils in ALA-PDT; the time course of their tumour localisation and circulating concentration will be determined from frozen sections and blood samples acquired at various time points after illumination. The influence of neutrophils on the effectiveness of long-term light fractionation will be determined by modulating the number of circulating neutrophils in the dark interval between the two fractions of a two-fold illumination scheme. In (b and c) we will investigate the presence of apoptotic cells after illumination with various single and two-fold illumination schemes in order to determine if the mode of cell death is different following different illumination schemes. POSSIBEL RESULTS / RELEVANCE FOR CANCER RESEARCH: The research proposed will contribute to the understanding of the mechanisms involved in ALA-PDT. This may result in a further increase in the effectiveness of clinical ALA-PDT for superficial skin cancer, actinic keratosis and Barrett's oesophagus through the design of optimal illumination schemes using long-term light fractionation schemes. Development of a treatment scheme that enables effective treatment of superficial tumours is essential for wider clinical acceptance of ALA-PDT.