Fluorescence distribution and photodynamic effect of ALA-induced PP IX in the DMH rat colonic tumour model

113

Summary An in vivo study of tissue distribution kinetics and photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA), chlorin e6 (Chl) and Photofrin (PII) was performed to evaluate the selectivity of porphyrin accumulation and tissue damage effects in a tumour model compared with normal tissue. C26 colon carcinoma of mice transplanted to the foot was used as a model for selectivity assessment. Fluorescence measurements of porphyrin accumulation in the foot bearing the tumour and in the normal foot were performed by the laser-induced fluorescence (LIF) system. A new high-intensity pulsed light delivery system (HIPLS) was used for simultaneous irradiation of both feet by light in the range of 600-800 nm, with light doses from 120 to 300 J cm-2 (0.6 J cm-2 per pulse, 1 Hz). Photoirradiation was carried out 1 h after injection of ALA, 3 h after injection of Chl and 24 h after injection of PII. A ratio of porphyrin accumulation in tumour vs normal tissue was used as an index of accumulation selectivity for each agent. PDT selectivity was determined from the regression analysis of normal and tumour tissue responses to PDT as a function of the applied light dose. A normal tissue damage index was defined at various values (50, 80 and 100%) of antitumour effect. The results of the LIF measurements revealed different patterns of fluorescence intensity in tumour and normal tissues for ALA-induced protoporphyrin IX (ALA-PpIX), Chl and PIT. The results of PDT demonstrated the differences in both anti-tumour efficiency and normal tissue damage for the agents used. The selectivity of porphyrin accumulation in the tumour at the time of photoirradiation, as obtained by the LIF measurements, was in the order ALA-PpIX > Chl > PII. PDT selectivity at an equal value of anti-tumour effect was in the order Chl > ALA-PpIX > Pll. Histological examination revealed certain differences in structural changes of normal skin after PDT with the agents tested. The results of PDT selectivity assessment with respect to differences in mechanisms of action for ALA, Chl and PII are discussed.

mentioning

confidence: 99%

A comparative study of tissue distribution and photodynamic therapy selectivity of chlorin e6, Photofrin II and ALA-induced protoporphyrin IX in a colon carcinoma model

Orenstein

Kostenich²,

Roitman³

et al. 1996

113

“…For example, various malignant tissues have an increased porphobilinogen deaminase activity, which converts ALA into porphobilinogen, and a decreased ferrochelatase activity, which converts PpIX into haem (van Hillegersberg et al, 1992). This alteration in enzymatic activities may cause an increased fluorescence in tumour tissue compared with normal tissue after ALA administration (van der Veen et al, 1994;Bedwell et al, 1992). Several normal tissues, especially those originating from ecto-and endoderm may also become photosensitised after exposure to ALA.…”

mentioning

confidence: 99%

Kinetics and localisation of PpIX fluorescence after topical and systemic ALA application, observed in skin and skin tumours of UVB-treated mice

Veen¹,

Bruijn²,

Berg³

et al. 1996

SummaryIn this study the kinetics and localisation of protoporphyrin IX (PpIX) fluorescence in skin and skin tumours were examined after topically (20% for 4 h) or systemically (200 mg kg-', i.p.) administered 5-aminolaevulinic acid (ALA). As a model we used hairless mice with skin lesions (actinic keratoses and squamous cell carcinoma), which were induced by daily UVB irradiation. The epidermis of the skin surrounding the tumours (T) was altered (AS); owing to the UVB irradiation, the epidermis was thicker and less elastic. Therefore, non-UVBirradiated mice were used to assess fluorescence of normal skin (NS). Light from a halogen lamp was used to excite at 500 + 20 nm and fluorescence was detected through a filter that passes light of 670 + 50 nm. Maximal fluorescence following i.p. ALA was observed 2 h post injection (p.i.) and was three times less than after topically applied ALA. Furthermore, after i.p. ALA a lower T selectively (T/NS) could be obtained than after topically applied ALA. Maximal fluorescence following topically applied ALA was achieved 6 h after the end of the 4 h application time. At that interval fluorescence of T was twice as high as directly after the application period. Furthermore, T selectivity (T/NS) after topical ALA at the interval of maximal fluorescence was higher than at the interval directly after application. With fluorescence cryomicroscopy localisation of fluorescence in the skin at the interval of maximal fluorescence was determined after both administration routes. For both cases fluorescence was mainly located in T, epidermis and hair follicles. Fluorescence in subcutis could only be observed at 2 h post i.p. ALA and at 6 h post topical ALA. No fluorescence could be observed in muscle. We conclude that, in this model and with these ALA doses, a higher fluorescence intensity and selectivity (T/NS) was achieved after topically applied ALA than after systemically administered ALA. These results make topically applied ALA more favourable for ALA-PDT of superficial skin tumours in this model. In general these results imply that by optimising the time after ALA application the efficacy and selectivity of topical ALA-PDT for skin tumours may be improved.

“…Several studies, both in vivo and in vitro, have reported encouraging results (Malik and Lugaci, 1987;Fukuda et al, 1989Fukuda et al, , 1992aDivaris et al, 1990;Bedwell et al, 1992;Fijan et al, 1995;Roberts and Cairnduff, 1995;Steinbach et al, 1995;Kriegmair et al, 1996). Selective endogenously generated photosensitization depends on differential cellular rates of synthesis and loss of the porphyrins.…”

mentioning

confidence: 99%

Stimulation of tetrapyrrole synthesis in mammalian epithelial cells in culture by exposure to aminolaevulinic acid

Washbrook

Fukuda²,

Battle³

et al. 1997

Summary Tetrapyrrole synthesis in CNCM-1221 cells exposed to 0.6 mm aminolaevulinic acid (ALA) was found to be approximately linear over a 6-h period of incubation. The rate was not significantly affected by cell density over a range of 0.015 to 0.15 x 106 cells cm-2 (final cell density). Tetrapyrrole synthesis was not affected by GABA or glutamic acid in concentrations up to 6 mm and 2.72 mm respectively, suggesting that these amino acids, which are similar in structure to ALA, do not competitively inhibit the ALA uptake pathway in these cells. Pre-exposure to haem arginate (up to 100 gM) was inhibitory, presumably by suppression (through the inhibition of ALA synthase) of an endogenous component of the response. The ALA-stimulated response was not modified by co-exposure to AIA (up to 100 mg ml-'). Despite significant reduction of protein synthesis, the porphyrinogenic response of cells exposed to ALA was unaffected by cycloheximide (10 gg ml-') or actinomycin D (10 gg ml-') even when cells were preincubated with these agents for 3 h before ALA exposure. Fetal bovine serum (10%) inhibited tetrapyrrole synthesis by 30% but increased the rate of porphyrin export by cells by a factor of 1.5. The uptake of [14C]ALA was shown to be strongly influenced by the density of the cultures. In dense cultures (final cell density of approximately 0.15 x 106 cells cm-2), the ALA uptake rate was less than 0.8 compared with a maximum rate of 4.2 fmol per cell h-' at a cell density of 0.02 x 106 cells cm-2. Since tetrapyrrole synthesis is less affected than ALA uptake by cell density, the resultant discrepancy in ALA incorporation occurrng in dense cultures implies that endogenous ALA synthesis is induced in these cells. ALA uptake was not affected by cycloheximide or actinomycin D in serum-free conditions. However, fetal bovine serum decreased external ALA uptake by about 50%. This effect was abrogated by preincubation with cycloheximide.