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TUMOUR MICROCIRCULATION – A TARGET FOR AMINOLAEVULINIC ACID BASED PHOTODYNAMIC THERAPY
 
F.R. Potter*, M.W.R. Reed & N.J. Brown
 
Microcirculation Research Unit
Section of Surgical & Anaesthetic Sciences
University of Sheffield
 
*ruth.potter@sheffield.ac.uk
 
 
 

INTRODUCTION

A well estabilished approach to photodynamic therapy (PDT) involves the administration of 5-aminolaevulinic acid (ALA) to induce an increased endogenous synthesis of protoporphyrin IX (PpIX), a potent photosensitiser, by exploiting the haem biosynthetic pathway (see Fig. 1).

 
 
Figure 1 ALA based PDT exploits the haem biosynthetic pathway

Studies have demonstrated a number of possible mechanisms of action of ALA based PDT (see Fig 2). There is evidence for direct cytotoxic effects within the tumour microenvironment, secondary to the production of intracellular reactive oxygen species (Dougherty et al., 1998) . It is suggested that the cellular membranes are an important target for damage , resulting in random necrosis (Schuitimaker et al., 1996). The production of ALA-induced PpIX within the mitochondria rapidly induces apoptosis both in vitro (Noodt et al., 1996., Kessel et al., 1998) and, in vivo (Webber et al., 1996). In addition to direct tumour cell killing, recent research by both our group and others, has focused on the microvasculature as a target for ALA based PDT (Boucher et al., 1994., Roberts et al., 1994., Leveckis et al., 1995., Dougherty et al., 1998). Convincing evidence suggests that damage to tumour and surrounding normal tissue microcirculation often plays a more critical role in tumour destruction than direct cytotoxicity.

 
Figure 2 Proposed mechanisms of ALA based PDT

Although ALA based PDT has proved to be clinically benefical for the treatment of certain cancers, including a variety of skin cancers (Kessel 1996., Dougherty et al., 1998), optimal tissue localisation remains a problem. Recent studies have begun to focus on the synthesis and use of ALA derivatives which are more lipophilic, to increase tissue levels of photosensitiser and thus treatment efficacy (Kloek et al., 1996., Gaullier et al., 1997., Kloek et al., 1998., Marti et al., 1999., Lange et al., 1999). We have begun to investigate the microcirculatory effects of ALA and ALA-hexyl ester based PDT, using in vivo microscopy (IVM).
 

MATERIALS & METHODS

Male Wistar rats (60-80g) were anaesthetised with hypnorm/diazepam (i.p). Tumour cells (Walker Rat Carcinoma – 2.5 x 105 cells) were implanted into the cremaster muscle at laporatomy.  The tumours allowed to grow for ~10-14 days.  ALA or ALA-hexyl ester (200mg/kg) were administered orally (for control animals this was replaced with 1ml of the drug vehicle PBS).  Approximately 4 hours later animals were anaesthetised, a tracheotomy performed, and a carotid cannula inserted for the administration of the fluorochrome FITC, and monitoring cardiovascular parameters.

An oesophageal thermistor probe was inserted and connected to a digital thermometer. The animal was then placed on a warming pad on a Perspex animal board in order to maintain body temperature (35-37 C) during the protocol (cremaster muscle temperature is also monitored).

The cremaster muscle was then exteriorised, and positioned on a glass microscope slide mounted on Perspex pegs attached to an animal board. The cremaster muscle preparation with intact neurovascular supply was moistened with physiological saline and covered with an impermeable membrane (Saran wrap) to prevent dehydration during the period of observation (Fig. 3).

The animal, warming pad and Perspx board were transferred to the stage of the microscope. Animals were allowed a stabilisation period of 30 minutes before baseline recording. Fig. 4 outlines the experiemntal protocol.

 

 Figure 3 Tumour cremaster preparation
 
Figure 4 Experimental protocol
 

The tumour vessels area to be treated was exposed to a green light source with a power density of 100mW/cm2 for ~10-12 mins, a total delivered energy of ~65J/cm2.

FITC-BSA is a fluorescent marker of plasma, and was administered intra-arterially (0.1ml/100g) 5 minutes before baseline recording. This allowed vessel integrity to be monitored via macromolecular leak. In a normal intact vessel this fluorescence is contained within the vessel, indicating vessel integrity.

The following experimental variables are studied:-

 
RESULTS

These preliminary studies demonstrate that ALA-PDT has little effect on tumour vessel diameter compared to control (no drug, no light).  ALA hexyl ester-PDT appears to reduce tumour vessel diameter (Fig.5).  There was also no effect on the normal vessel diameter observed with either ALA or ALA-ester PDT compared to control (Fig. 6).
 

Figure 5 Effect of ALA & ALA hexyl ester based PDT on tumour vessel diameter
 
 
 Figure 6 Effect of ALA & ALA hexyl ester based PDT on normal vessel diameter

ALA-PDT results in a small increase in the level of macromolecular leak observed compared with control.  In comparison ALA hexyl ester-PDT substantially increases the level of macromolecular leak compared to both control and ALA-PDT studies. (Fig. 7).  There is no effect seen on the level of macromolecular leak from the normal vessels with either ALA or ALA hexyl ester-PDT, compared to control.
 

Figure 7 Effect of ALA & ALA hexyl ester based PDT on tumour vessel macromolecular leak
 
Figure 8 Effect of ALA & ALA hexyl ester based PDT on normal vessel macromolecular leak
 
9(a)  Before PDT
 
9(a) After PDT
 

Figure 9 (a) & (b) Images to illustrate and demonstrate the effect of ALA hexyl ester based PDT tumour macrovasculature.  In particular, the increased level of macromolecular leak (a) showed by the fluorescent flare within the interstitium, and the formation of what appears to be a thrombus (b).
 

SUMMARY

Preliminary data show that both ALA and ALA-hexyl ester based PDT result in substantial tumour microvascular changes and damage in vivo.  This is demonstrated by a number of factors including;  an increase in macromolecular leak, suggested decreased vessel integrity and damage;  decreased blood flow, vascular stasis, and thrombus formation (see Fig. 9b), all of which appear to be more profound with the ALA-ester compared to ALA based PDT.  Decreased vessel diameter was also observed with ALA hexyl ester-PDT.

These tumour microvascular changes and damage may contribute to the mechanism of action and toxicity of both ALA and ALA hexyl ester based PDT.  It is hoped that further acute, and chronic studies will confirm and establish the importance, and significance of this preliminary data.
 

ACKNOWLEDGEMENTS

I would like to thank, the Special Trustees for the Former Sheffield United Hospital for funding these studies which form part of my PhD, and the Centre for Photobiology and Photodynamic Therapy, University of Leeds, for kindly providing the ALA hexyl ester.
 

REFERENCES
 
 
BOUCHER, N.R., BROWN, N.J. & REED, M.W.R. (1995). Microcirculatory effects of aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX)-mediated photodynamic therapy (PDT) in vivo. B J Cancer, 72, 23.

DOUGHERTY, T.J., GOMER, G.J., HENDERSON, B.W., JORI, G., KESSEL, D., KORBELIK, M., MOAN, J. & PENG, Q. (1998). Photodynamic therapy. J Natl Cancer Inst., 90, 889-905.

GAULLIER, J.M., BERG, K., PENG, Q., ANHOLT, H., SELBO, P.K., MA, L.W., MOAN, J. (1997). Use of 5-aminolevulinic acid esters to improve photodynamic therapy on cells in culture. Cancer Res. 57, 1481-1486.

KESSEL, D. (1996). Photodynamic therapy of neoplastic disease. Drugs of today, 32, 385-396.

KESSEL, D. & LUO, Y. (1998). Mitochondrial photodamage and PDT-induced apoptosis. J Photochem Photobiol B., 42, 89-95.

KLOEK, J., AKKERMANS, W., BEIJERSBERGEN VAN HENEEGOUWEN, G.M.J. (1998). Derivatives of 5-aminolaevulinic acid esters for photodynamic therapy: enzymatic conversion into protoporphyrin. Photochem. Photobiol. 67, 150-154.

KLOEK, J., BEIJERSBERGEN VAN HENEEGOUWEN, G.M.J. (1996). Prodrugs of 5-aminolevulinic acid for photodynamic therapy. Photochem. Photobiol. 64, 994-1000.

LANGE, N., JICHLINSKI, P., ZELLWEGER, M., FORRER, M., MARTI, A., GUILLOU, L., KUCERA, P., WAGNIERES, G., VAN DEN BERGE, H. (1999). Photodetection of early human bladder cancer baesed on the fluorescence of 5-aminolevulinic acid hexyl ester induced protoporphyrin IX: apilot study. Br J Cancer, 80, 185-193.

LEVECKIS, J., BROWN, N.J. & REED, M.W.R. (1995). The effect of aminolaevulinic acid-induced, protoporphyrin IX-mediated photodynamic therapy on the cremaster muscle microcirculation in vivo. Br J Cancer, 72, 1113-1119.

MARTI, A., LANGE, N., VAN DEN BERGH, H., SEDMERA, D., KUCERA, P. (1999). Optimailisation of the formation and distribution of protoporphyrin IX in the urothelium: an in vitro approach. J Urology., 162, 546-552.

NOODT, B.B., BERG, K., STOKKE, T., PENG, Q. & NESLAND, J.M. (1996). Apoptosis and necrosis induced with light and 5-aminolaevulinic acid-derived protoporphyrin IX. Br J Cancer, 74, 22-29.

ROBERTS, D.J.H., CAIRNDUFF, F., DRIVER, I., DIXON, B. & BROWN, S.B. (1994). Tumour vascular shutdown following photodynamic therapy based on polyhaematoporphyrin or 5-aminolaevulinic acid. Int J Oncol., 5, 763-768.

SCHUITMAKER, J.J., BAAS, P., VAN LEENGOED, H.L.L.M., VAN DERN MEULEN, F.W., STAR, W.M. & VAN ZANDWIJK, N. (1996). Photodynamic therapy:a promising new modality for the treatment of cancer. J Photochem Photobiol B., 34, 3-12.

WEBBER, J., LUO, Y., CRILLY, R., FROMM, D. & KESSEL, D. (1996). An apoptotic response to photodynamic therapy with endogenous protoporphyrin in vivo. J Photochem Photobiol B., 35, 209-211.