Introduction
Although hematoporphyrin derivative (HPD), as “first generation” photosensitizer, has been used in experimental PDT of tumours in animal and human for several years, it could not be considered an ideal photosensitizer and its drawbacks are well documented ⁽¹⁾. Therefore some “natural” photosensitizers like derivatives of chlorophyll, a (pheophorbid-a, pyropheophorbide-a, chlorine-6 ) and bacteriochlorophyll-a as “second generation” photosensitizers have been under investigations because of their advantageous photoproperties including relatively high photostability, high oxygen quantum yields and the good absorption capacities in the spectral range above 630 nm together with simple and inexpensive preparation. The development of photosensitizers with strong absorption around 700 nm offer the advantages of the optimum light penetration through the tissue at these wavelengths ⁽²⁾. In this context, the investigated 13²-hydroxy-bacteriopheophorbidea methyl ester (13²-OH-BPME) seems to be one of the promising compounds of the class of bacteriochlorophyll-a derivative. The photodynamic potential of of 13²-OH-BPME has been extensively described on the photophysical ⁽³⁾, as well as the cellular level ⁽⁴⁾.
The present paper deals with the different tissue pharmacokinetics biodistribution of 13²- OH-BPME using the method of absorption spectroscopy in order to define the optimum time for effective PDT according to the incubation time of the sensitizer.

Material and Methods

Chemicals

The potential photosensitizer 13²-OH BPME was prepared according to Moser & his colleuges ⁽⁴⁾. It is dissolved in Dulbeccos phosphate buffered saline (PBS), [Biochrom Serored, Berlin, Germany], with 1% Tween 80 [Sigma chemical, Germany] and sterilized by filtration before in vivo administration.

Animals and tumour model
B6D2F1 mice bearing ovarian carcinoma inoculated subcutaneously in the flank (0.1 ml tumour cell suspension) were used as animal model. After a steady tumour growth over one week, the tumour showed an average diameter of about 1 cm. Every experimental group consisted of 3 animals for each time interval.

Pharmacokinetic studies
P13²-OH-BPME was administered iv at a dose of 5 mg/kg body weight, the incubation periods in darkness were 2h, 4h, 8h, 12h, 24h, 48h and 168h, then the animals were sacrificed and the tissues of different organs were stored in the gas phase of liquid nitrogen. The 13²-OH-BPME recovery was studied in tumour, lung, liver, spleen, kidney, muscle and skin.

Chemical extraction and spectroscopic evaluation
Tissue samples were thawed, weighed wet, refrozen and thoroughly homogenized in 1-2 ml Methanol/Acetone (1:1). The homogenates were centrifuged at 800-1000 for 10 min. The supernatants were centrifuged again at 1800- 2000. Absorption spectra of the extracts were analysed spectrophotometrically (Perkin Elmer UV/Vis-Lambda 2) in the spectral range of 200- 1000 nm. Concentrations were calculated using the absorption coefficient of λ (748 nm) = 15800 l./M-1 cm-1.

Results
The data presented in Table 1  showed rapid accumulation of 13²-OH-BPME in paren-chymatous organs. Relatively high accumulations rates were detected in the lung and maintained for 12h after administration of the sensitizer. The 13²-OH-BPME concentrations measured in the tumour revealed its maximum value after 8h post injection. A long time retention until 168h post injection was not detected in any of the investigated tissues since the concentrations was below the minimal detectable concentration of 0.023nmol/g tissue. The muscle tissue which represented the surrounding healthy tissues for the tumour presented very low concentrations all through the incubation time. The concentrations in the skin showed its maximum concentration at 4h and its minimum concentration was at 8h post injection period. After 48h examination time no 13²-OH-BPME was detected in the skin. The maximum tumour-/ muscle ratio of 31 and tumour-/ skin ratio of 23 were detected at 8h after administration of the sensitizer.

Discussion

The preferential uptake of the photosensitizer by the neoplastic tissue is an crucial parameter in PDT process. The pharmacokinetic of photosensitizer is a specific feature. PDT of course will be more effective if the light energy is applied when the concentration of the photosensitizer in the tumour tissue is higher than in the adjacent normal tissue ^(3-12). Thus, it is important to quantify the photosensitizer concentration in normal and neoplastic tissues ⁽¹²⁾ in order to evaluate the biodistribution patterns, as well as the elimination rates in various tissues and organs ⁽⁴⁾, to improve the PDT planning.

The parenchymatous organs and the lung, unlike other normal tissues, accumulated very high concentrations of the photosensitizer. The data presented in this study show a high uptake of OH-BPME in liver, spleen, lung and kidney. These values remained high until 12 hours incubation time and decreased dramatically later on until 48 hours incubation time, after 48 hours OH-BPME cannot be detected except in the liver where a very minimal concentration detected. Also, muscle and skin show a low uptake during the whole incubation period. Considerable concentrations in the tumour tissue were recorded in this study with a maximum value of 9.5 nmol/g at 8 hours incubation time. After 48 hours no retention in the tumour tissue could be detected. A high tumour / muscle ratio of 31 and tumour / skin ratio of 23 after 8 hours were recorded. These high ratios resulted from high 13² OH-BPME uptake by the tumour compared with muscle and skin tissues. These high ratios might be an advantage of 13² OH-BPME in its application in the PDT procedure, as the muscle represents the normal surrounding tissues to the inoculated tumour and this high ratio during light application will leads to minimal surrounding tissue destruction. Also the high tumour/ skin ratio offers an advantage of minimising skin photosensitization side effects after therapy. In relation to the pheophorbide-a photosensitizer group, Yano et al., have recorded a pheophorbidea tumour/ muscle ratio of 100 after 4 hours post injection⁽¹³⁾. Iwai and Kimura⁽¹²⁾, compared the pharmacokinetics of pheophorbid-a and pheophorbide dimmer in mice bearing FM3A tumours and they recorded the time of maximum pheophorbide dimmer concentration between 9-12 hours and between 18-24 hours when pheoforbid-a was used. They also reported tumour-/ muscle ratios of more than 20 at this corresponding time for the both sensitizers. Röder et al^(3)., investigated the pharmacokinetics of 13² OH- BPME in mice bearing Lewis lung carcinoma using the fluorescence spectroscopy as photosensitizer detection method and she suggested that PDT using 13² OH- BPME could be effective post sensitizer administration time at 12h (3). Chan et al.⁽²⁾ reported tumour / muscle- skin ratios for aluminium sulfonated phthalocyanines varying from 10 to 2. Richter et al.⁽⁵⁾ reported a benzoporphyrin derivatives tumour/ muscle ratio of 4 and Reddi et al. ⁽¹⁴⁾, reported a ratio of 7.5 with zinc phthalocyanines between 18-24h post sensitizer administration.

Because of the high 13² OH-BPME tumour accumulation and the high tumour/ muscleskin ratios recorded in the present study and in comparison with other studies, 13² OH-BPME might be considered a good candidate for its application in PDT.

Acknowledgements
Many thanks for Dr. C. Dressler for technical help in the present study and for professor H. P. Berlin. This study is a part of the program “Photodynamische Lasertherapie” granted by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie of the Federal Republic of Germany.

References

1. Bown S, Tralau C, Coleridge S, et al. Photodynamic therapy with porphyrin and phthalocyanine sensitisation: Quantitative studies in normal rat liver. Br J Cancer 1986; 54: 43-52.

2. Chan W, Marshall J, Svenson J. Effect of sulfonation on the cell and tissue distribution of the photosensitizer aluminium phthalocyanine. Cancer Res. 1990; 50: 4533-4538.

3. Röder B, Derssler C, Hagemann R, et al. On the pharmacokinetics of 13²-hxdroxybacteriopheophorbide- a methyl ester studied by fluorescence spectroscopy on Lewis lung carcinoma bearing mice. SPIE 1994; 2078: 427-437.

4. Moser J, Herchenbach B, Evenschor K, et al. Biotechnology of bacteriopheoforbides, naturally occurring 2nd. generation photosensitizers. Laser in Med Sci 1992; 7: 272.

5. Richter A. Characterisation of benzoporphyrin derivatives, a new photosensitizer SPIE 1988; 997: 132-138.

6. Basil J, Berlien H.-P. Basics of photodynamic therapy: In Applied Laser Medicine; 252 -288; Berlien H.-P. and Muller G., Springer Publischer, Berlin, Germany. 2003.

7. Ismail M S, and Phillip C. Laser in Gynaecology. In Applied Laser Medicine 2003; 346 -378; Berlien H.-P. and Muller G., Springer Publischer, Berlin, Germany.

8. Hornung R, Fehr M K, Walt H, et al. PEGm-THPCmediated photodynamic effects on normal rat tissues. Photochemistery and Photobiology, 2000; 72 (5): 696 – 670.

9. Schlosser V, Koechli RO, Cattaneo R, et al. Phptodynamic effects in vitro in fresh gynaecological tumours analyzed with bioluminescence method. Clinical and Laboratory medicine 1999; 37 (2): 115 120.

10. Whaitcre CM, Feyes DK, Satoh T. et al. : Photodynamic therapy with phthalocyanine photosensetiser of SW 480 human colon cancer xenografts in athymic mice. Cli Cancer Res 2000; 6: 2021 – 2027.

11. Ahmed N and Mokhtar M: Mechanism of photodynamic therapy induced cell death. Methods enzymology 2000; 312: 342 – 385.

12. Iwai K and Kimura S. Efficiency of pheophorbidedimmer in photodynamic therapy of mouse tumour. J Clin Biochem Nutr 1988; 5: 145-149.

13. Yano T, Uozumi T, Kawamoto K, et al. Photodynamic therapy for rat pituitary tumour in vitro and in vivo using pheoforbide-a and white light. Laser in surgery and medicine 1991; 11: 174-182.

14. Reddi E, Castro G, Biolo R, et al. Pharmacokinetic studies with zinc II phthalocyanine in tumour-bearing mice. Br J Cancer 1987; 56: 597-600.