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132-hydroxy bacteriopheophorbide a methylester specific uptake ratio in mice bearing lewis lung carcinoma
Lasermedizin 13: 41-44 (1997) © by Gustav Fischer Verlag _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
13 2-Hydroxy Bacteriopheophorbide a MethyLester Specific Uptake Ratio in Mice Bearing Lewis Lung Carcinoma 13 2-Hydroxy-Bacteriopheophorbid a MethyLester spezifische Aufnahmerate bei Mausen mit Lewis lung Karzinom M. S. ISMAIL I.2,3, C. DRESSLER4, P. KOEPPE 6 , C. PHILIPpI, B. RbDER 5 , H.WEITZEL2 and H.-P. BERLIEN I,4 Dept. of Laser Medicine, Neukolln Hospital (Head of Dept. Prof. Dr. H.-P' BERLIEN), Berlin Dept. of Gyn. and Obst., University Clinic B. Franklin (Head of Dept. Prof. Dr. H. WEITZEL), Berlin 3 Dept. of Gyn. and Obst. AI-Azhar University (Head of Dept. Prof. Dr. N. YONIS), Cairo, Egypt 4 Laser and Medicine Technology Centre (GmbH), Berlin 5 Dept. of Photobiophysics, Humboldt University, Berlin 6 Institute for Med.rrechn. Physic, University Clinic B. Franklin, Berlin I
2
Received: March 1997 . Revised: March 1997
The ratio of the accumulated photosensitizer concentration to its injected dose is named the specific uptake ratio (SUR). Investigations on a photosensitizer's biodistribution using the parameter SUR offer a better understanding of the pharmacokinetic behaviours of different photo sensitizers and render the comparison of different photosensitizers possible by overcoming the problem of using different sensitizer doses in different studies. The SUR of 13 2-OH-BPME was recorded in mice bearing Lewis lung carcinoma after i.v administration of 7.8 f,lmole/kg body weight at different incubation time intervals. The photosensitizer was chemically extracted from selected tissues and concentrations were measured by absorption spectroscopy. The parenchymatous organs liver, spleen, and kidney as well as the lung showed maximum 13 2-OH-BPME uptake ratios of 1.5 up 5.4 between 2 hand 4 h incubation times. The malignant tissue showed high 13 2-OHBPME SUR between 2 hand 12 h post injection with a maximum SUR of 1.2 at 8 h incubation time. The 13 2-OH-BPME SUR in muscle tissue, which represented the normal tumour surrounding tissue, and in the skin were very low and never exceeded 0.09 and 0.3 in muscle and skin, respectively. These results suggest that PDT using 13 2-OH-BPME could be effective at a photosensitizer incubation time of about 8 h post injection, when the tumour 13 2-OH-BPME SUR is maximum value, while muscle and skin exhibit minimum 13 2-OH-BPME uptake. Therefore, phototoxic side effects on normal tissues in the tumour surrondings should not be induced.
Keywords: absorption spectroscopy, pharmacokinetics, photosensitizer, photodynamic therapy (PDT), 132 -hydroxy bacteriopheophorbide a methylester (13 2 -OH-BPME), specific uptake ratio
Die in einem Gewebe akkumulierte Konzentration eines Photosensibilisators im Verhaltnis zur verabreichten Gesamtdosis wird als Spezifische Aufnahmerate (specific uptake ratio, SUR) bezeichnet. Untersuchungen zur Sensibilisator-Bioverteilung unter Verwendung des Parameters SUR ermoglichen einen objektiven Vergleich der Pharmakokinetiken verschiedener Sensibilisatoren, indem das Problem der unterschiedlichen Dosen, die in den unterschiedlichen Studien beschrieben wurden, somit umgangen wird. Die SUR-Kinetik des 13 2-Hydroxy-Bacteriopheophorbid a-Methylesters (13 2-OH-BPME) wurde hier am Tumormodell des Lewis lung-Karzinoms in der Maus nach intravenoser Injektion von 7.8 f,lmollkg Korpergewicht tiber einen Inkubationszeitraum von 168 h untersucht. Dafiir wurde 13 2-OH-BPME aus ausgewiihlten Organen bzw. Geweben chemisch extrahierty, und die Farbstoffkonzentrationen absorptionsspektroskopisch bestimmt. In den parenchymatOsen Organen Leber, Milz und Niere sowie in der Lunge wurden maximale SUR-Werte von 1,5 bis 5,4 zwischen 2 h und 4 h nach Injektion detektiert. Das Tumor-
42
M. S. ISMAIL et al.
gewebe zeigte hohe 13 2-OH-BPME-SUR wahrend einer Inkubationsdauer von 2 h bis 12 h mit einer maximalen SUR von 1.2 nach 8 h Inkubation. In Muskelgewebe, das hier das Normalgewebe der Tumorumgebung darstellte, und in der Haut wurden sehr geringe SUR-Werte bestimmt, die zu keinem der untersuchten Inkubationsintervalle 0.09 (Muskel) bzw. 0.3 (Haut) iiberschritten. Aufgrund dieser Daten sollte eine effektive PDT unter Verwendung von 13 2-OH-BPME ca. 8 h post injectionem durchgefiihrt werden konnen, da nach diesem Intervall die SUR im Tumor einen Maximalwert erreicht, wahrend die Aufnahmerate in Haut und Muskelgewebe minimal ist. Somit sollten keine phototoxischen Schlidigungen der Normalgewebe in der Tumorumgebung erwartet werden konnen.
Schliisselworte: Absorptionsspektroskopie, Pharmakokinetik, Photosensibilisator, Photodynamische Therapie (PDT), J3 2 -Hydroxy-Bacteriopheophorbid a-Methylester (J3 2 -OH-BPME), speziJische Aufnahmerate
Introduction Uptake, toxicity and carcinogenicity in normal tissues are the limiting factors for the commonly used chemotherapeutic drugs (1). In PDT the preferential uptake of the photosensitizer by the neoplastic tissue in relation to the healthy tissue is one of the most important parameters in achieving maximum success with this type of therapy (3). Several strategies can be full owed to construct sensitizers with a more selective tumour uptake and / or retention (1). The ratio of the photosensitizer concentration to its injected dose in the body is known as specific uptake ratio (SUR) (4). In the last years some "natural" photosensitizers such as derivatives of chlorophyll a (pheophorbide a, pyrophorbide a, chlorine e6) and bacteriochlorophyll a as "second generation" photo sensitizers have been under investigation not only because of their photoproperties including a relative high photostability but also because of their relatively simple and inexpensive preparation. The development of photosensitizers with high absorption around 700 nm offers the further advantage of optimum light penetration through the tissue within this range (9). The 13 2-hydroxy bacteriopheophorbide a methylester (13 2 -OHBPME) would appear to be one of the most promising compounds of the class of bacteriochlorophyll a derivatives. The specific uptake ratio of 13 2-OH-BPME was recorded in mice bearing Lewis lung carcinoma.
cutaneously in the flank. After steady tumour growth during the first week the tumour had an average diameter of about I cm. Every experimental group was made up 3 animals. Pharmacokinetics studies: 13 2-OH-BPME was administered i.v. at a dose of 7.8 /lmole/kg body weight (b.w.). The incubation periods in darkness were 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 168 h, after which the animals were sacrificed. Specimen of different organs were stored under liquid nitrogen. The 13 2-OH-BPME recovery was studied in tumour, lung, liver, spleen, kidney, muscle and skin. Chemical extraction and spectroscopic evaluation: Tissue samples were thawed, weighed, refrozen and thoroughly homogenized in 1-2 ml methanol/acetone (1: I). The homogenates were centrifuged at 800-1000/min- 1 for 10 min. The supernatants were then centrifuged again at 1800-2000/min for 5 min. Absorption spectra of the extracts were analysed spectrophotometric ally (Perkin Elmer UVNis-Lambda 2) in the spectral range of 200-1000 nm. Concentrations were calculated using the absorption coefficient of Ie at 748 nm = 15800 L.M- I cm- I . Specific uptake ratio: The biodistribution results are then expressed in terms of the specific uptake ratio (SUR) (3). SUR=C/D where C is the photosensitizer (13 2-OH-BPME) concentration in nmole/g tissue and D is the 13 2-OH-BPME injected dose in /lmole/kg body weight. A SUR above one marks an active enrichment in the accumulating tissues, whereas a SUR value below one is characteristic for lower availability in the tissue compared to the dose per kg b.w. At a SUR of 1, the measured concentration of the photosensititzer in the tissue per g matches the given dose per kg b.w.
Results Table 1 and figures 1, 2 show a rapid uptake of the 13 2 _ OH-BPME by the parenchymatous organs, liver and kidney. Relatively high uptaks were recorded in the lung where the SUR values between 5.4 and 2.6 were maintained for the first 12 h incubation time. The 13 2-OH BPME uptake measured in the tumour revealed its maximum value of 1.2 at 8 h incubation time. The uptake of 13 2-OH BPME in the tumour was continued until 48 hand at 168 h, no photosensetizer uptake was recorded. Muscle Table 1. 13 2-OH BPME SUR in different tissues of mice bearing Lewis lung carcinoma Organ
Material & methods Chemicals: The potential photosensitizer 13 2-OH-BPME was dissolved III Dulbecco's phosphate buffered saline (PBS), [Biochrom Seromed, Berlin Germany], with 1% Tween 80 [Sigrna, Germany] and sterilised by filtration before in vivo administration. Animals and tumour model: B6D2Fl mice bearing Lewis lung carcinoma were used as animal model. 0.1 ml Lewis lung carcinoma tumour cell suspension (106 cells/ml) was inoculated sub-
Liver Spleen Lung Kidney Muscle Skin Tumour
Time 2h
4h
8h
12 h
24h
48 h
2.6 0.6 5.4 1.2 0.09 0.08 0.7
1.3 0.3 2.6 1.5 0.04 0.28 0.43
I 0.3 3.2 0.57 0.03 0.05 1.2
0.8 0.27 2.9 0.32 0.025 0.1 0.7
0.16 0 0.76 0.25 0 0.08 0.2
0.2 0 0.01 0.05 0.006 0.08 0.2
Specific Uptake Ratio in Mice Bearing Lewis Lung Carcinoma
43
6 1,2
5 4
§
en
\
.~\
-------- Lung \
\
0,8
~Iiver
3
0:: en
:::::> 0,6
------- Kidney
2
----0---
0,4
Sp leen
\
\
Wll
Turn
0
r
\
oJ
""
------- Mus c Ie ~--o"I---
Ski n
~ ---~~<':> Tim e (hJ
Time (hI
Fig. 1. I 32-OH BPME SUR in lung, liver, kidney, and spleen.
Fig. 2. 13 2-OH BPME SUR in tumour, skin, and muscle.
OH-BPM Tumor/Muscle -'Skin Ratio
40
35 30 25 20 15 10
5
Tumor/Mus le Rat
O~~~~ Fig. 3. 13 2-OH BPME tumour/skinmuscle ratio.
Time (h)
tissue which represented the tumour surrounding healthy tissue, had a very low uptake level allthrough the examined periods and did not exceeded 0.09 at any time and could not be detected after 48 h incubation time. The uptake in the skin showed its maximum ratio of 0.28 at 4 h and its minimum ratio of 0.05 at 8 h incubation period. After 48 h incubation time, no 13 2 -OH BPME uptake could be detected in the skin. The maximum tumour-I muscle ratio of 40: 1 and tumour-/skin ratio of 23: 1 were detected at 8 h incubation time (figure 3).
Discussion An essential parameter in PDT is the preferential uptake of the photosensitizer by the neoplastic tissue. PDT is most effective if the light energy is delivered when the concentration of the sensitizer in the tumour tissue is higher than that of the adjacent normal tissue (2, 4, 6, 8). In order to improve the PDT planning, it is very important to quantify
48
168
the photosensitizer concentrations in normal and neoplastic tissues (6) and to evaluate the biodistribution pattern as well as the elimination rates in various tissues and organs (6).
The ratio of the photosensitizer concentration extraction to its injected dose in the body is defeined as the specific uptake ratio (SUR) (4). Photosensitizer pharmacokinetics evaluation using the specific uptake ratio offers a better understanding of the pharmacokinetics behaviour of different photosensitizers as well as the possibility of comparison of different photo sensitizers through overcoming the problem of different photosensitizers doses used in different studies. The specific uptake ratio (SUR) of I 32-OH-BPME was recorded on the Lewis lung carcinoma in mice after i.v administration of 7.8 /lmole/kg body weight at different incubation intervals. The parenchymatous organs, unlike other normal tissues showed high uptake of 13 2 -OHBPME. The data presented in this study showed a high uptake of 13 2-OH-BPME in liver, lung and kidney. These
44
M. S. ISMAIL et al.
values remained high until 12 h incubation time after which they decreased dramatically until 48 h incubation time and after that no 13 2-OH-BPME uptake could be detected. The muscle and skin showed very low uptake during the whole incubation period. A relatively high uptake in the tumour tissue was recorded in this study with a maximum value at 8 h incubation time. After 48 h no 13 2-OHBPME could be detected in the tumour tissue. High tumour-/muscle ratios of 40: 1 and tumour-/skin ratio of 23:1 at 8 h were both recorded. These high ratios resulted from a high 13 2-OH-BPME uptake in tumour compared to its uptake in muscle and skin tissues. These high ratios added an advantage for 13 2-OH-BPME in its application in PDT, as the muscle representing the normal tumour surrounding tissues and the high ratio during light application will offer a minimal destruction of the surrounding tissue. Also the high tumour/skin ratio offers an advantage of minimizing skin photosensitization during and after PDT. YANO et al. (10) have recorded a pheophorbide a tumour/muscle ratio of 100: 1 at 4 h incubation time whereby at Ih was the maximum time for the pheophorbide a uptake. IWAI & KIMURA (3), compared the pharmacokinetics of pheophorbide a and pheophorbide dimer in mice bearing FM3A tumours and reported that the 9 h-12 h is the time of maximum pheophorbide dimer concentrations and 18 h-24 h is the time of maximum pheophorbide a concentrations. They also reported tumour-/muscle ratios of more than 20 at this corresponding times. RODER et al. (9), also investigated the pharmacokinetics of 13 2-OH BPME in mice bearing Lewis lung carcinoma using the fluorescence spectroscopy and suggested that PDT using 13 2-OH BPME could be effective at a photosensitizer incubation time of about 12 h. Most of the tumour! muscle ratios reported in previous studies in relation to the other photo sensitizers were lower than the ratios which reported for the pheophorbide group. CHAN et al. (2) reported relative ratios for aluminium sulfonated phthalocyanines varying from 10: 1 to 2: 1. RICHTER et aI. (8) have reported a benzoporphyrin derivatives tumour-/muscle ratio of 4.23:1. Also, REDD! et al. (7), have reported a ratio of7.5: 1 with zinc phthalocyanine at 18-24 h. Because of the high 13 2 -OH-BPME tumour uptake and the high tumour-/muscle-skin ratios which recorded in the present study and in comparison with other studies, 13 2_
OH-BPME can be considered a good candidate photosensetizer for its application in the photodynamic processes, especially at a time interval between 8 hours and 12 hours post injection.
References 1. CASTRO D. J., SAXON R. E., LUFKIN R. B.: Future direction of
2. 3. 4.
5. 6.
7. 8. 9.
10.
laser phototherapy for diagnosis and treatment of malignancies. Laryngoscope 10 I: 1-10 (1991) CHAN W., MARSHALL J., SVENSON J.: Effect of sulfonation on the cell and tissue distribution of the photosensitizer aluminium phthalocyanine. Cancer Res. 50: 4533-4538 (1990) IWAI K., KIMURA S.: Efficiency of pheophorbide-dimer in photodynamic therapy of mouse tumour. J. Clin. Biochem. Nutr. 5: 145-149 (1988) JEEVES W., WILSON B., FIRNAU G., BROWN K.: Studies of HPD and radio labelled HPD in vivo and in vitro. In: Advances in experimental medicine and biology: Methods in porphyrin photosensitization. DAVID KESSEL (ed.), 193: 51-67 (1985) MOAN J.: Properties for optimal PDT sensitizers. J. Photoch. Photibiol.: B: 5: 521-524 (1990) MOSER J., HERCHENBACH B., EVENSCHOR K., KIRSCH D., SCHOCH S.: Biotechnology of bacteriopheophorbides, naturally occurring 2nd. generation photosensitizers. Laser in Med. Sci. 7: 272 (1992) REDDI E., CASTRO G., BIOLO R., JORI G.: Pharmacokinetic studies with zinc II phthalocyanine in tumour-bearing mice. Br. J. Cancer 56: 597-600 (1987) RICHTER A.: Characterisation of benzoporphyrin derivatives, a new photosensitizer SPIE 997: 132-138 (1988) RODER B., DRESSLER c., HAGEMANN R., FUCHS B., BERLIEN H.-P., NOWAK CH., MOSER J.: On the pharmacokinetics of 13 2-hydroxy-bacteriopheophorbide a methyl ester studied by fluorescence spectroscopy on lewis lung carcinoma bearing mice. SPIE 2078: 427-437 (1994) YANO T., UOZUMI T., KAWAMOTO K., ONDA J., FUJIMOTO N.: Photodynamic therapy for rat pituitary tumour in vitro and in vivo using pheophorbide a and white light. Laser in surgery and medicine 11: 174-182 (1991)
Correspondence address: Dr. med. M. S. ISMAIL, Dept. of Laser Medicine, Neukolln Hospital, Rudower Str. 48, D - 12351 Berlin, Germany. Tel: 0049130/60043832; Fax: 0049/30/60043870
13 2-Hydroxy Bacteriopheophorbide a MethyLester Specific Uptake Ratio in Mice Bearing Lewis Lung Carcinoma 13 2-Hydroxy-Bacteriopheophorbid a MethyLester spezifische Aufnahmerate bei Mausen mit Lewis lung Karzinom M. S. ISMAIL I.2,3, C. DRESSLER4, P. KOEPPE 6 , C. PHILIPpI, B. RbDER 5 , H.WEITZEL2 and H.-P. BERLIEN I,4 Dept. of Laser Medicine, Neukolln Hospital (Head of Dept. Prof. Dr. H.-P' BERLIEN), Berlin Dept. of Gyn. and Obst., University Clinic B. Franklin (Head of Dept. Prof. Dr. H. WEITZEL), Berlin 3 Dept. of Gyn. and Obst. AI-Azhar University (Head of Dept. Prof. Dr. N. YONIS), Cairo, Egypt 4 Laser and Medicine Technology Centre (GmbH), Berlin 5 Dept. of Photobiophysics, Humboldt University, Berlin 6 Institute for Med.rrechn. Physic, University Clinic B. Franklin, Berlin I
2
Received: March 1997 . Revised: March 1997
The ratio of the accumulated photosensitizer concentration to its injected dose is named the specific uptake ratio (SUR). Investigations on a photosensitizer's biodistribution using the parameter SUR offer a better understanding of the pharmacokinetic behaviours of different photo sensitizers and render the comparison of different photosensitizers possible by overcoming the problem of using different sensitizer doses in different studies. The SUR of 13 2-OH-BPME was recorded in mice bearing Lewis lung carcinoma after i.v administration of 7.8 f,lmole/kg body weight at different incubation time intervals. The photosensitizer was chemically extracted from selected tissues and concentrations were measured by absorption spectroscopy. The parenchymatous organs liver, spleen, and kidney as well as the lung showed maximum 13 2-OH-BPME uptake ratios of 1.5 up 5.4 between 2 hand 4 h incubation times. The malignant tissue showed high 13 2-OHBPME SUR between 2 hand 12 h post injection with a maximum SUR of 1.2 at 8 h incubation time. The 13 2-OH-BPME SUR in muscle tissue, which represented the normal tumour surrounding tissue, and in the skin were very low and never exceeded 0.09 and 0.3 in muscle and skin, respectively. These results suggest that PDT using 13 2-OH-BPME could be effective at a photosensitizer incubation time of about 8 h post injection, when the tumour 13 2-OH-BPME SUR is maximum value, while muscle and skin exhibit minimum 13 2-OH-BPME uptake. Therefore, phototoxic side effects on normal tissues in the tumour surrondings should not be induced.
Keywords: absorption spectroscopy, pharmacokinetics, photosensitizer, photodynamic therapy (PDT), 132 -hydroxy bacteriopheophorbide a methylester (13 2 -OH-BPME), specific uptake ratio
Die in einem Gewebe akkumulierte Konzentration eines Photosensibilisators im Verhaltnis zur verabreichten Gesamtdosis wird als Spezifische Aufnahmerate (specific uptake ratio, SUR) bezeichnet. Untersuchungen zur Sensibilisator-Bioverteilung unter Verwendung des Parameters SUR ermoglichen einen objektiven Vergleich der Pharmakokinetiken verschiedener Sensibilisatoren, indem das Problem der unterschiedlichen Dosen, die in den unterschiedlichen Studien beschrieben wurden, somit umgangen wird. Die SUR-Kinetik des 13 2-Hydroxy-Bacteriopheophorbid a-Methylesters (13 2-OH-BPME) wurde hier am Tumormodell des Lewis lung-Karzinoms in der Maus nach intravenoser Injektion von 7.8 f,lmollkg Korpergewicht tiber einen Inkubationszeitraum von 168 h untersucht. Dafiir wurde 13 2-OH-BPME aus ausgewiihlten Organen bzw. Geweben chemisch extrahierty, und die Farbstoffkonzentrationen absorptionsspektroskopisch bestimmt. In den parenchymatOsen Organen Leber, Milz und Niere sowie in der Lunge wurden maximale SUR-Werte von 1,5 bis 5,4 zwischen 2 h und 4 h nach Injektion detektiert. Das Tumor-
42
M. S. ISMAIL et al.
gewebe zeigte hohe 13 2-OH-BPME-SUR wahrend einer Inkubationsdauer von 2 h bis 12 h mit einer maximalen SUR von 1.2 nach 8 h Inkubation. In Muskelgewebe, das hier das Normalgewebe der Tumorumgebung darstellte, und in der Haut wurden sehr geringe SUR-Werte bestimmt, die zu keinem der untersuchten Inkubationsintervalle 0.09 (Muskel) bzw. 0.3 (Haut) iiberschritten. Aufgrund dieser Daten sollte eine effektive PDT unter Verwendung von 13 2-OH-BPME ca. 8 h post injectionem durchgefiihrt werden konnen, da nach diesem Intervall die SUR im Tumor einen Maximalwert erreicht, wahrend die Aufnahmerate in Haut und Muskelgewebe minimal ist. Somit sollten keine phototoxischen Schlidigungen der Normalgewebe in der Tumorumgebung erwartet werden konnen.
Schliisselworte: Absorptionsspektroskopie, Pharmakokinetik, Photosensibilisator, Photodynamische Therapie (PDT), J3 2 -Hydroxy-Bacteriopheophorbid a-Methylester (J3 2 -OH-BPME), speziJische Aufnahmerate
Introduction Uptake, toxicity and carcinogenicity in normal tissues are the limiting factors for the commonly used chemotherapeutic drugs (1). In PDT the preferential uptake of the photosensitizer by the neoplastic tissue in relation to the healthy tissue is one of the most important parameters in achieving maximum success with this type of therapy (3). Several strategies can be full owed to construct sensitizers with a more selective tumour uptake and / or retention (1). The ratio of the photosensitizer concentration to its injected dose in the body is known as specific uptake ratio (SUR) (4). In the last years some "natural" photosensitizers such as derivatives of chlorophyll a (pheophorbide a, pyrophorbide a, chlorine e6) and bacteriochlorophyll a as "second generation" photo sensitizers have been under investigation not only because of their photoproperties including a relative high photostability but also because of their relatively simple and inexpensive preparation. The development of photosensitizers with high absorption around 700 nm offers the further advantage of optimum light penetration through the tissue within this range (9). The 13 2-hydroxy bacteriopheophorbide a methylester (13 2 -OHBPME) would appear to be one of the most promising compounds of the class of bacteriochlorophyll a derivatives. The specific uptake ratio of 13 2-OH-BPME was recorded in mice bearing Lewis lung carcinoma.
cutaneously in the flank. After steady tumour growth during the first week the tumour had an average diameter of about I cm. Every experimental group was made up 3 animals. Pharmacokinetics studies: 13 2-OH-BPME was administered i.v. at a dose of 7.8 /lmole/kg body weight (b.w.). The incubation periods in darkness were 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 168 h, after which the animals were sacrificed. Specimen of different organs were stored under liquid nitrogen. The 13 2-OH-BPME recovery was studied in tumour, lung, liver, spleen, kidney, muscle and skin. Chemical extraction and spectroscopic evaluation: Tissue samples were thawed, weighed, refrozen and thoroughly homogenized in 1-2 ml methanol/acetone (1: I). The homogenates were centrifuged at 800-1000/min- 1 for 10 min. The supernatants were then centrifuged again at 1800-2000/min for 5 min. Absorption spectra of the extracts were analysed spectrophotometric ally (Perkin Elmer UVNis-Lambda 2) in the spectral range of 200-1000 nm. Concentrations were calculated using the absorption coefficient of Ie at 748 nm = 15800 L.M- I cm- I . Specific uptake ratio: The biodistribution results are then expressed in terms of the specific uptake ratio (SUR) (3). SUR=C/D where C is the photosensitizer (13 2-OH-BPME) concentration in nmole/g tissue and D is the 13 2-OH-BPME injected dose in /lmole/kg body weight. A SUR above one marks an active enrichment in the accumulating tissues, whereas a SUR value below one is characteristic for lower availability in the tissue compared to the dose per kg b.w. At a SUR of 1, the measured concentration of the photosensititzer in the tissue per g matches the given dose per kg b.w.
Results Table 1 and figures 1, 2 show a rapid uptake of the 13 2 _ OH-BPME by the parenchymatous organs, liver and kidney. Relatively high uptaks were recorded in the lung where the SUR values between 5.4 and 2.6 were maintained for the first 12 h incubation time. The 13 2-OH BPME uptake measured in the tumour revealed its maximum value of 1.2 at 8 h incubation time. The uptake of 13 2-OH BPME in the tumour was continued until 48 hand at 168 h, no photosensetizer uptake was recorded. Muscle Table 1. 13 2-OH BPME SUR in different tissues of mice bearing Lewis lung carcinoma Organ
Material & methods Chemicals: The potential photosensitizer 13 2-OH-BPME was dissolved III Dulbecco's phosphate buffered saline (PBS), [Biochrom Seromed, Berlin Germany], with 1% Tween 80 [Sigrna, Germany] and sterilised by filtration before in vivo administration. Animals and tumour model: B6D2Fl mice bearing Lewis lung carcinoma were used as animal model. 0.1 ml Lewis lung carcinoma tumour cell suspension (106 cells/ml) was inoculated sub-
Liver Spleen Lung Kidney Muscle Skin Tumour
Time 2h
4h
8h
12 h
24h
48 h
2.6 0.6 5.4 1.2 0.09 0.08 0.7
1.3 0.3 2.6 1.5 0.04 0.28 0.43
I 0.3 3.2 0.57 0.03 0.05 1.2
0.8 0.27 2.9 0.32 0.025 0.1 0.7
0.16 0 0.76 0.25 0 0.08 0.2
0.2 0 0.01 0.05 0.006 0.08 0.2
Specific Uptake Ratio in Mice Bearing Lewis Lung Carcinoma
43
6 1,2
5 4
§
en
\
.~\
-------- Lung \
\
0,8
~Iiver
3
0:: en
:::::> 0,6
------- Kidney
2
----0---
0,4
Sp leen
\
\
Wll
Turn
0
r
\
oJ
""
------- Mus c Ie ~--o"I---
Ski n
~ ---~~<':> Tim e (hJ
Time (hI
Fig. 1. I 32-OH BPME SUR in lung, liver, kidney, and spleen.
Fig. 2. 13 2-OH BPME SUR in tumour, skin, and muscle.
OH-BPM Tumor/Muscle -'Skin Ratio
40
35 30 25 20 15 10
5
Tumor/Mus le Rat
O~~~~ Fig. 3. 13 2-OH BPME tumour/skinmuscle ratio.
Time (h)
tissue which represented the tumour surrounding healthy tissue, had a very low uptake level allthrough the examined periods and did not exceeded 0.09 at any time and could not be detected after 48 h incubation time. The uptake in the skin showed its maximum ratio of 0.28 at 4 h and its minimum ratio of 0.05 at 8 h incubation period. After 48 h incubation time, no 13 2 -OH BPME uptake could be detected in the skin. The maximum tumour-I muscle ratio of 40: 1 and tumour-/skin ratio of 23: 1 were detected at 8 h incubation time (figure 3).
Discussion An essential parameter in PDT is the preferential uptake of the photosensitizer by the neoplastic tissue. PDT is most effective if the light energy is delivered when the concentration of the sensitizer in the tumour tissue is higher than that of the adjacent normal tissue (2, 4, 6, 8). In order to improve the PDT planning, it is very important to quantify
48
168
the photosensitizer concentrations in normal and neoplastic tissues (6) and to evaluate the biodistribution pattern as well as the elimination rates in various tissues and organs (6).
The ratio of the photosensitizer concentration extraction to its injected dose in the body is defeined as the specific uptake ratio (SUR) (4). Photosensitizer pharmacokinetics evaluation using the specific uptake ratio offers a better understanding of the pharmacokinetics behaviour of different photosensitizers as well as the possibility of comparison of different photo sensitizers through overcoming the problem of different photosensitizers doses used in different studies. The specific uptake ratio (SUR) of I 32-OH-BPME was recorded on the Lewis lung carcinoma in mice after i.v administration of 7.8 /lmole/kg body weight at different incubation intervals. The parenchymatous organs, unlike other normal tissues showed high uptake of 13 2 -OHBPME. The data presented in this study showed a high uptake of 13 2-OH-BPME in liver, lung and kidney. These
44
M. S. ISMAIL et al.
values remained high until 12 h incubation time after which they decreased dramatically until 48 h incubation time and after that no 13 2-OH-BPME uptake could be detected. The muscle and skin showed very low uptake during the whole incubation period. A relatively high uptake in the tumour tissue was recorded in this study with a maximum value at 8 h incubation time. After 48 h no 13 2-OHBPME could be detected in the tumour tissue. High tumour-/muscle ratios of 40: 1 and tumour-/skin ratio of 23:1 at 8 h were both recorded. These high ratios resulted from a high 13 2-OH-BPME uptake in tumour compared to its uptake in muscle and skin tissues. These high ratios added an advantage for 13 2-OH-BPME in its application in PDT, as the muscle representing the normal tumour surrounding tissues and the high ratio during light application will offer a minimal destruction of the surrounding tissue. Also the high tumour/skin ratio offers an advantage of minimizing skin photosensitization during and after PDT. YANO et al. (10) have recorded a pheophorbide a tumour/muscle ratio of 100: 1 at 4 h incubation time whereby at Ih was the maximum time for the pheophorbide a uptake. IWAI & KIMURA (3), compared the pharmacokinetics of pheophorbide a and pheophorbide dimer in mice bearing FM3A tumours and reported that the 9 h-12 h is the time of maximum pheophorbide dimer concentrations and 18 h-24 h is the time of maximum pheophorbide a concentrations. They also reported tumour-/muscle ratios of more than 20 at this corresponding times. RODER et al. (9), also investigated the pharmacokinetics of 13 2-OH BPME in mice bearing Lewis lung carcinoma using the fluorescence spectroscopy and suggested that PDT using 13 2-OH BPME could be effective at a photosensitizer incubation time of about 12 h. Most of the tumour! muscle ratios reported in previous studies in relation to the other photo sensitizers were lower than the ratios which reported for the pheophorbide group. CHAN et al. (2) reported relative ratios for aluminium sulfonated phthalocyanines varying from 10: 1 to 2: 1. RICHTER et aI. (8) have reported a benzoporphyrin derivatives tumour-/muscle ratio of 4.23:1. Also, REDD! et al. (7), have reported a ratio of7.5: 1 with zinc phthalocyanine at 18-24 h. Because of the high 13 2 -OH-BPME tumour uptake and the high tumour-/muscle-skin ratios which recorded in the present study and in comparison with other studies, 13 2_
OH-BPME can be considered a good candidate photosensetizer for its application in the photodynamic processes, especially at a time interval between 8 hours and 12 hours post injection.
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Correspondence address: Dr. med. M. S. ISMAIL, Dept. of Laser Medicine, Neukolln Hospital, Rudower Str. 48, D - 12351 Berlin, Germany. Tel: 0049130/60043832; Fax: 0049/30/60043870