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Uptake and distribution of intravenously or intravesically administered photosensitizers in the rat
CANCER LETTERS Cancer Letters 75 (1993) 65-70
Uptake
and distribution of intravenously or intravesically administered photosensitizers in the rat
Torgny
Windahl*a, Qian Pengd, Johan Moand, Sverker Hellstenb, Bengt Axelssonc, Lennart Liifgren”
‘tDepurtmmr
of Biophysics, Institute jtir
“Deparm~mt
of’ ENT,
Cancer Reseurch. Montc&llo,
Long Islund Jewish Mrdicul
(Received 19 January 1993: revision received 3 September
O.slo. Notwo!
Center. NY.
1993: accepted
LISA
28 September
1993)
Abstract Photodynamic therapy using i.v. injected porphyrin photosensitizers have been used to treat selected cases of superficial bladder cancer. Since cutaneous photosensitivity. lasting 6-8 weeks, is a well known undesirable side effect of this therapy, we instilled the photosensitizers intravesically in rats and compared the uptake of photosensitizers in different tissues by this route of administration with the uptake after intravenous injection. The intravesical mode of delivery enhanced photosensitizer uptake in the bladder wall. while giving low concentrations in extravesical organs. Intravesical instillation of the photosensitizers may therefore increase their efficacy and reduce phototoxicity as compared with intravenous injection. Comparing the results obtained by two assays, one based on porphyrin fluorescence and the other based on the application of radioactively labelled photosensitizers, it was concluded that the i.v. administration route may result in tissue uptake of significant amounts of aggregated non-fluorescent, supposedly inactive drug, while the intravesical administration led to less uptake of aggregates relative to active drug monomers. KeJ’ rcordst Photodynamic
therapy;
Hematoporphyrins;
1. Introduction Photodynamic therapy has been used in the management of various malignant tumors [13], including urothelial carcinoma of the bladder [3. 12.16]. Selected cases of superficial bladder cancer, particularly carcinoma in situ [?I], were shown to benefit from photodynamic therapy. The method, 0304-3835/93/$06.00 SSDI
0
lntravesical
instillation
however. is associated with significant local complications, including frequency of micturition, urgency, dysuria and in some cases pronounced reduction of bladder capacity [8,11], possibly with risk of development of a contracted bladder. The diminution of bladder capacity is probably due to undesirable activation of the administered photosensitizer in the muscular layers of the bladder wall
1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved.
0304-3835(93)03194-A
66
[20]. Cutaneous photosensitivity lasting 6-8 weeks is another well known side effect after systemic administration of photosensitizer. In order to avoid adverse phototoxicity it would seem appropriate to give the photosensitizer intravesically rather than intravenously, provided that the intravesical route does not result in impaired accumulation of the drug within the urothelium. The aim of the present study was 4-fold; firstly to assess the uptake of hematoporphyrin derivatives in the bladder wall after intravesical instillation compared with intravenous injection, secondly to study the distribution of these drugs in the systemic circulation and extravesical organs, thirdly to observe if an enhancer (polysorbate 80) could improve the uptake of intravesically instilled drugs and fourthly to compare results obtained from measurements of porphyrin fluorescence with radioactively labelled substances. The latter study was performed to address the problem of porphyrin aggregation. Porphyrin aggregates may be taken up by tumors, but do not fluoresce and may be uncompletely disaggregated in the assay procedures. For our studies we chose a hematoporphyrin derivative (HPD) and its purified form dihematoporphyrin ether (DHE) termed Photofrin II (P II). These drugs have been characterized by other investigators [3.4]. 2. Materials and methods 2.1. Photosmsitixrs HPD was obtained from The Queen Elizabeth Pharmacy Department, Woodville Hospital, South, Australia, as an aqueous solution at a concentration of 5.0 mgiml. ‘“C-labelled HPD and lJC-labelled DHE were supplied from the University of Leeds, Industrial Service Ltd., as a freeze-dried powder. In both cases the drugs were dissolved in isotonic saline at a concentration of 5.0 mgiml containing 1.0 PCilabelled substance per ml. The porphyrins, both the labelled and the unlabelled were prepared with the same methods as earlier described in the work of Lipson et al. [4] The labelled carbon atoms were all in the ring of the porphyrin and not in the side chains. 2.2. Experimental protocol Female Wistar rats (72) were used in the present
study (mean body weight 236 g). In the case of labelled drugs. 40 animals were divided into 4 groups (10 for each group) and subjected to either intravesical instillation or intravenous injection of ‘3C-labelled HPD or 14C-P II following bilateral ligation of the ureters under general anesthesia with intraperitoneal mebumal. They were sacrilited 1 h later. In another group (also 10 rats), the animals were killed 48 h after an intravenous injection of 14C-labelled HPD without previous ureteral ligation. The instilled volume was 0.4 ml and the concentrations of 14C-labelled HPD and P II in the instilled solutions were 5.0 mgiml. The instillate was kept in the bladder for 1 h. The same volume was used for intravenous injection, corresponding to approximately 10 mg drug/kg body weight. In addition, 5 animals were used as controls with only intravesical administration of 0.4 ml saline solution. Blood samples were drawn from the aorta. The instillate was recovered by aspiration from the exposed bladder. Thereafter, the bladder was incised, carefully washed in saline and dried on filter paper. The lungs, heart. liver, spleen, kidneys, ovaries, oral mucosa and samples from abdominal muscle were removed and stored at -70°C together with the bladder tissue until examined. The organs were then thawed. allowed to dry at room temperature for 24 h and combusted in a Tri-Carb sample oxidizer. The radioactive material was collected in 21 ml CarboSorbiPermafluor (8:13 v/v) and analyzed in a liquid scintillation counter. In the experiments with unlabelled HPD, the animals were divided into 3 groups (5 for each) and sacrificed 1 h after HPD had been given intravenously, intravesically or intravesically plus 10% surface detergent (polysorbate 80), respectively. The concentrations of HPD in different tissues were then assessed with spectrofluorometry as previously described [6.18]. Two animals served as controls receiving only 0.4 ml saline solution intravesically. 3. Results 3.1. Intrawnous injection The distribution of HPD and P II in tissues after intravenous injection is shown in Fig. la-c. The pattern of uptake after 1 h was similar to that after
T. Windahl et al. /Cancer
Lett. 75 (1993) 65-70
Muscle
,og/g
Heart
67
Oral mucosa
Kidney
Ovary
Spleen
Ovary
Spleen
Liver
wet ttssue
. -90 80 70 @I 50 40 30 20 10 0
+ Muscle
Kidney
Heart
Bladder
Lung
Liver
mucosa fl9/g 90 80 70
60
tissue
1
n
14C-HpD (lhj
q
lJC-PII (Ihj
T
’
r
T
f
llB lkk T
l-
r
Muscle
Fig. 1. (a-c)
Heart
Distribution of hematoporphyrin ing and spectrofluorometric
Oral mucosa
Kidney
Bladder
Ovary
Spieen
LWT
Liver
derivative (HPD) and dihematoporphyrin ether (P II) determined by radioactive measurements after intravenous injection of the photosensitizers.
count.
68
n
Fluoresence-HpD
1
14C-HpD “x-Pll
Muscle Fig. 2. Distribution
ofthe
Heart
photosensitizers
Kidney
Bladder x 10
Ovary
HPD and P II determined by radioactive I h after lntraceslcal instillation.
Spleen countmg
Lung
Liver
and ~pcctrotluorometrlc
measurements
48 h for both substances.
A slight decrease in absolute values occurred between 1 and 48 h in muscle, heart and lung, and some increase of the values in oral mucosa, kidneys, spleen and liver. The values obtained with the radioactivity method were between twice (muscle) and six times (ovary) as high as those obtained with the fluorescence method.
ations with intraperitoneal injection in mice [1,7,18,19]. Our fluorescence values for HPD were in good agreement with the findings of other investigators 3 h after intravenous injection of 10 mg P II/kg body weight in mice [ 171. In the liver, for instance, the respective values were 25 and 27 pg/g, in the spleen I1 and 12 pgig and in the kidney 8 and 10
3.2. htravesical instillatkwz Analysis of the tissue distribution of HPD and P II after intravesical instillation of the drugs (Fig. 2) showed a very high uptake by the bladder wall. The fluorescence measurements of HPD agreed with the i4C-HPD values, whereas uptake of P II was significantly lower. In extravesical organs this mode of drug delivery resulted in very low concentrations, close to the detection limit.
flgig. There are some possible reasons for the discrepancy between the fluorescence and radioisotope measurements after intravenous injection. One explanation is that both HPD and P II contain a large fraction of aggregates with a very low fluorescence quantum yield [ 14). Chromatographic analysis has indicated that such aggregates are preferentially taken up by tumors [14.22]. High uptake of aggregated material is to be expected also in other tissues with high endocytic activity, such as liver and spleen. As the aggregates are measured by the radioisotopic, but not the fluorescence method, this selective uptake of aggregated substance may explain why radioactive counting gave the higher values. Complete disaggregation of porphyrins probably does not occur in solutions selected for this purpose in fluorescence measure-
4. Discussion When either hematoporphyrin derivative or its partially purified version, dihematoporphyrin ether was given intravenously, the uptake per gram of tissue decreased in the following order: liver > spleen, ovary, kidney, lung > heart > muscle. This order is in agreement with previous observ-
T. Windahl et al. /Cancer
69
Lett. 75 (1993) 65-70
ments. Action spectroscopy, fluorescence excitation spectroscopy and absorption spectroscopy have shown that the photosensitizing effect of HPD and P II was due to the fluorescencing fraction, the aggregates being virtually inactive [15]. Thus, data obtained from fluorescence may be more relevant to photodynamic therapy than data obtained with radiolabelled substances. When the drugs were given intravesically, there was less uptake of P II than of HPD in the bladder wall. The reason for this is not known. One possible explanation is that P II contains a larger fraction of aggregates than HPD, and the aggregates may penetrate less well than monomers into tissues. The data for other tissues agree with this suggestion. Thus, in contrast to the data for intravenous administration, the uptake values after intravesical instillation were higher using the fluorescence than the radioisotope method. Entry to the circulation is probably easier for the fluorescent monomers than for the aggregates. Our analyses do not permit any quantitative assessment of the photosensitizer in the different layers of the bladder wall. It might also be possible to further increase the uptake of photosensitizers after intravesical instillation by addition of an enhancer such as polysorbate 80 to the instillate as tried in conjunction with intravesical instillation of doxyrubicin [5,9,10]. So far, however, this has not been successful in our experiment with HPD. 5. Conclusions The intravesical mode of delivering the photosensitizers enhanced their uptake within the bladder wall as compared with intravenous administration, while giving very low uptake in various extravesical organs. Our study, however, does not permit conclusions concerning depth of penetration of the photosensitizers into or beneath the urothelium. Further studies are required to assess if intravesical administration of photosensitizers can increase the efficacy of photodynamic therapy in patients with superficial bladder cancer and also reduce the associated phototoxicity. 6. Acknowledgments The study
was supported
by grants
from
the
Swedish Cancer Council.
Foundation
and ijrebro
County
7. References I
Amano, T., Prom, tumour
2
3
4
5
6
7
8
injection
G.R. and Lin, C-W. (1988) Intraas a more effective means of porphyrin
administration for photodynamic therapy. J. Ural., 139. 392-395. Benson, R.C. (1985) Treatment of diffuse transitional cell carcinoma in situ by whole bladder hematoporphyrin derivative photodynamic therapy. J. Urol., 134. 675-678. Dougherty, T.J.. Potter, W.R. and Weishaupt. K.R. (1984) The structure of the active component of hematoporphyrin derivative. In: Porphyrin localization and treatment of tumors. pp. 301-314. Editors: D.R. Doiron and C.J. Comer. Alan R. Lisslnc., New York. Lipson,R.L.,Baldes.E.J.andOlsen.A.M.(1961)Theuse of a derivative of haematoporphyrin in tumor detection. J. Natl. Cancer Inst.. 26, l-8. Eksborg. S., Edsmyr, F. and Naslund. 1. (1982) Intravesical instillation with adriamycin + Tween 80 in patients with superficial bladder tumors. Eur. Ural.. 8, 213-215. Gomer, C.J., Jester, J.V., Razum, N.J., Szirth. B.C. and Murphree A.L. (1985) Photodynamic therapy of intraocular tumors: examination of hematoporphyrin derivative distribution and long-term damage in rabbit ocular tissue. Cancer Res., 45. 3718-3725. Gomer. C.J. and Dougherty, T.J. (1979) Determination of ‘H and 14C hematoporphyrin derivative distribution in malignant and normal tissue. Cancer Res.. 39, 145-151. Harty. J.I., Mohammad. A., Wieman, T.J.. Tseng, M.T.. Ackerman, D. and Broghamer, W. (1989) Complications of whole bladder dihematoporphyrin ether photodynamic therapy. J. Ural.. 141, 1341-1346. Hellsten, S.. Eksborg, S. and Axelsson. B. (1987) Absorption of Adriamycin into the systemic circulation after intravesical instillation with or without Tween 80 in the rat. Ural. Res., IS. 15-21. Hellsten, S.. Axelsson, B.. Eksborg. S. and Lindkvist. C. (1988) Organ distribution of adriamycin after intravesical instillation with or without Tween 80 in the rat. Ural. Res., 16, 325-327. Jocham. D, Beer. M., Baumgartner, R., Staehler. G. and Unsold, E. (1989) Long term experience with integral photodynamic therapy of TIS bladder carcinoma. In: Photosensitizing compounds: their Chemistry. Biology and Clinical Use, pp. 198-208. Editor: Wiley. Chichester (Ciba Foundation symposium 146). Kelly, J.F. and Snell, M.E. (1976) Hematoporphyrin derivative: a possible aid in the diagnosis and therapy of carcinoma of the bladder. J. Ural.. 115. 150. Marcus. S.L. (1990) Photodynamic therapy of human cancer: clinical status, potential and needs. In: Future directions and applications in photodynamic therapy. SPIE Institutes for Advanced Optical Technologies. pp. 3-56, vol. 156. Editor: C.J. Gomer. Washington: SPIE Optical Engineering Press.
70 14
15
16
17
18
T Win&hi Moan, J. and Sommer, S. (1983) Uptake of the components of hematophorphyrin derivative by cells and tumours. Cancer Lett., 21. 167-174. Moan, J. and Sommer. S. (1984) Action spectra for hematoporphyrin derivative and photofrin II with respect to sensitization of human cells in vitro to photoinactivation. Photochem. Photobiol., 40. 631-634. Naito, K., Hisazumi, H., Uchibayashi. T., Amano, T., Hirata. A., Komatsu, K.. Ishida, T. and Miyoshi. N. (1991) Integral laser photodynamic treatment of refractory multifocal bladder turnouts. J. Ural.. 146. 1541-1545. Pantelides, M.L., Moore, J.V. and Blacklock, N.J. (1989) A comparison of serum kinetics and tissue distribution of photofrin II following intravenous and intraperitoneal injection in the mouse. Photochem. Photobiol., 49, 67-70. Peng, Q., Evensen. J.F.. Rimington. C. and Moan. J. (1987) A comparison of different photosensitizing dyes with respect to uptake C3H-tumors and tissues of mice. Cancer Lett., 36, l-10.
19
20
21
22
Ed ul. /Cancer
Lrti.
75 (19931 65-70
Peng. Q., Moan, J.. Kongshaug, M., Evensen. J.F., Anholt. H. and Rimington. C. (199 I) Sensitizer for photodynamic therapy of cancer: a comparison of the tissue distribution of photofrin II and aluminium phtalocyanine tetrasulfonate in nude mice bearing a human malignant tumor. Int. J. Cancer, 48, 258-264. Pope. A.J. and Bown. SC. (1991) The morphological and functional changes in rat bladder following photodynamic therapy with phtalocyanine photosensitization. J. Ural.. 145, 1064-1070. Shumaker. B.P. and Hertzel, F.W. (1987) Clinical laser photodynamic therapy in the treatment of bladder carcinoma. Photochem. Photobiol., 46. 899-901. Sommer. S., Moan. J.. Christensen, T. and Evensen, J.F. (1984) A Chromatographic study of hematoporphyrin derivatives. In: Porphyrins in tumour phototherapy, pp. 81-91. Plenum Publ. Corp.
Uptake
and distribution of intravenously or intravesically administered photosensitizers in the rat
Torgny
Windahl*a, Qian Pengd, Johan Moand, Sverker Hellstenb, Bengt Axelssonc, Lennart Liifgren”
‘tDepurtmmr
of Biophysics, Institute jtir
“Deparm~mt
of’ ENT,
Cancer Reseurch. Montc&llo,
Long Islund Jewish Mrdicul
(Received 19 January 1993: revision received 3 September
O.slo. Notwo!
Center. NY.
1993: accepted
LISA
28 September
1993)
Abstract Photodynamic therapy using i.v. injected porphyrin photosensitizers have been used to treat selected cases of superficial bladder cancer. Since cutaneous photosensitivity. lasting 6-8 weeks, is a well known undesirable side effect of this therapy, we instilled the photosensitizers intravesically in rats and compared the uptake of photosensitizers in different tissues by this route of administration with the uptake after intravenous injection. The intravesical mode of delivery enhanced photosensitizer uptake in the bladder wall. while giving low concentrations in extravesical organs. Intravesical instillation of the photosensitizers may therefore increase their efficacy and reduce phototoxicity as compared with intravenous injection. Comparing the results obtained by two assays, one based on porphyrin fluorescence and the other based on the application of radioactively labelled photosensitizers, it was concluded that the i.v. administration route may result in tissue uptake of significant amounts of aggregated non-fluorescent, supposedly inactive drug, while the intravesical administration led to less uptake of aggregates relative to active drug monomers. KeJ’ rcordst Photodynamic
therapy;
Hematoporphyrins;
1. Introduction Photodynamic therapy has been used in the management of various malignant tumors [13], including urothelial carcinoma of the bladder [3. 12.16]. Selected cases of superficial bladder cancer, particularly carcinoma in situ [?I], were shown to benefit from photodynamic therapy. The method, 0304-3835/93/$06.00 SSDI
0
lntravesical
instillation
however. is associated with significant local complications, including frequency of micturition, urgency, dysuria and in some cases pronounced reduction of bladder capacity [8,11], possibly with risk of development of a contracted bladder. The diminution of bladder capacity is probably due to undesirable activation of the administered photosensitizer in the muscular layers of the bladder wall
1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved.
0304-3835(93)03194-A
66
[20]. Cutaneous photosensitivity lasting 6-8 weeks is another well known side effect after systemic administration of photosensitizer. In order to avoid adverse phototoxicity it would seem appropriate to give the photosensitizer intravesically rather than intravenously, provided that the intravesical route does not result in impaired accumulation of the drug within the urothelium. The aim of the present study was 4-fold; firstly to assess the uptake of hematoporphyrin derivatives in the bladder wall after intravesical instillation compared with intravenous injection, secondly to study the distribution of these drugs in the systemic circulation and extravesical organs, thirdly to observe if an enhancer (polysorbate 80) could improve the uptake of intravesically instilled drugs and fourthly to compare results obtained from measurements of porphyrin fluorescence with radioactively labelled substances. The latter study was performed to address the problem of porphyrin aggregation. Porphyrin aggregates may be taken up by tumors, but do not fluoresce and may be uncompletely disaggregated in the assay procedures. For our studies we chose a hematoporphyrin derivative (HPD) and its purified form dihematoporphyrin ether (DHE) termed Photofrin II (P II). These drugs have been characterized by other investigators [3.4]. 2. Materials and methods 2.1. Photosmsitixrs HPD was obtained from The Queen Elizabeth Pharmacy Department, Woodville Hospital, South, Australia, as an aqueous solution at a concentration of 5.0 mgiml. ‘“C-labelled HPD and lJC-labelled DHE were supplied from the University of Leeds, Industrial Service Ltd., as a freeze-dried powder. In both cases the drugs were dissolved in isotonic saline at a concentration of 5.0 mgiml containing 1.0 PCilabelled substance per ml. The porphyrins, both the labelled and the unlabelled were prepared with the same methods as earlier described in the work of Lipson et al. [4] The labelled carbon atoms were all in the ring of the porphyrin and not in the side chains. 2.2. Experimental protocol Female Wistar rats (72) were used in the present
study (mean body weight 236 g). In the case of labelled drugs. 40 animals were divided into 4 groups (10 for each group) and subjected to either intravesical instillation or intravenous injection of ‘3C-labelled HPD or 14C-P II following bilateral ligation of the ureters under general anesthesia with intraperitoneal mebumal. They were sacrilited 1 h later. In another group (also 10 rats), the animals were killed 48 h after an intravenous injection of 14C-labelled HPD without previous ureteral ligation. The instilled volume was 0.4 ml and the concentrations of 14C-labelled HPD and P II in the instilled solutions were 5.0 mgiml. The instillate was kept in the bladder for 1 h. The same volume was used for intravenous injection, corresponding to approximately 10 mg drug/kg body weight. In addition, 5 animals were used as controls with only intravesical administration of 0.4 ml saline solution. Blood samples were drawn from the aorta. The instillate was recovered by aspiration from the exposed bladder. Thereafter, the bladder was incised, carefully washed in saline and dried on filter paper. The lungs, heart. liver, spleen, kidneys, ovaries, oral mucosa and samples from abdominal muscle were removed and stored at -70°C together with the bladder tissue until examined. The organs were then thawed. allowed to dry at room temperature for 24 h and combusted in a Tri-Carb sample oxidizer. The radioactive material was collected in 21 ml CarboSorbiPermafluor (8:13 v/v) and analyzed in a liquid scintillation counter. In the experiments with unlabelled HPD, the animals were divided into 3 groups (5 for each) and sacrificed 1 h after HPD had been given intravenously, intravesically or intravesically plus 10% surface detergent (polysorbate 80), respectively. The concentrations of HPD in different tissues were then assessed with spectrofluorometry as previously described [6.18]. Two animals served as controls receiving only 0.4 ml saline solution intravesically. 3. Results 3.1. Intrawnous injection The distribution of HPD and P II in tissues after intravenous injection is shown in Fig. la-c. The pattern of uptake after 1 h was similar to that after
T. Windahl et al. /Cancer
Lett. 75 (1993) 65-70
Muscle
,og/g
Heart
67
Oral mucosa
Kidney
Ovary
Spleen
Ovary
Spleen
Liver
wet ttssue
. -90 80 70 @I 50 40 30 20 10 0
+ Muscle
Kidney
Heart
Bladder
Lung
Liver
mucosa fl9/g 90 80 70
60
tissue
1
n
14C-HpD (lhj
q
lJC-PII (Ihj
T
’
r
T
f
llB lkk T
l-
r
Muscle
Fig. 1. (a-c)
Heart
Distribution of hematoporphyrin ing and spectrofluorometric
Oral mucosa
Kidney
Bladder
Ovary
Spieen
LWT
Liver
derivative (HPD) and dihematoporphyrin ether (P II) determined by radioactive measurements after intravenous injection of the photosensitizers.
count.
68
n
Fluoresence-HpD
1
14C-HpD “x-Pll
Muscle Fig. 2. Distribution
ofthe
Heart
photosensitizers
Kidney
Bladder x 10
Ovary
HPD and P II determined by radioactive I h after lntraceslcal instillation.
Spleen countmg
Lung
Liver
and ~pcctrotluorometrlc
measurements
48 h for both substances.
A slight decrease in absolute values occurred between 1 and 48 h in muscle, heart and lung, and some increase of the values in oral mucosa, kidneys, spleen and liver. The values obtained with the radioactivity method were between twice (muscle) and six times (ovary) as high as those obtained with the fluorescence method.
ations with intraperitoneal injection in mice [1,7,18,19]. Our fluorescence values for HPD were in good agreement with the findings of other investigators 3 h after intravenous injection of 10 mg P II/kg body weight in mice [ 171. In the liver, for instance, the respective values were 25 and 27 pg/g, in the spleen I1 and 12 pgig and in the kidney 8 and 10
3.2. htravesical instillatkwz Analysis of the tissue distribution of HPD and P II after intravesical instillation of the drugs (Fig. 2) showed a very high uptake by the bladder wall. The fluorescence measurements of HPD agreed with the i4C-HPD values, whereas uptake of P II was significantly lower. In extravesical organs this mode of drug delivery resulted in very low concentrations, close to the detection limit.
flgig. There are some possible reasons for the discrepancy between the fluorescence and radioisotope measurements after intravenous injection. One explanation is that both HPD and P II contain a large fraction of aggregates with a very low fluorescence quantum yield [ 14). Chromatographic analysis has indicated that such aggregates are preferentially taken up by tumors [14.22]. High uptake of aggregated material is to be expected also in other tissues with high endocytic activity, such as liver and spleen. As the aggregates are measured by the radioisotopic, but not the fluorescence method, this selective uptake of aggregated substance may explain why radioactive counting gave the higher values. Complete disaggregation of porphyrins probably does not occur in solutions selected for this purpose in fluorescence measure-
4. Discussion When either hematoporphyrin derivative or its partially purified version, dihematoporphyrin ether was given intravenously, the uptake per gram of tissue decreased in the following order: liver > spleen, ovary, kidney, lung > heart > muscle. This order is in agreement with previous observ-
T. Windahl et al. /Cancer
69
Lett. 75 (1993) 65-70
ments. Action spectroscopy, fluorescence excitation spectroscopy and absorption spectroscopy have shown that the photosensitizing effect of HPD and P II was due to the fluorescencing fraction, the aggregates being virtually inactive [15]. Thus, data obtained from fluorescence may be more relevant to photodynamic therapy than data obtained with radiolabelled substances. When the drugs were given intravesically, there was less uptake of P II than of HPD in the bladder wall. The reason for this is not known. One possible explanation is that P II contains a larger fraction of aggregates than HPD, and the aggregates may penetrate less well than monomers into tissues. The data for other tissues agree with this suggestion. Thus, in contrast to the data for intravenous administration, the uptake values after intravesical instillation were higher using the fluorescence than the radioisotope method. Entry to the circulation is probably easier for the fluorescent monomers than for the aggregates. Our analyses do not permit any quantitative assessment of the photosensitizer in the different layers of the bladder wall. It might also be possible to further increase the uptake of photosensitizers after intravesical instillation by addition of an enhancer such as polysorbate 80 to the instillate as tried in conjunction with intravesical instillation of doxyrubicin [5,9,10]. So far, however, this has not been successful in our experiment with HPD. 5. Conclusions The intravesical mode of delivering the photosensitizers enhanced their uptake within the bladder wall as compared with intravenous administration, while giving very low uptake in various extravesical organs. Our study, however, does not permit conclusions concerning depth of penetration of the photosensitizers into or beneath the urothelium. Further studies are required to assess if intravesical administration of photosensitizers can increase the efficacy of photodynamic therapy in patients with superficial bladder cancer and also reduce the associated phototoxicity. 6. Acknowledgments The study
was supported
by grants
from
the
Swedish Cancer Council.
Foundation
and ijrebro
County
7. References I
Amano, T., Prom, tumour
2
3
4
5
6
7
8
injection
G.R. and Lin, C-W. (1988) Intraas a more effective means of porphyrin
administration for photodynamic therapy. J. Ural., 139. 392-395. Benson, R.C. (1985) Treatment of diffuse transitional cell carcinoma in situ by whole bladder hematoporphyrin derivative photodynamic therapy. J. Urol., 134. 675-678. Dougherty, T.J.. Potter, W.R. and Weishaupt. K.R. (1984) The structure of the active component of hematoporphyrin derivative. In: Porphyrin localization and treatment of tumors. pp. 301-314. Editors: D.R. Doiron and C.J. Comer. Alan R. Lisslnc., New York. Lipson,R.L.,Baldes.E.J.andOlsen.A.M.(1961)Theuse of a derivative of haematoporphyrin in tumor detection. J. Natl. Cancer Inst.. 26, l-8. Eksborg. S., Edsmyr, F. and Naslund. 1. (1982) Intravesical instillation with adriamycin + Tween 80 in patients with superficial bladder tumors. Eur. Ural.. 8, 213-215. Gomer, C.J., Jester, J.V., Razum, N.J., Szirth. B.C. and Murphree A.L. (1985) Photodynamic therapy of intraocular tumors: examination of hematoporphyrin derivative distribution and long-term damage in rabbit ocular tissue. Cancer Res., 45. 3718-3725. Gomer. C.J. and Dougherty, T.J. (1979) Determination of ‘H and 14C hematoporphyrin derivative distribution in malignant and normal tissue. Cancer Res.. 39, 145-151. Harty. J.I., Mohammad. A., Wieman, T.J.. Tseng, M.T.. Ackerman, D. and Broghamer, W. (1989) Complications of whole bladder dihematoporphyrin ether photodynamic therapy. J. Ural.. 141, 1341-1346. Hellsten, S.. Eksborg, S. and Axelsson. B. (1987) Absorption of Adriamycin into the systemic circulation after intravesical instillation with or without Tween 80 in the rat. Ural. Res., IS. 15-21. Hellsten, S.. Axelsson, B.. Eksborg. S. and Lindkvist. C. (1988) Organ distribution of adriamycin after intravesical instillation with or without Tween 80 in the rat. Ural. Res., 16, 325-327. Jocham. D, Beer. M., Baumgartner, R., Staehler. G. and Unsold, E. (1989) Long term experience with integral photodynamic therapy of TIS bladder carcinoma. In: Photosensitizing compounds: their Chemistry. Biology and Clinical Use, pp. 198-208. Editor: Wiley. Chichester (Ciba Foundation symposium 146). Kelly, J.F. and Snell, M.E. (1976) Hematoporphyrin derivative: a possible aid in the diagnosis and therapy of carcinoma of the bladder. J. Ural.. 115. 150. Marcus. S.L. (1990) Photodynamic therapy of human cancer: clinical status, potential and needs. In: Future directions and applications in photodynamic therapy. SPIE Institutes for Advanced Optical Technologies. pp. 3-56, vol. 156. Editor: C.J. Gomer. Washington: SPIE Optical Engineering Press.
70 14
15
16
17
18
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