Journal ofPhotochemistry
and Photobiology,
B: Biology, 5 (1990)
519
- 525
519
News and Views
Photosensitizing properties of phorbides BEATE
RdDER
Division of Experimental Biophysics and Spectroscopy, Department Humboldt University Berlin, Invalidenstr. 42, 1040 Berlin (G.D.R.)
of Physics,
The high photosensitizing efficacy of porphyrins has been known for more than 50 years and, in the 194Os, certain porphyrins were demonstrated to be selectively accumulated by tumour tissues. These two properties have been used in fluorescence diagnostics and the photodynamic therapy (PDT) of tumours. The most commonly used sensitizer is a complex mixture of porphyrins, derived from haematoporphyrin, and thus termed haematoporphyrin derivative (HPD, Photofrin I and II, Photodyn) [l]. This mixture is far from ideal. It is difficult to reproduce its composition in different batches, and it has poor tumour selectivity and weak molar absorbance in the red region of the spectrum where tissue penetration is high. Skin sensitization in animals and humans is observed for about 30 days after treatment [ 11. For these reasons, we decided to look more closely at another group of porphyrin-based sensitizers, the porphines. One particular class of porphines - the phorbides - are of interest because of their relatively simple and inexpensive extraction from chlorophylls and their high degree of purity. Phorbides are characterized by intense So + S 1 electronic transitions at wavelengths around 660 nm, where the penetration depth of light into tissues is higher than at 630 nm (used in PDT with HPD). However, like haematoporphyrins, porphyrins have high triplet quantum yields (up to 0.9), whereas the yields of porphines are 0.6 - 0.7. The most frequently studied phorbide is pheophorbide a, and the photophysical parameters of this compound have recently been investigated in ethanol (monomers) and water (aggregates). The fluorescence lifetimes of monomeric and aggregated pheophorbide a [2], the dimerization constant and energy [3], the triplet state lifetimes and quantum yields of both monomers and aggregates [4] and the singlet oxygen quantum yields [3,4] have all been determined. The triplet quantum yield of monomeric pheophorbide a is about 0.75, and its singlet oxygen yield about 0.6. However, when irradiated in water, aggregated pheophorbide a displays very Elsevier
Sequoia/Printed
in The Netherlands
520
NEWS AND VIEWS
poor singlet oxygen generation (i.e. 0.01) [4] and generates high numbers of oxygen radicals [5]. Consequently, although it is probable that monomers only act via the type II photosensitization pathway [4], it cannot be excluded that aggregates also promote type I mechanisms [ 51. In a series of in vitro studies involving both cells [6, 71 and cell membranes [ 81, it has been shown that pheophorbide a sensitizes in vitro tumour cells about 20 times more efficiently than HPD. The in uiuo photodynamic activity of pheophorbide a has been studied in mice with Ehrlich ascites [9] and Lewis lung carcinoma [4]. On topical administration of pheophorbide a 30 min before laser irradiation (670 nm), about 58% of the animals are cured, compared with 30% of the HPDtreated mice. Moreover, pheophorbide a accumulation in rat skin tumour tissue is about ten times higher than in normal tissue [lo]. Pheophorbide a also exhibits powerful photodynamic activity towards psoriatic plaques in humans [ 91. In addition to pheophorbide a, several other phorbides have been found to be powerful photosensitizers. The oral administration of pheophorbide a, pyropheophorbide a and lo-hydroxy-pheophorbide a photosensitizes rats to visible light [ll]. Pheophorbide b has been shown to be much less photoactive in uiuo than pheophorbide a [ 121. Lastly, the in vitro photodynamic activity of a pheophorbide dimer (two molecules of pyropheophorbide cross-linked with arginine) is lower than that of pheophorbide a [13]. On the basis of these observations, the synthesis of a variety of phorbides and phorbide dimers has been started and their photodynamic activity is being tested in both cell cultures and tumour-bearing animals. In conclusion, it appears quite possible that some phorbides, especially pheophorbide a, will be used in PDT in the future, because of their selective accumulation by tumours, low toxicity and high absorbance at wavelengths around 660 nm. A very important parameter for the use of sensitizers in clinical PDT will be the metabolism of photosensitizing drugs in humans. On the one hand, the sensitizer must be retained long enough for the phototreatment of tumour tissues and on the other, it must be released rapidly to minimize side effects. In the best variant, the sensitizer is metabolized by the organism. We believe that this aspect will become decisive in selecting clinically useful sensitizers. It is currently impossible to predict which of the investigated compounds or group of compounds has the best parameters since this problem has not been studied in detail as yet.
1 D. Kessel; HP and HPD - photophysics, photochemistry and phototherapy, Photo&em. Photobiol., 39 (1984) 851 - 859. 2 B. Rtider and H. Wabnitz, Time-resolved fluorescence spectroscopy of hematoporphyrin, mesoporphyrin. pheophorbide a and chlorin e in ethanol and aqueous solution J. Photochem. Photobiol. B, 1 (1987) 476 - 503.
NEWS AND VIEWS
521
3 I. Marlow, F. Marlow and B. Roder, Bestimmung der Aggregationswtime von Phaophorbid a in w&rigem Milieu: 2. Chem., 26 (1986) 338 - 339. 4 A. A. Krasnovsky, Jr., K. V. Neverov, S. Yu. Egorov, B. Roder and T. Lewald, Photophysical studies on pheophorbide a and pheophytin a - phosphorescence and photosensitized singlet oxygen luminescence, J. Photochem. Photobiol. B, 5 (1990) 245 _ 254. 5 R. Haseloff, B. Ebert and B. Roder, Generation of free radicals by photoactivation of pheophorbide a, hematoporphyrin and protoporphyrin, J. Photochem. Photobiol. B, 3 (1989) 593 - 602. 6 S. Takahashi; Laser-excited photochemical effects on biological cells containing pheophorbide a. I. Generation of singlet molecular oxygen and effect on cultured cancer cells, J. Jpn. Sot. Laser Med., 4 (1984) 99 - 104. ‘7 B. Rlider Pheophorbide a - a new sensitizer for PDT, Stud. Biophys., 114 (1986) 183 - 186. 8 M. Kuwabara, T. Yamamoto, 0. Inanami and F. Sato, Mechanism of photosensitization by pheophorbide a studied by photohemolysis of erythrocytes and electron spin resonance spectroscopy, Photochem. Photobiol., 49 (1989) 37 - 42. 9 B. Ridder, G. Wischnewsky, S. Nicklisch, H. Slawaticky and H. Meffert, Pheophorbide a or b in treatment of skin diseases and various tumors, DDR Patent, DD 248282 Al, 61 K 31/395 (8.10.1984); Chem. Abstr., 108 2163423, 1988. 10 N. Maeda, K. Ichikawa, T. Kobayishi and N. Mizuno, Pheophorbide a PDT for chemically induced carcinoma of rat skin, Clin. Res., 33 (1985) A633. 11 H. Endo. H. Hosoya, T. Koyama and M. Ichioka, Isolation of lo-hydroxypheophorbide a as a photosensitizing pigment from alcohol-treated Chlorella cells, Agric. Biol. Chem., 46 (1982) 2183 - 2188. 12 B. A. Tapper, E. Lohery, E. Hove and R. M. Allison, Photosensitivity from chlorophyll-derived pigments, J. Sci. Food Agric., 26 (1975) 277 - 281. 13 K. Iwai and S. Kimura, Efficiency of pheophorbide-dimer in photodynamic therapy of mouse tumor, J. Clin. Biochem. Nutr., 5 (1988) 145 - 149.
Properties for optimal PDT sensitizers J. MOAN Institute for Cancer Research, (Norway)
The Norwegian
Radium
Hospital,
Montebello,
0310 Oslo 3
1. Introduction The momentum of research in the field of photodynamic therapy (PDT) of cancer is now so large and the results so promising that this mode of treatment will most probably make its way into cancer hospitals world wide. To a large extent the progress of PDT will depend on the development of sensitizers with optimal properties. Since “cancer” is a large family of diseases with widely different clinical patterns, it is unlikely that a single sensitizer can serve all purposes. For instance, in some cases a highly selective uptake of the sensitizer in the tumour is required, whereas in other cases the photosensitizing efficiency of the sensitizer may be the crucial factor. In the treatment of tumours with a thickness larger than 0.5 - 0.7 cm,
and Photobiology,
B: Biology, 5 (1990)
519
- 525
519
News and Views
Photosensitizing properties of phorbides BEATE
RdDER
Division of Experimental Biophysics and Spectroscopy, Department Humboldt University Berlin, Invalidenstr. 42, 1040 Berlin (G.D.R.)
of Physics,
The high photosensitizing efficacy of porphyrins has been known for more than 50 years and, in the 194Os, certain porphyrins were demonstrated to be selectively accumulated by tumour tissues. These two properties have been used in fluorescence diagnostics and the photodynamic therapy (PDT) of tumours. The most commonly used sensitizer is a complex mixture of porphyrins, derived from haematoporphyrin, and thus termed haematoporphyrin derivative (HPD, Photofrin I and II, Photodyn) [l]. This mixture is far from ideal. It is difficult to reproduce its composition in different batches, and it has poor tumour selectivity and weak molar absorbance in the red region of the spectrum where tissue penetration is high. Skin sensitization in animals and humans is observed for about 30 days after treatment [ 11. For these reasons, we decided to look more closely at another group of porphyrin-based sensitizers, the porphines. One particular class of porphines - the phorbides - are of interest because of their relatively simple and inexpensive extraction from chlorophylls and their high degree of purity. Phorbides are characterized by intense So + S 1 electronic transitions at wavelengths around 660 nm, where the penetration depth of light into tissues is higher than at 630 nm (used in PDT with HPD). However, like haematoporphyrins, porphyrins have high triplet quantum yields (up to 0.9), whereas the yields of porphines are 0.6 - 0.7. The most frequently studied phorbide is pheophorbide a, and the photophysical parameters of this compound have recently been investigated in ethanol (monomers) and water (aggregates). The fluorescence lifetimes of monomeric and aggregated pheophorbide a [2], the dimerization constant and energy [3], the triplet state lifetimes and quantum yields of both monomers and aggregates [4] and the singlet oxygen quantum yields [3,4] have all been determined. The triplet quantum yield of monomeric pheophorbide a is about 0.75, and its singlet oxygen yield about 0.6. However, when irradiated in water, aggregated pheophorbide a displays very Elsevier
Sequoia/Printed
in The Netherlands
520
NEWS AND VIEWS
poor singlet oxygen generation (i.e. 0.01) [4] and generates high numbers of oxygen radicals [5]. Consequently, although it is probable that monomers only act via the type II photosensitization pathway [4], it cannot be excluded that aggregates also promote type I mechanisms [ 51. In a series of in vitro studies involving both cells [6, 71 and cell membranes [ 81, it has been shown that pheophorbide a sensitizes in vitro tumour cells about 20 times more efficiently than HPD. The in uiuo photodynamic activity of pheophorbide a has been studied in mice with Ehrlich ascites [9] and Lewis lung carcinoma [4]. On topical administration of pheophorbide a 30 min before laser irradiation (670 nm), about 58% of the animals are cured, compared with 30% of the HPDtreated mice. Moreover, pheophorbide a accumulation in rat skin tumour tissue is about ten times higher than in normal tissue [lo]. Pheophorbide a also exhibits powerful photodynamic activity towards psoriatic plaques in humans [ 91. In addition to pheophorbide a, several other phorbides have been found to be powerful photosensitizers. The oral administration of pheophorbide a, pyropheophorbide a and lo-hydroxy-pheophorbide a photosensitizes rats to visible light [ll]. Pheophorbide b has been shown to be much less photoactive in uiuo than pheophorbide a [ 121. Lastly, the in vitro photodynamic activity of a pheophorbide dimer (two molecules of pyropheophorbide cross-linked with arginine) is lower than that of pheophorbide a [13]. On the basis of these observations, the synthesis of a variety of phorbides and phorbide dimers has been started and their photodynamic activity is being tested in both cell cultures and tumour-bearing animals. In conclusion, it appears quite possible that some phorbides, especially pheophorbide a, will be used in PDT in the future, because of their selective accumulation by tumours, low toxicity and high absorbance at wavelengths around 660 nm. A very important parameter for the use of sensitizers in clinical PDT will be the metabolism of photosensitizing drugs in humans. On the one hand, the sensitizer must be retained long enough for the phototreatment of tumour tissues and on the other, it must be released rapidly to minimize side effects. In the best variant, the sensitizer is metabolized by the organism. We believe that this aspect will become decisive in selecting clinically useful sensitizers. It is currently impossible to predict which of the investigated compounds or group of compounds has the best parameters since this problem has not been studied in detail as yet.
1 D. Kessel; HP and HPD - photophysics, photochemistry and phototherapy, Photo&em. Photobiol., 39 (1984) 851 - 859. 2 B. Rtider and H. Wabnitz, Time-resolved fluorescence spectroscopy of hematoporphyrin, mesoporphyrin. pheophorbide a and chlorin e in ethanol and aqueous solution J. Photochem. Photobiol. B, 1 (1987) 476 - 503.
NEWS AND VIEWS
521
3 I. Marlow, F. Marlow and B. Roder, Bestimmung der Aggregationswtime von Phaophorbid a in w&rigem Milieu: 2. Chem., 26 (1986) 338 - 339. 4 A. A. Krasnovsky, Jr., K. V. Neverov, S. Yu. Egorov, B. Roder and T. Lewald, Photophysical studies on pheophorbide a and pheophytin a - phosphorescence and photosensitized singlet oxygen luminescence, J. Photochem. Photobiol. B, 5 (1990) 245 _ 254. 5 R. Haseloff, B. Ebert and B. Roder, Generation of free radicals by photoactivation of pheophorbide a, hematoporphyrin and protoporphyrin, J. Photochem. Photobiol. B, 3 (1989) 593 - 602. 6 S. Takahashi; Laser-excited photochemical effects on biological cells containing pheophorbide a. I. Generation of singlet molecular oxygen and effect on cultured cancer cells, J. Jpn. Sot. Laser Med., 4 (1984) 99 - 104. ‘7 B. Rlider Pheophorbide a - a new sensitizer for PDT, Stud. Biophys., 114 (1986) 183 - 186. 8 M. Kuwabara, T. Yamamoto, 0. Inanami and F. Sato, Mechanism of photosensitization by pheophorbide a studied by photohemolysis of erythrocytes and electron spin resonance spectroscopy, Photochem. Photobiol., 49 (1989) 37 - 42. 9 B. Ridder, G. Wischnewsky, S. Nicklisch, H. Slawaticky and H. Meffert, Pheophorbide a or b in treatment of skin diseases and various tumors, DDR Patent, DD 248282 Al, 61 K 31/395 (8.10.1984); Chem. Abstr., 108 2163423, 1988. 10 N. Maeda, K. Ichikawa, T. Kobayishi and N. Mizuno, Pheophorbide a PDT for chemically induced carcinoma of rat skin, Clin. Res., 33 (1985) A633. 11 H. Endo. H. Hosoya, T. Koyama and M. Ichioka, Isolation of lo-hydroxypheophorbide a as a photosensitizing pigment from alcohol-treated Chlorella cells, Agric. Biol. Chem., 46 (1982) 2183 - 2188. 12 B. A. Tapper, E. Lohery, E. Hove and R. M. Allison, Photosensitivity from chlorophyll-derived pigments, J. Sci. Food Agric., 26 (1975) 277 - 281. 13 K. Iwai and S. Kimura, Efficiency of pheophorbide-dimer in photodynamic therapy of mouse tumor, J. Clin. Biochem. Nutr., 5 (1988) 145 - 149.
Properties for optimal PDT sensitizers J. MOAN Institute for Cancer Research, (Norway)
The Norwegian
Radium
Hospital,
Montebello,
0310 Oslo 3
1. Introduction The momentum of research in the field of photodynamic therapy (PDT) of cancer is now so large and the results so promising that this mode of treatment will most probably make its way into cancer hospitals world wide. To a large extent the progress of PDT will depend on the development of sensitizers with optimal properties. Since “cancer” is a large family of diseases with widely different clinical patterns, it is unlikely that a single sensitizer can serve all purposes. For instance, in some cases a highly selective uptake of the sensitizer in the tumour is required, whereas in other cases the photosensitizing efficiency of the sensitizer may be the crucial factor. In the treatment of tumours with a thickness larger than 0.5 - 0.7 cm,
