Photochemistry and Photobiology Vol. 52, No. 2, pp. 439-443, 1990 Printed in Great Britain. All rights reserved
003 1-8655/YO $03 .00+0.00 Copyright 0 1990 Pergamon Press plc
REVIEW ARTICLE
PHOTOSENSITIZED PROCESSES in vivo: PROPOSED PHOTOTHERAPEUTIC 'APPLICATIONS* GIULIO JORI Department of Biology, University of Padova, Via Loredan 10, 35131 Padova, Italy (Received 17 August 1989; accepted 18 October 1989)
Abstract-The use of photosensitizing dyes having intense absorption bands in the 600-900 nm spectral interval opens up new prospects in the field of photochemotherapy, because it allows the illumination of relatively large tissue volumes with no significant damage to photosensitizer-free tissues. Special interest is currently focused on the photodynamic therapy of solid tumors, because of the property of several dyes with a macrocyclic chemical structure (porphyrins, chlorins, phthalocyanines, xanthenes) to accumulate in significant amounts and be retained for prolonged periods of time by neoplastic lesions. Strategies are being developed for enhancing the selectivity of tumor targeting by photosensitizers through the exploitation of functional or biochemical differences between normal and malignant cells.
absorbing dyes can biological systems be damaged by such wavelengths. At the same time, the decreased importance of light-scattering processes at longer wavelengths allows deep penetration of red light into tissues (Eichler et al., 1977).
INTRODUCTION
Although the use of light as a tool in the treatment of human diseases was proposed in ancient times (see Spikes, 1985), until very recently the development of phototherapeutic modalities was limited by the lack of sufficient information on the optical properties of biological tissues, as well as by the belief that the penetration power of IJV and visible light into tissues was extremely poor. Nowadays, the availability of sophisticated electrooptical techniques and the advance of knowledge on photophysical and photochemical processes occurring in microheterogeneous systems have drastically changed the overall picture. A variety of phototherapeutic and photochemotherapeutic approaches has been elaborated, some of which have currently reached the stage of widespread clinical application (Regan and Parrish, 1982; Hasan, 1988). While phototherapy takes advantage of the light absorption by endogenous constituents of cells and tissues (e.g. bilirubin in the phototherapy of neonatal jaundice), photochemotherapy involves the administration of a photosensitizing drug which is accumulated in significant amounts by the diseased areas; the latter are then illuminated by wavelengths specifically absorbed by the photosensitizer. In the last few years, noticeable progress in the field of photochemotherapy has been made through the introduction of photosensitizers with absorption bands in the 600-900 nm interval. The molecules normally present in animal tissues, with the exception of melanin, exhibit no appreciable absorbance in this spectral region (Spikes and Jori, 1987): consequently, only upon administration of red-light
RED-LIGHT ABSORBING PHOTOSENSITIZERS
Table 1 lists some photosensitizers which have been used in vivo and which display significant absorption above 600 nm. They are all characterized by a polycyclic chemical structure; the extensively delocalized electron cloud lowers the energy gap between the ground state and the first excited singlet state, so that electronic excitation may also be achieved by irradiation with low-energy light wavelengths. In general, the position and intensity of the absorption bands is slightly affected by the nature of the peripheral substituents protruding from the macrocycle. On the other hand, hypochromicity and broadening of the absorption bands is often induced by aggregation of the dyes, as usually occurs in aqueous media (Reddi and Jori, 1988). In most cases, aggregation also shortens the lifetime of the lowest excited singlet and triplet states, thus depressing their photosensitizing efficiency. Consequently, the extent of monomerization of photosensitizers in vivo may represent a critical factor for the success of a given phototherapeutic application. In general, the factors hindering the aggregation of macrocyclic dyes may be summarized as follows: (i) the presence of charged substituents (e.g. carboxylate or sulfonate groups) destabilizes the dimeric species by electrostatic repulsion: the octacarboxylic porphyrin uroporphyrin is essentially monomeric in aqueous solutions at 10 p M concentration, while the corresponding dicarboxylic porphyrin protoporphyrin is extensively aggregated even at
*Presented at the Interdisciplinary Meeting on Luminescence: Fundamentals and Applications, 29/30 May 1989, Bologna, Italy. 439
GIULIO JORI
440
Table 1. Spectroscopic properties of photosensitizers used in phototherapeutic treatments with red light (Hasan, 1988; Neckers, 1988) Photosensitizer class
Porphyrins Porphycencs Chlorins Purpurins Phthalocyanincs Naphthalocyanines Chalcogenopyrilium dyes ~
Specific example
Hematoporphyrin derivative Tetra-n-propylporphycene Monoaspartylchlorin e6 Sn( 1V)-ctiopurpurin Zn(I1)-phthalocyanine with 0 - 4 S03H groups Si(1V)-naphthalocyanine
Absorption range in the red (nm)
Extinction coefficient* at max (M-I cm-')
620-640 610-650 670-700 670-7 10 670-720
3500 50 OOO 120 000 42 000 150 OOO
780-810 780-820
180 000 150 000-300 OOO
~~
~~
*Values reported Are typical for each photosensitizer class, and were determined on a variety of media.
1 pM (Jori and Spikes, 1984); (ii) the presence of ligands perpendicular to the flat aromatic molecule of metal-dye complexes can sterically inhibit aggregation, e.g. Al(II1) phthalocyanine with a chloride ion coordinated to the metal is largely monomeric in aqueous solution, while Zn(I1) or Mg(I1) derivatives have a great tendency to undergo aggregation (Spikes, 1986); (iii) the binding of the dyes to macromolecular structures such as proteins or to low-polarity systems, including micelles, oil emulsions and liposomes, overcomes the hydrophobic intramolecular interactions which are mainly responsible for dye aggregation: thus, although 10 IJ.Mhematoporphyrin is less than 50% monomeric in aqueous solution, it is completely monomerized upon complexation with serum albumin or incorporation into phospholipid vesicles (Jori and Spikes, 1984).
Other desirable properties of photosensitizers to be used for therapeutic purposes are: a high degree of chemical purity [as compared with the heterogeneous nature of most hematoporphyrin derivatives (HpD)*]; photostability to red-light irradiation, in order to avoid the generation of by-products having unknown chemical structure and biological effects; and low systemic toxicity at therapeutically useful doses. In the case of hematoporphyrin, both acute and chronic toxicity are negligible in the dose range 1-10 mg/kg body weight, which is used in most clinical applications (Dougherty, 1987). It is likely that similar conclusions obtain for other photosensitizers, such as chlorins or phthalocyanines, which are usually administered at doses smaller than those used with porphyrins, because of the greater extinction coefficient in the red spectral region. *Abbreviations: HpD, hematoporphyrin derivative; LDL, low-density lipoproteins; PDT, photodynamic therapy.
USE OF PHOTOSENSITIZED PROCESSES IN TUMOR THERAPY
Until very recently, most phototherapeutic applications with red-light absorbing photosensitizers involved the use of porphyrins, mainly because porphyrin-sensitized photoprocesses have been extensively studied (Spikes, 1975) and some naturally occurring porphyrins have been found to be accumulated by tumors (Auler and Banzer, 1942) and by psoriatic areas (Silver, 1937). Porphyrins are still widely used in photochemotherapy , although several second-generation photosensitizers with better spectroscopic properties in the 6OCL900 nm range are being investigated (Table 1). Some diseases for which phototherapy with the abovementioned photosensitizers has been at least proposed at an experimental level are shown in Table 2 (see Spikes and Jori, 1987, and references therein). Certainly, the most widespread clinical applications of these phototherapeutic techniques have been in the field of solid tumors, owing to the property of specific porphyrins (especially some chemical HpD or known under the commercial names of Photofrin I and 11) to be accumulated in significant amounts and retained for prolonged periods of time by a variety of neoplastic lesions (Dougherty, 1987). The treatment is generally defined as photodynamic therapy (PDT), because photodamage of tumor tissues only occurs in the presence of oxygen. A typical PDT protocol is summarized in Scheme 1. The intravenously injected HpD (or Photofrin 11) reaches maximal tumor concentrations (of the order of a few pg of porphyrin per gram of tissue) within about 3 h; however, irradiation of the tumor area is most frequently performed 48-72 h after administration of the drug. At this time the ratio of photosensitizer concentration between tumor and peritumoral tissues is particularly large. Notable exceptions are the components of the reticuloendo-
44 1
Review Article Table 2. Selected diseases for which treatment by PDT is presently under investigation (Regan and Parrish, 1982; Spikes and Jori. 1987) Present state of development of phototherapeutic treatment Investigations are at phase 11-111 clinical level in several countries A limited number of clinical trials has been carried out at: Dermatological Clinic, Humboldt University, East Berlin, and University of California at Irvine Accumulation of fluorescent photosensitizers by atheromatous plaques in experimental animals may be used for early diagnosis and control of plaque development Several bacteria, mycoplasma, yeasts and viruses are photoinactivated by exposure to photosensitizers and red light. PDT may be used for sterilization of surgical beds
Disease Solid tumors Psoriasis Atheromas Viral and microbial infections
INJECTION
OF
(Intravenously, PHOTOSENSITIZER ELIMINATION
1 3 - 72 h
NORMAL TISSUES
1 ca. 4 SKIN
1
PHOTOSENSITIZER - 5 mo/kg)
1
1 - 3 h
ACCUMULATION OF
c c
THE
1 SERUM
TISSUES
PHOTOSENSITIZER
frwn ~~
weeks
8Y
2
-
4 weeks
~~~
3 h - 1 week! TUMOR
TISSUES
1 40 - 72
h
IRRADIATION OF TUMOR AREA (50 - 800 J/an2)
4
24 h after
PO7
APPEARANCE OF TUMOR RESPONSE TO PDT
Scheme 1 . Typical protocol for the P D T of tumors.
thelial system (including liver and spleen) and skin, which may retain detectable amounts of porphyrins for several weeks (Kessel, 1984). As a consequence, the onset of general skin photosensitivity is often an undesired side-effect of PDT (Dougherty, 1987). The total dose of light delivered to the neoplastic areas ranges between about 50 and 800 J/cm2, depending on the size and optical properties of the tumor. In all cases, the exposure rate must be carefully controlled and possibly not exceed 200 mW/cm2,in order to reduce the effects of hyperthermia which could overlap with photochemical effects (Svaasand, 1984). The response of the tumor to PDT usually appears as massive hemorrhagic tissue necrosis, reflecting vascular damage as the predominant primary effect of tumor photosensitization (Zhou, 1989). This is in agreement with the observed localization of a significant fraction of tumor-associated HpD in the vascular stroma (Dougherty, 1987). Photodynamic therapy has been
applied so far to a few thousand patients all over the world, most of whom have objectively benefited from the treatment. Besides the eradication of tumors of relatively small size, PDT may be used for palliative purposes and for sterilization of the surgical bed after resection of the tumoral mass (Dougherty, 1984). All these forms of PDT have been applied in Italy, during a multi-center clinical trial which lasted 3 yr and involved 88 patients (Corti et al., 1985; Perria et a l . , 1988). The results are currently being evaluated by the Italian Ministry of Health for possible official approval of PDT as a new therapeutic modality for tumors. The outcome of PDT was successful in a very large percentage of treated cases, although the patients selected had almost always been previously exposed to radiotherapy and/or chemotherapy, with negative results. Interestingly, a few patients with multiple neoplastic lesions underwent repeated PDT sessions at about
GIULIOJORI
442
2-month intervals. Thus, these findings indicate that the previous use of other therapeutic regimens does not interfere with the efficacy of PDT on tumors; moreover, the repeatability of photodynamic treatment emphasizes the safety of PDT for normal tissues. NEW DIRECTIONS IN THE PHOTODYNAMIC THERAPY
(Oseroff et al., 1986) that different endotissular compartments of tumors may be labeled by selected photosensitizers through the concerted use of appropriate delivery systems. Acknowledgement-This work was supported by the Consiglio Nazionale delle Ricerche (Italy), under the special project “Oncologia”, contract number.
OF TUMORS
At present, the main limitations of PDT are poor efficiency of HpD photoactivation by red light and the relatively limited selectivity of tumor targeting by photosensitizing dyes. While we hope that the former problem will be solved by replacing HpD with photosensitizers strongly absorbing at 700-800 nm (Neckers, 1988), the latter aspect has not been sufficiently addressed until now, partly because of incomplete elucidation of the factors controlling the accumulation in and release of photosensitizers from tumor tissues in v i v o . Some approaches have been proposed to enhance the specificity of tumor loading by photosensitizers. All of them exploit some differences existing between malignant and normal cells: ( 9 photosensitizers may be covalently coupled to monoclonal antibodies directed towards antigens which are specifically present on the surface of the malignant tumor cells (Mew et al., 1983); (ii) the mitochondria of various malignant and benign tumor cells accumulate larger amounts of certain lipophilic cationic dyes, such as rhodamines and other xanthenes, as compared with the mitochondria of normal cells. The process is driven by the internally negative membrane potential of this organelle, which allows penetration of the positive photosensitizer into the mitochondrion, in accordance with the Nernst equation (Beckman et al., 1987). The actual potential of this approach, although promising, cannot be defined until the photosensitization mechanism of these dyes (which may not be of photodynamic type) has been elucidated; (iii) several types of hyperproliferating cells, including tumor cells, express a particularly large number of membrane receptors for lowdensity lipoproteins (LDL). The protein-receptor complex undergoes endocytosis via the formation of coated pits (Goldstein et al., 1979). Thus, hydrophobic photosensitizers may be incorporated into the lipid moiety of LDL and released inside the malignant cells at the level of the lysosomes (Barel et al., 1986). The potential of these novel procedures of photosensitizer delivery to increase the efficacy of PDT must still be defined. It has been proposed
REFERENCES
Auler, H. and G. Banzer (1942) Untersuchungen iiber die rolle der porphyrine bei geschwulstkranken menschen und tieren. Z. Krebsforsch. 53, 65-73. Barel, A,, G. Jori, A. Perin, P. Romandini, A. Pagnan and S. Biffanti (1986) Role of high-, low- and very lowdensity lipoproteins in the transport and tumour delivery of hematoporphyrin in vivo. Cancer Lett. 32, 145-150. Beckman, W. C., S. K. Powers, J. T. Brown, G. Y. Gillespie, D . D. Bigner and J. L. Camps (1987) Differential retention of rhodamine-123 by avian sarcoma virus-induced glioma and normal brain tissue of the rat in vivo. Cancer 59, 266-270. Corti, L., L. Tomio, F. Calzavara, G. Mandoliti, P. L. Zorat and T. Tsanov (1985) Evaluation of hematoporphyrin photodynamic therapy to treat malignant tumours. In Photodynamic Therapy of Tumours and Other Diseases (Edited by G . Jori and C. A. Perria), pp. 317-320. Ed. Libreria Progetto, Padova. Dougherty, T. J. (1984) Photodynamic therapy (PDT) of malignant tumours. CRC Crit. Rev. Oncol. Hematol. 2, 83-116. Dougherty, T. J. (1987) Photosensitizers: therapy and detection of malignant tumours. Photochem. Photobiol. 45, 879-889. Eichler, J., J. Knof and H. Henz (1977) Measurements of the depth of penetration of light in tissue. Rad. Environ. Biophys. 14, 239-245. Goldstein, J. L., R. G. W. Anderson and M: S. Brown (1979) Coated pits, coated vesicles and receptormediated endocytosis. Nature 279, 679-685. Hasan, T.(Ed.) (1988) Advances in Photochemotherapy, SPIE-International Society of Optical Engineers, Bellingham, WA. Jori, G. and J. D. Spikes (1984) Photobiochemistry of porphyrins. In Topics in Photomedicine (Edited by K. C. Smith), pp. 183-318. Plenum Press, New York. Kessel, D. (1984) Hematoporphyrin and HpD: photophysics, photochemistry and phototherapy. Photochem. Photobiol. 39, 851-859. Mew, D.,C. K. Wat, G. H . N. Towers and J. G. Levy (1983) Photoimmunotherapy: treatment of animal tumours with tumor-specific monoclonal antibodieshematoporphyrin conjugates. J. Zmmunol. 130, 1473-1477. Neckers, D. C. (Ed.) (1988) New Directions in Photodynamic Therapy. Proc. SPIE 847, Bellingham, WA. Oseroff, A. R., D. Ohuoha, T . Hasan, J. C . Bommer and M. L. Yarmush (1986) Antibody-targeted photolysis: selective photodestruction of human T-cell leukemia cell using monoclonal antibody-chlorin e6 conjugates. Proc. Nail. Acad. Sci. USA 83, 8744-8748. Perria, C., M. Carai, A. Falzoi, G. Orunesu, A. Rocca, G. Massarelli and G. Jori (1988) Photodynamic therapy of malignant brain tumours: clinical results of, difficulties with, questions about, and future prospects for the neurosurgical applications. Neurosurgery 23, 557-563. Reddi, E. and G. Jori (1988) Steady-state and timeresolved spectroscopic studies of photodynamic sensitizers: porphyrins and phthalocyanines. Rev. Chem. Interm.-tO, 241-268.
Review Article Regan, J. D. and J. A. Parrish (Eds) (1982) The Science of Photomedicine. Plenum Press, New York. Silver, H. (1937) Psoriasis vulgaris treated with hematoporphyrin. Arch. Dermatol. Syphilol. 36, 1118-1119. Spikes, J. D. (1975) Porphyrins and related compounds as photodynamic sensitizers. Ann. N . Y. Acad. Sci. 244, 495-508. Spikes, J. D. (1985) The historical development of ideas on applications of photosensitized reactions in the health sciences. In Primary Photo-processes in Biology and Medicine (Edited by R. V. Bensasson, G. Jori, E. J. Land and T. G. Truscott), pp. 209-227. Plenum Press, New York. Spikes, J. D. (1986) Phthalocyanines as photosensitizers
443
in biological systems and for the photodynamic therapy of tumours. Photochem. Photobiol. 43, 691-699. Spikes, J. D. and G. Jori (1987) Photodynamic therapy of tumours and other diseases using porphyrins. Lasers Med. Sci. 2, 3-15. Svaasand, L. 0. (1984) Thermal and optical dosimetry for photoradiation therapy of malignant tumours. In Porphyrins in Tumour Phototherapy (Edited by A. Andreoni and R. Cubeddu), pp. 261-279. Plenum Press, New York. Zhou, C. (1989) Mechanisms of tumor necrosis induced by photodynamic therapy. J. Photochem. Photobiol. B: Biol. 3, 299-318.
003 1-8655/YO $03 .00+0.00 Copyright 0 1990 Pergamon Press plc
REVIEW ARTICLE
PHOTOSENSITIZED PROCESSES in vivo: PROPOSED PHOTOTHERAPEUTIC 'APPLICATIONS* GIULIO JORI Department of Biology, University of Padova, Via Loredan 10, 35131 Padova, Italy (Received 17 August 1989; accepted 18 October 1989)
Abstract-The use of photosensitizing dyes having intense absorption bands in the 600-900 nm spectral interval opens up new prospects in the field of photochemotherapy, because it allows the illumination of relatively large tissue volumes with no significant damage to photosensitizer-free tissues. Special interest is currently focused on the photodynamic therapy of solid tumors, because of the property of several dyes with a macrocyclic chemical structure (porphyrins, chlorins, phthalocyanines, xanthenes) to accumulate in significant amounts and be retained for prolonged periods of time by neoplastic lesions. Strategies are being developed for enhancing the selectivity of tumor targeting by photosensitizers through the exploitation of functional or biochemical differences between normal and malignant cells.
absorbing dyes can biological systems be damaged by such wavelengths. At the same time, the decreased importance of light-scattering processes at longer wavelengths allows deep penetration of red light into tissues (Eichler et al., 1977).
INTRODUCTION
Although the use of light as a tool in the treatment of human diseases was proposed in ancient times (see Spikes, 1985), until very recently the development of phototherapeutic modalities was limited by the lack of sufficient information on the optical properties of biological tissues, as well as by the belief that the penetration power of IJV and visible light into tissues was extremely poor. Nowadays, the availability of sophisticated electrooptical techniques and the advance of knowledge on photophysical and photochemical processes occurring in microheterogeneous systems have drastically changed the overall picture. A variety of phototherapeutic and photochemotherapeutic approaches has been elaborated, some of which have currently reached the stage of widespread clinical application (Regan and Parrish, 1982; Hasan, 1988). While phototherapy takes advantage of the light absorption by endogenous constituents of cells and tissues (e.g. bilirubin in the phototherapy of neonatal jaundice), photochemotherapy involves the administration of a photosensitizing drug which is accumulated in significant amounts by the diseased areas; the latter are then illuminated by wavelengths specifically absorbed by the photosensitizer. In the last few years, noticeable progress in the field of photochemotherapy has been made through the introduction of photosensitizers with absorption bands in the 600-900 nm interval. The molecules normally present in animal tissues, with the exception of melanin, exhibit no appreciable absorbance in this spectral region (Spikes and Jori, 1987): consequently, only upon administration of red-light
RED-LIGHT ABSORBING PHOTOSENSITIZERS
Table 1 lists some photosensitizers which have been used in vivo and which display significant absorption above 600 nm. They are all characterized by a polycyclic chemical structure; the extensively delocalized electron cloud lowers the energy gap between the ground state and the first excited singlet state, so that electronic excitation may also be achieved by irradiation with low-energy light wavelengths. In general, the position and intensity of the absorption bands is slightly affected by the nature of the peripheral substituents protruding from the macrocycle. On the other hand, hypochromicity and broadening of the absorption bands is often induced by aggregation of the dyes, as usually occurs in aqueous media (Reddi and Jori, 1988). In most cases, aggregation also shortens the lifetime of the lowest excited singlet and triplet states, thus depressing their photosensitizing efficiency. Consequently, the extent of monomerization of photosensitizers in vivo may represent a critical factor for the success of a given phototherapeutic application. In general, the factors hindering the aggregation of macrocyclic dyes may be summarized as follows: (i) the presence of charged substituents (e.g. carboxylate or sulfonate groups) destabilizes the dimeric species by electrostatic repulsion: the octacarboxylic porphyrin uroporphyrin is essentially monomeric in aqueous solutions at 10 p M concentration, while the corresponding dicarboxylic porphyrin protoporphyrin is extensively aggregated even at
*Presented at the Interdisciplinary Meeting on Luminescence: Fundamentals and Applications, 29/30 May 1989, Bologna, Italy. 439
GIULIO JORI
440
Table 1. Spectroscopic properties of photosensitizers used in phototherapeutic treatments with red light (Hasan, 1988; Neckers, 1988) Photosensitizer class
Porphyrins Porphycencs Chlorins Purpurins Phthalocyanincs Naphthalocyanines Chalcogenopyrilium dyes ~
Specific example
Hematoporphyrin derivative Tetra-n-propylporphycene Monoaspartylchlorin e6 Sn( 1V)-ctiopurpurin Zn(I1)-phthalocyanine with 0 - 4 S03H groups Si(1V)-naphthalocyanine
Absorption range in the red (nm)
Extinction coefficient* at max (M-I cm-')
620-640 610-650 670-700 670-7 10 670-720
3500 50 OOO 120 000 42 000 150 OOO
780-810 780-820
180 000 150 000-300 OOO
~~
~~
*Values reported Are typical for each photosensitizer class, and were determined on a variety of media.
1 pM (Jori and Spikes, 1984); (ii) the presence of ligands perpendicular to the flat aromatic molecule of metal-dye complexes can sterically inhibit aggregation, e.g. Al(II1) phthalocyanine with a chloride ion coordinated to the metal is largely monomeric in aqueous solution, while Zn(I1) or Mg(I1) derivatives have a great tendency to undergo aggregation (Spikes, 1986); (iii) the binding of the dyes to macromolecular structures such as proteins or to low-polarity systems, including micelles, oil emulsions and liposomes, overcomes the hydrophobic intramolecular interactions which are mainly responsible for dye aggregation: thus, although 10 IJ.Mhematoporphyrin is less than 50% monomeric in aqueous solution, it is completely monomerized upon complexation with serum albumin or incorporation into phospholipid vesicles (Jori and Spikes, 1984).
Other desirable properties of photosensitizers to be used for therapeutic purposes are: a high degree of chemical purity [as compared with the heterogeneous nature of most hematoporphyrin derivatives (HpD)*]; photostability to red-light irradiation, in order to avoid the generation of by-products having unknown chemical structure and biological effects; and low systemic toxicity at therapeutically useful doses. In the case of hematoporphyrin, both acute and chronic toxicity are negligible in the dose range 1-10 mg/kg body weight, which is used in most clinical applications (Dougherty, 1987). It is likely that similar conclusions obtain for other photosensitizers, such as chlorins or phthalocyanines, which are usually administered at doses smaller than those used with porphyrins, because of the greater extinction coefficient in the red spectral region. *Abbreviations: HpD, hematoporphyrin derivative; LDL, low-density lipoproteins; PDT, photodynamic therapy.
USE OF PHOTOSENSITIZED PROCESSES IN TUMOR THERAPY
Until very recently, most phototherapeutic applications with red-light absorbing photosensitizers involved the use of porphyrins, mainly because porphyrin-sensitized photoprocesses have been extensively studied (Spikes, 1975) and some naturally occurring porphyrins have been found to be accumulated by tumors (Auler and Banzer, 1942) and by psoriatic areas (Silver, 1937). Porphyrins are still widely used in photochemotherapy , although several second-generation photosensitizers with better spectroscopic properties in the 6OCL900 nm range are being investigated (Table 1). Some diseases for which phototherapy with the abovementioned photosensitizers has been at least proposed at an experimental level are shown in Table 2 (see Spikes and Jori, 1987, and references therein). Certainly, the most widespread clinical applications of these phototherapeutic techniques have been in the field of solid tumors, owing to the property of specific porphyrins (especially some chemical HpD or known under the commercial names of Photofrin I and 11) to be accumulated in significant amounts and retained for prolonged periods of time by a variety of neoplastic lesions (Dougherty, 1987). The treatment is generally defined as photodynamic therapy (PDT), because photodamage of tumor tissues only occurs in the presence of oxygen. A typical PDT protocol is summarized in Scheme 1. The intravenously injected HpD (or Photofrin 11) reaches maximal tumor concentrations (of the order of a few pg of porphyrin per gram of tissue) within about 3 h; however, irradiation of the tumor area is most frequently performed 48-72 h after administration of the drug. At this time the ratio of photosensitizer concentration between tumor and peritumoral tissues is particularly large. Notable exceptions are the components of the reticuloendo-
44 1
Review Article Table 2. Selected diseases for which treatment by PDT is presently under investigation (Regan and Parrish, 1982; Spikes and Jori. 1987) Present state of development of phototherapeutic treatment Investigations are at phase 11-111 clinical level in several countries A limited number of clinical trials has been carried out at: Dermatological Clinic, Humboldt University, East Berlin, and University of California at Irvine Accumulation of fluorescent photosensitizers by atheromatous plaques in experimental animals may be used for early diagnosis and control of plaque development Several bacteria, mycoplasma, yeasts and viruses are photoinactivated by exposure to photosensitizers and red light. PDT may be used for sterilization of surgical beds
Disease Solid tumors Psoriasis Atheromas Viral and microbial infections
INJECTION
OF
(Intravenously, PHOTOSENSITIZER ELIMINATION
1 3 - 72 h
NORMAL TISSUES
1 ca. 4 SKIN
1
PHOTOSENSITIZER - 5 mo/kg)
1
1 - 3 h
ACCUMULATION OF
c c
THE
1 SERUM
TISSUES
PHOTOSENSITIZER
frwn ~~
weeks
8Y
2
-
4 weeks
~~~
3 h - 1 week! TUMOR
TISSUES
1 40 - 72
h
IRRADIATION OF TUMOR AREA (50 - 800 J/an2)
4
24 h after
PO7
APPEARANCE OF TUMOR RESPONSE TO PDT
Scheme 1 . Typical protocol for the P D T of tumors.
thelial system (including liver and spleen) and skin, which may retain detectable amounts of porphyrins for several weeks (Kessel, 1984). As a consequence, the onset of general skin photosensitivity is often an undesired side-effect of PDT (Dougherty, 1987). The total dose of light delivered to the neoplastic areas ranges between about 50 and 800 J/cm2, depending on the size and optical properties of the tumor. In all cases, the exposure rate must be carefully controlled and possibly not exceed 200 mW/cm2,in order to reduce the effects of hyperthermia which could overlap with photochemical effects (Svaasand, 1984). The response of the tumor to PDT usually appears as massive hemorrhagic tissue necrosis, reflecting vascular damage as the predominant primary effect of tumor photosensitization (Zhou, 1989). This is in agreement with the observed localization of a significant fraction of tumor-associated HpD in the vascular stroma (Dougherty, 1987). Photodynamic therapy has been
applied so far to a few thousand patients all over the world, most of whom have objectively benefited from the treatment. Besides the eradication of tumors of relatively small size, PDT may be used for palliative purposes and for sterilization of the surgical bed after resection of the tumoral mass (Dougherty, 1984). All these forms of PDT have been applied in Italy, during a multi-center clinical trial which lasted 3 yr and involved 88 patients (Corti et al., 1985; Perria et a l . , 1988). The results are currently being evaluated by the Italian Ministry of Health for possible official approval of PDT as a new therapeutic modality for tumors. The outcome of PDT was successful in a very large percentage of treated cases, although the patients selected had almost always been previously exposed to radiotherapy and/or chemotherapy, with negative results. Interestingly, a few patients with multiple neoplastic lesions underwent repeated PDT sessions at about
GIULIOJORI
442
2-month intervals. Thus, these findings indicate that the previous use of other therapeutic regimens does not interfere with the efficacy of PDT on tumors; moreover, the repeatability of photodynamic treatment emphasizes the safety of PDT for normal tissues. NEW DIRECTIONS IN THE PHOTODYNAMIC THERAPY
(Oseroff et al., 1986) that different endotissular compartments of tumors may be labeled by selected photosensitizers through the concerted use of appropriate delivery systems. Acknowledgement-This work was supported by the Consiglio Nazionale delle Ricerche (Italy), under the special project “Oncologia”, contract number.
OF TUMORS
At present, the main limitations of PDT are poor efficiency of HpD photoactivation by red light and the relatively limited selectivity of tumor targeting by photosensitizing dyes. While we hope that the former problem will be solved by replacing HpD with photosensitizers strongly absorbing at 700-800 nm (Neckers, 1988), the latter aspect has not been sufficiently addressed until now, partly because of incomplete elucidation of the factors controlling the accumulation in and release of photosensitizers from tumor tissues in v i v o . Some approaches have been proposed to enhance the specificity of tumor loading by photosensitizers. All of them exploit some differences existing between malignant and normal cells: ( 9 photosensitizers may be covalently coupled to monoclonal antibodies directed towards antigens which are specifically present on the surface of the malignant tumor cells (Mew et al., 1983); (ii) the mitochondria of various malignant and benign tumor cells accumulate larger amounts of certain lipophilic cationic dyes, such as rhodamines and other xanthenes, as compared with the mitochondria of normal cells. The process is driven by the internally negative membrane potential of this organelle, which allows penetration of the positive photosensitizer into the mitochondrion, in accordance with the Nernst equation (Beckman et al., 1987). The actual potential of this approach, although promising, cannot be defined until the photosensitization mechanism of these dyes (which may not be of photodynamic type) has been elucidated; (iii) several types of hyperproliferating cells, including tumor cells, express a particularly large number of membrane receptors for lowdensity lipoproteins (LDL). The protein-receptor complex undergoes endocytosis via the formation of coated pits (Goldstein et al., 1979). Thus, hydrophobic photosensitizers may be incorporated into the lipid moiety of LDL and released inside the malignant cells at the level of the lysosomes (Barel et al., 1986). The potential of these novel procedures of photosensitizer delivery to increase the efficacy of PDT must still be defined. It has been proposed
REFERENCES
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