Tumor-localizing and radiosensitizing properties of meso -tetra(4- nido -carboranylphenyl)porphyrin (H 2 TCP)
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Journal of Porphyrins and Phthalocyanines
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J. Porphyrins Phthalocyanines 2008; 12: 866-873
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Tumor-localizing and radiosensitizing properties of mesotetra(4-nido-carboranylphenyl)porphyrin (H2TCP) Marina Soncina, Elisabetta Frisoa, Giulio Joria, Erhong Haob, M. Graça H. Vicenteb, Giovanni Miottoc, Paolo Colauttid, Davide Morod, Juan Espositod, Giancarlo Rosie and Clara Fabris*a Department of Biology, University of Padova, 35131 Padova, Italy Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA c Department of Biochemistry, University of Padova, 35131 Padova, Italy d INFN, Laboratori Nazionali di Legnaro, 35020 Legnaro, Pd, Italy e ENEA C.R. Casaccia, Roma, Italy a
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Received 20 June 2008 Accepted 16 July 2008 ABSTRACT: Background and Purpose: Boron Neutron Capture Therapy (BNCT) is a binary cancer treatment that exploits the short range particles released from a nuclear fission reaction involving the non-radioactive 10B nucleus and low-energy neutrons for the destruction of tumor cells. In this pers pective, porphyrins and phthalocyanines can represent a vehicle for the transport of significant amounts of boron to the neoplastic lesion. Material and Methods: B16F1 melanotic melanoma subcutaneously transplanted in C57/BL6 mice has been used as an in vivo model. Pharmacokinetic studies were performed by intratumoral and intravenous injection of a meso-substituted porphyrin containing 36 B atoms per molecule (H2TCP) and the distribution of H2TCP in the tumor was assessed by fluorescence microscopy analysis. The tumor-bearing mice were exposed to the radiation field for 20 min at a reactor power of 5 kW. Results: At 0.5 h after intratumoral administration or at 24 h after intravenous injection, the amount of 10B in the tumor was found to be about 60 ppm and about 6 ppm, respectively. In spite of the different amounts of 10B in the tumor at the time of irradiation, a very similar delay in tumor growth (5-6 days) was induced by neutron irradiation in the two groups of injected mice with respect to control mice. Conclusions: Our results demonstrate that a suitable boron-loaded porphyrin displays a significant affinity for subcutaneous tumors, and upon activation by thermal neutrons, can promote an important response even in a fairly aggressive and generally radioresistant tumor such as melanotic melanoma. Copyright © 2008 Society of Porphyrins & Phthalocyanines. KEYWORDS: porphyrin, melanotic melanoma, boron neutron capture therapy, fluorescence.
INTRODUCTION Boron neutron capture therapy (BNCT) repre sents an interesting modality for the treatment of a variety of solid tumors, which is currently under going clinical trials for the treatment of brain tumors and peripheral melanoma [1]. In general, patients af SPP full
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*Correspondence to: Clara Fabris, email: clara.fabris@ unipd.it, fax. +39 049-8276344 Copyright © 2008 Society of Porphyrins & Phthalocyanines
fected by cutaneous melanoma are characterized by a very poor prognosis, except for early stage cases where patients are liable to undergo radical surge ry. In BNCT, 10B-carrying compounds are activated by irradiation with thermal neutrons obtained from a nuclear reactor; this results in the emission of α particles and 7Li atoms by the 10B (n,α) 7Li reaction. These densely ionizing particles deposit their ener gies within a spatial range of 7-10 μm, i.e., on di mension scales smaller than most cell diameters and
Published on web 08/25/2008
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RADIOSENSITISING PROPERTIES OF 10B-ENRICHED H2TCP
can often eradicate tumor cells without serious injury to the surrounding normal tissue [2]. At present, only two BNCT agents are in Phase I-II clinical trials, namely disodium mercapto-closododecaborate (BSH) [3, 4] and L-4-dihydroxyborylphenylalanine (BPA) [5, 6, 7]. Even though BSH and BPA have been shown to be safe and effective in animal models, both these agents display only moderate selectivity for neoplastic cells and short re tention times in tumors. Furthermore, BSH has limi ted chemical stability due to its tendency to undergo air-oxidation [8]. On the other hand, BPA, although chemically non-toxic, contains only a low percen tage of boron by weight (5%); hence large amounts of this drug are needed in order to achieve thera peutically useful boron concentrations in the tumor tissue [7, 9, 10]. Recently, new perspectives have been opened in this field by the synthesis of chemically pure boronlabelled porphyrins and phthalocyanines [11], with high boron content (> 20% by weight) where the te trapyrrolic derivative acts as a vehicle for the trans port of significant amounts of boron to the neoplastic lesion. In BNCT, the efficient and selective accumulation of boron in the tumor is critical for therapy success. A precise measurement of the boron concentration in the tumor and venous blood is essential, to calculate the doses delivered to the tumor and normal tissues. In this regard, porphyrins and phthalocyanines, in comparison with BPA and BSH, have the advan tage of being more easily detected and quantified by fluorescence spectroscopic techniques. Preliminary investigations from our laboratory pointed out that a meso-tetra(4-nido-carboranylphenyl)porphyrin (H2TCP), whose chemical structure is shown in Fig. 1, is accumulated in significant amounts by a sub H Na H H H Na
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Fig. 1. Chemical structure of H2TCP Copyright © 2008 Society of Porphyrins & Phthalocyanines
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cutaneously transplanted melanotic melanoma and exerts its photosensitizing action against this type of malignant cell [12]. The studies were performed by using C57BL/6 mice for which the B16 melano ma is syngenic, thus avoiding the use of immunosuppression. To properly analyze the effect of the radiation field on such a tumor model in view of BNCT studies, an experimental approach has been devised using a new thermal column called HYTHOR (HYbrid Thermal spectrum sHifter TapirO Reactor) inserted in the fast nuclear reactor Tapiro at Enea Casaccia, Italy [13]. Based on our previous results, our investigations were pursued with a two-fold aim: (a) to identify a mo re effective H2TCP formulation for intravenous and intratumoral injection, which enhances the efficien cy of 10B accumulation in the melanotic melanoma, since a significant response of the tumor to BNCT is claimed to require boron concentrations as high as 15-30 ppm [14] and (b) to test the efficacy of BNCT on our tumor model through experimental sessions performed at the Hythor thermal column.
EXPERIMENTAL Synthesis B-enriched H2TCP [tetra(4-nido-carboranylphe nyl)porphyrin] was prepared using a similar procedu re to that which we have previously reported, for the non-enriched analog [12, 15], as described below. A solution of 10B-enriched 4-(closo-carboranyl)benzal dehyde [16] (240 mg, 1 mmol) and freshly distilled pyrrole (0.07 mL, 1 mmol) in dry dichloromethane (100 mL) was purged with argon for 30 min. BF3.OEt2 (0.1 mmol) was added and the mixture was stirred at room temperature under argon for 8 h. DDQ (171 mg, 0.75 mmol) was added and the final reaction mix ture was stirred at room temperature for 30 min. The resulting solution was concentrated under vacuum and the resulting residue was purified by column chromatography using dichloromethane/hexane 1:2 for elution. The first and major fraction was collected and re-crystallized from dichloromethane/methanol, yielding 151 mg (52% yield) of 10B-enriched tetra(4closo-carboranylphenyl)porphyrin. mp > 300 °C. MS (MALDI): m/z 1151.07 [M + H]+. 1H NMR (CDCl3): δ, ppm -2.84 (br, 2H, NH), 1.7-3.4 (br, 40H, BH), 4.32 (br s, 4H, o-carborane-CH ), 7.92 (d, 8H, Ar-H), 8.20 (d 8H, Ar-H), 8.82 (s, 8H, β-H). UV‑vis (CHCl3): λmax, nm 418, 514, 550, 590 and 646. 10B-enriched H2TCP as the tetrasodium salt was prepared by dis solving the 10B-enriched tetra(4-closo-carboranylphe nyl)porphyrin (115 mg, 0.1 mmol) in a 3:1 mixture of pyridine and piperidine (12 mL) and stirring at 10
J. Porphyrins Phthalocyanines 2008; 12: 866-873
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room temperature in the dark for 36 h, under argon. The solvent was completely removed under vacuum and the residue re-dissolved in 40% aqueous acetone after which it was slowly passed through a Dowex 50WX2-100 resin in the sodium form. The porphyrin fraction was collected and diluted with 70% aqueous acetone and again passed through the ion-exchange resin. After removal of the solvent under vacuum, the tetraanionic porphyrin was re-crystallized from methanol/diethyl ether giving a yield of 110 mg (92%) of 10B-enriched H2TCP. MS (MALDI): m/z 1201.6. 1H NMR (acetone-d6): δ, ppm -2.71 (s, 2H, NH) , -2.45 to -1.90 (br, 4H, BH), 0.5-2.50 (br, 32H, BH), 2.60 (br s, 4H, nido-carborane-CH), 7.69 (d, 8H, J = 8.0 Hz), 7.99 (d, 8H, J = 8.0 Hz), 8.90 (s, 8H, β-H). UV-vis (acetone): λmax, nm 420, 516, 554, 594 and 650. Animals and tumor Female C57BL/6 mice (20-22 g body weight) were supplied by Charles River Laboratories (Como, Italy) and kept in standard cages with free access to tap water and standard dietary chow. Animal care was performed according to the guidelines established by the Italian Committee for humane treatment of expe rimental animals. The B16F1 melanotic melanoma, a pigmented variant of murine melanoma B16 [17] which differs because of its highly metastatic poten tial, was subcutaneously transplanted into the upper flank of the mice by injecting 20 µL (106 cells) of a sterile cell suspension in phosphate-buffered sa line (PBS). The cell line was cultured in Dulbecco’s modified minimal essential medium (DMEM, Sig ma, St. Louis USA) containing 10% heat-inactivated fetal calf serum (FCS, Gibco, Auckland, N.Z.) and supplemented with penicillin (100 units.mL‑1), strep tomycin (100 µg.mL‑1), amphotericin (0.25 µg.mL‑1) and 2 mM glutamine (Sigma). The cell culture was maintained at 37 °C in a humidified atmosphere of 5% CO2 in air. All pharmacokinetic and therapeutic studies were carried out on the seventh day after transplantation, when the tumor volume was about 0.02 cm3. When necessary, the mice were anaesthetized by i.p. injec tion of Zoletil® (20 mg/kg). The tumor underwent no spontaneous remission. Pharmacokinetic studies At 7 days after transplantation, the B16F1 tumorbearing mice were injected (a) intravenously with 10 mg/kg of the H2TCP (2.6 mg/kg boron) dissolved in 200 µL of a formulation (1% methanol, 19% di methylsulfoxide, 30% polyethyleneglycol 400, and 50% water) favoring the solubility and monomeric character of the porphyrin, or (b) intratumorally with 1 mg/kg of the H2TCP (0.26 mg/kg boron) in 20 µL Copyright © 2008 Society of Porphyrins & Phthalocyanines
of the same solution. At predetermined times after porphyrin injection, the mice (5 animals at each time point) were sacrificed by prolonged exposure to CO2 vapors. The H2TCP content in the serum and selec ted tissues was determined by spectrophotofluorime tric procedures. Briefly, the blood was centrifuged (3000 rpm for 15 min) to remove the erythrocytes and appropriately diluted with 2% aqueous sodium dodecyl sulfate (SDS). The tissue specimens were ho mogenized in 2% SDS, suitably diluted in the same solvent and the resulting solutions were analyzed for their porphyrin content by fluorescence spectros copy (excitation at 410 nm, emission at 600-800 nm) using a calibration plot built with known concentra tions of H2TCP in the same solvent. The data were converted into boron concentrations on the basis of the known stoichiometry of boron atoms per porphy rin molecule. Previous studies [12] had shown that the covalent bonds between the carborane and the porphyrin are not split in vivo. This procedure was shown to extract at least 90% of the porphyrin from the tissue specimens [18]. In order to define the pathway of porphyrin ex cretion from the organism, a group of five mice, af ter i.v. injection of H2TCP at a dose of 10 mg/kg bo dy weight, was maintained in a metabolic cage and fed a special chlorophyll-free diet since the intensive fluorescence of this compound could interfere with the spectrophotofluorimetric porphyrin detection. The urine and the fecis of the mice were collected from 24 h until 1 week after H2TCP injection. After dilu tion in 2% SDS the urine was analyzed spectropho tofluorimetrically. Moreover, the porphyrin content in the fecis was determined after suitable dilution of homogenate in 2% SDS. Fluorescence microscopy analyses After intratumoral or intravenous injection of H2TCP, the mice were sacrificed and the tumor with the overlying skin was excised, frozen in liquid ni trogen after dipping in isopentane and sectioned with a cryostat at -30 °C. The sections thus obtained (thickness 7-10 µM) were collected and placed on gelatin-coated slides, hydrated with PBS and finally mounted by using a glycerol-PBS mixture (1:1, v/v). The sections were observed by means of an Olym pus IMT-2 fluorescence microscope equipped with a refrigerated CCD camera (Micromax, Princeton Ins truments). A 75-W Xenon lamp was used as the ex citation source. Fluorescence images obtained with a 10× objective (Olympus) were acquired and analy zed with the imaging software Metamorph (Universal Imaging). For the porphyrin fluorescence detection, a set of filters with 400 nm excitation and 620 nm emission was used. J. Porphyrins Phthalocyanines 2008; 12: 866-873
RADIOSENSITISING PROPERTIES OF 10B-ENRICHED H2TCP
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BNCT studies When the tumor volume reached about 0.02 cm3, the mice were intratumorally injected with 1 mg/kg H2TCP or intravenously with 10 mg/kg H2TCP dis solved in the above-described formulation. After 0.5 and 24 h, respectively, the animals were exposed to the thermal beam of the Hythor column, for 20 min at the operating power level of 5.0 kW. In order to shield some particularly sensitive organs, the mouse abdomen was protected with a thermal neutron ab sorber layer which is a kind of pant made of 0.14 g.cm‑2 of 98% boric acid enriched with 10B. After irradiation, the tumor size was measured daily by means of a calliper. Individual tumor volu mes (V) were calculated by assuming a hemiellipsoi dal structure for the tumor nodule and measuring the two perpendicular axes (a and b) and the height (c). The application of the relationship; V = 2/3 p (a/2 b/2 c) provided the tumor volume. The number of days taken for the tumor volume to grow from its initial value of 0.02 ± 0.005 cm3 to a value of 0.8-1 cm3 was calculated for the individual neoplastic lesions. The mice were sacrificed by euthanasia before the tumor development became too large in order to mi nimize the risk of undue suffering and spreading of the malignancy across the body through lung or liver metastases. The effectiveness of the treatment was evaluated by comparing the rate of tumor growth of the irradia ted mice with that observed for control mice which had been simultaneously transplanted but not injec ted with H2TCP and not exposed to the thermal neu tron radiation.
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recovery was measured in the melanotic lesion, the skin (peritumoral tissue), lung (which can frequen tly entrap colloidal particles), kidney and liver (to identify the main pathway of porphyrin elimination from the body), and spleen (since hydrophobic drugs are known to be efficiently taken up by the compo nents of the reticuloendothelial system) [18]. The bo ron concentration in the tumor was found to be about 6 ppm, a quantity relatively close to the 15-30 ppm that are generally assumed to be necessary to achie ve an extensive therapeutic effect upon irradiation with thermal neutrons [14]. As expected, liver and spleen represent the main sites of porphyrin accumu lation, in agreement with what has been observed for a variety of porphyrin derivatives [18]. In order to reduce the risk of non-selective dama ge following exposure to the thermal neutron beam, a group of animals was intratumorally injected with 1 mg/kg of H2TCP. As one can see in Fig. 2B, at 30 min after administration, the recovery of 10B in the tumor was about 60 ppm; under these conditions only
RESULTS Pharmacokinetic studies The distribution of H2TCP in the melanotic me lanoma-bearing mice was studied using two admi nistration modalities, namely intravenous and intra tumoral. The addition of a minimal quantity of me thanol (1%) to a formulation previously developed in our laboratory [12] was found to be well tolerated by the animals and allowed a significant increase of H2TCP recovery in the tumor at 24 h after i.v. injec tion of 10 mg/kg porphyrin. This time interval was selected on the basis of the almost complete clea rance of the porphyrin from the blood circulation system and the satisfactory selectivity of tumor tar geting as compared with the surrounding skin. The amount of 10B recovered after i.v. injection from dif ferent organs is shown in Fig. 2A: in particular, the Copyright © 2008 Society of Porphyrins & Phthalocyanines
Fig. 2. Recovery of boron from tumor and selected normal tissues of C57BL/6 mice bearing a subcutaneously trans planted B16F1 melanotic melanoma. The mice were i.v.injected with 10 mg/kg H2TCP (Fg 2A) or i.t.-injected with 1 mg/kg H2TCP (Fg 2B). The histograms represent mean ± standard deviation of recoveries from 5 mice at each time point J. Porphyrins Phthalocyanines 2008; 12: 866-873
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traces of porphyrin were detected in serum, liver and spleen, indicating that only minor amounts of the ra diosensitizing agent leak into the general blood cir culation. The analysis of the porphyrin excretion from the organism indicates that H2TCP is preferentially ex creted through the feces. The maximum amount of porphyrin in the feces was found at 24-48 h after in jection and traces of photosensitizer were detectable after 1 week. No H2TCP was found in the urine of treated mice. The predominance of the bile-gut path way for the clearance of porphyrin derivatives was already demonstrated for other hydrophobic com pounds based on the tetrapyrrolic macrocycle and is in agreement with the accumulation of large H2TCP amounts in the liver. Fluorescence microscopy analyses Sections obtained from tumor with the overlying skin were analyzed at 0.5 and 24 h after intratumo ral or intravenous injection of H2TCP. The results are shown in Figs 3 and 4. As one can observe in Fig. 3, at 0.5 h after intratumoral administration, the fluorescence signal of H2TCP appears to be mostly distributed in a defined area of the malignant lesion which is likely to correspond to the injection site of the dye. Surrounding tumoral cells, as well as the skin overlying the melanoma, exhibited no fluorescence signals typical of porphyrins. In the specimens collected at 24 h after intrave nous injection of the porphyrin (Fig. 4), the distribu
tion pattern of fluorescence appears to be markedly different. The neoplastic cells show the presence of a fluorescence signal distributed throughout the tu mor; as expected the emission is two times less in tense than that observed in intra-tumorally injected mice. The porphyrin fluorescence is detectable in the epidermal layers as well as in the connective capsu le surrounding the neoplastic bulk [19]. Analyses of samples obtained from mice not injected with the H2TCP, did not show fluorescence signal typical of porphyrin. BNCT studies The experiments of thermal neutron irradiation on a subcutaneously transplanted melanotic melano ma in C57BL/6 mice were performed at 0.5 h after intratumoral administration of 1 mg/kg H2TCP or at 24 h after intravenous injection of 10 mg/kg H2TCP. The mice exposed to the HYTHOR radiation field at a reactor power of 5 kW received an absorbed dose composed as follows: dose Dn delivered by neutrons through thermal neutron capture reactions with nitrogen nuclei 14N(n,p)14C; proton recoils from 1 H(n,n’)1H elastic reactions and heavier recoils due to elastic and anelastic fast neutron reactions; dose Dg delivered by gamma rays from the reactor source; and dose Dbnc delivered by charged particles emer ging from neutron-capture reactions 10B(n,a)7Li [13]. As shown in Fig. 5, no significant difference in the rate of tumor growth was observed between control untreated mice and mice that had been exposed to the
Fig. 3. Micrographs of melanotic melanoma with the overlying skin collected from mice at 30 min after intratumoral injection of 1 mg/kg H2TCP. In image A, a weak fluorescence signal is detectable only in the tumor cells underlying the epidermal layers. Image B shows an intense fluorescence in a defined area of the tumor whereas in the surrounding tissue no signals are detected (image C). Images D, E and F represent the bright field of micrographs A, B and C, respectively Copyright © 2008 Society of Porphyrins & Phthalocyanines
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RADIOSENSITISING PROPERTIES OF 10B-ENRICHED H2TCP
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Fig. 4. Micrographs of melanotic melanoma with the overlying skin collected from mice at 24 h after intravenous injection of 10 mg/kg H2TCP. In image A, a dotted fluorescence signal is detectable in the epidermal layers. Image B shows fluorescence of H2TCP distributed through the tumor cells. The connective capsule surrounding the tumor bulk appears to be the main site of fluorescence emission (image C). Images D, E and F represent the bright field of micrographs A, B and C, respectively
discussion
Fig. 5. Rate of tumor growth for C57BL/6 mice bearing a subcutaneously transplanted B16F1 melanotic melanoma exposed to thermal neutrons (5 kW) for 20 min. Uninjected and unirradiated control mice (full squares), only irradiated mice (clear squares), irradiated 0.5 h after i.t-injection of 1 mg/kg H2TCP (full circles), irradiated 24 h after i.vinjection of 10 mg/kg H2TCP (full triangles). Each point represents the average of 12 mice ± standard error
thermal neutron flux for 20 min without prior injec tion of H2TCP. On the other hand, a significant delay in tumor growth (5-6 days) was induced by thermal neutron irradiation in the two groups of porphyrininjected mice. The delay was practically identical for the intratumorally and intravenously injected mice in spite of different amounts of 10B in the tumor at the time of irradiation. Copyright © 2008 Society of Porphyrins & Phthalocyanines
Several porphyrins have been shown to exhibit a large affinity for and a prolonged retention by a varie ty of solid tumors [20]. However, some concern has been raised about the effect of heavy loading of the porphyrin molecule with boron clusters on its phar macokinetic properties, including the extent and se lectivity of accumulation by neoplastic tissues [21]. The results presented in this paper fully confirm the findings obtained in our [22] and other [23, 24] la boratories showing that porphyrin derivatives bea ring up to four carborane cages can be systemical ly injected in vivo with no detectable toxic effects, while their overall biodistribution is closely similar to that typical of non-boronated porphyrins. In par ticular, the amount of 10B-loaded porphyrin which is accumulated in the melanotic melanoma is sufficient to induce an appreciable delay in tumor growth after thermal neutron irradiation. Interestingly, there is no detectable difference in the tumor response to radiation treatment after intra tumoral or intravenous administration of the H2TCP in spite of an about ten-fold difference in the endo tumoral boron concentration, as expressed in ppm. It is unlikely that this identical behavior reflects a satu ration of 10B effect above the 6 ppm threshold since excellent responses of tumor to BNCT treatment have been found to occur in the presence of boron concen trations as large as 15-30 ppm [3, 14]. It appears rea sonable to hypothesize that the inhomogeneous dis J. Porphyrins Phthalocyanines 2008; 12: 866-873
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tribution of the porphyrin among the various com partments of the melanoma, especially in the case of the intratumorally delivered radiosensitizer, leads to tissue damage preferentially localized in the micro environment of the boronated porphyrin owing to the short lifetime of the radiogenerated toxic species (see Introduction). We have previously shown that, the presence of particularly large amounts of a boronated phthalocyanine in subcellular membranes of mali gnant cells was found to promote an important delay in the rate of tumor growth after BNCT treatment, even though the amount of boron was of the order of 1 ppm [22]. In this connection, our present findings suggest that intravenous administration of the boronated por phyrin is preferable owing to a more scattered distri bution in different subcellular sites. The albeit partial response of a normally radioresistant tumor [25], such as the melanotic melanoma, to BNCT carried out at 24 h after injection of H2TCP, is certainly encoura ging. Further improvement of the overall outcome of BNCT in this tumor model can be pursued by using two approaches: (a) optimization of the individual pa rameters modulating the pharmacokinetic behavior of H2TCP, as well as the effect of thermal neutrons; and (b) exploring the possible combination of BNCT with photodynamic therapy (PDT) owing to the wellknown property of porphyrins, to be electronically excited upon irradiation with selected visible light wavelengths, thereby causing tumor damage via ge neration of hyper-reactive oxygen species, namely by a mechanism which is deeply different from that typical of BNCT [26]. The latter option would be particularly attractive since two different therapeutic modalities can be applied by using one single radioand photo-sensitizing agent. Acknowledgements This research has been financially supported by the Italian Institute for Nuclear Physics (INFN) and by the University of Padova Project (Progetti di Ate neo) No. CPDA048247. The synthesis of 10B-enri ched H2TCP was partially funded by the US National Institutes of Health, grant number CA098902.
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RADIOSENSITISING PROPERTIES OF 10B-ENRICHED H2TCP
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J. Porphyrins Phthalocyanines 2008; 12: 866-873
J. Porphyrins Phthalocyanines 2008.12:866-873. Downloaded from www.worldscientific.com by ISTITUTO NAZIONALE DI FISICA NUCLEARE (INFN) - LNL LABORATORI NAZIONALE DI LEGNARO on 10/10/12. For personal use only.
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