Tumor-localizing and radiosensitizing properties of meso -tetra(4- nido -carboranylphenyl)porphyrin (H 2 TCP)

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

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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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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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