Photodiagnosis and Photodynamic Therapy (2006) 3, 205—213

Photodynamic therapy in Argentina Adriana Casas, Alcira Batlle ∗ Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP), CONICET and Hospital de Cl´ınicas Jos´ e de San Mart´ın, University of Buenos Aires, C´ ordoba 2351 1er Subsuelo; Buenos Aires 1120, Argentina Available online 27 July 2006 KEYWORDS ALA; 5-Aminolevulinic acid; PDT; Photodynamic therapy; History; Argentina

Summary The use of endogenous Protoporphyrin IX generated through the heme biosynthetic pathway after administration of 5-aminolevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). In Buenos Aires, Argentina, the Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP), reported for the first time, in 1975, porphyrin synthesis from ALA in highly dividing plant tissues. Increased porphyrin synthesis in tumours as well as cell photosensitisation was reported soon after. Our group is also interested in studying the use of new synthetic lipophilic derivatives of ALA as well as ALA delivery in liposomes. We have elucidated the mechanism of ALA transport in mammalian and yeast cells. The interactions between ALA-PDT and nitric oxide were investigated in three murine adenocarcinoma cell lines. In the National University of R´ıo Cuarto, C´ ordoba, a group is devoted to the synthesis of new porphyrin-derived photosensitisers to study their effects on photoinactivation of bacterial and mammalian cells death by PDT. At the Centre of Electron Microscopy of the Cordoba National University, a prototype of a 630 nm noncoherent light source was designed and constructed. Cost of the light source and scarce knowledge of the benefits of PDT by physicians limit the spread of the treatment throughout the country. © 2006 Elsevier B.V. All rights reserved.

The beginnings In Buenos Aires, Argentina, the Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP), was Abbreviations: ALA, 5-aminolevulinic acid; Me-ALA, ALAmethyl ester; DMSO, dimethylsulphoxide; GABA, ␥-aminobutyric acid; He-ALA, ALA-hexyl ester; i.p., intraperitoneal; i.t., intratumour; THP-ALA, R; S-2-(hydroxymethyl)tetrahydropyranylALA; PDT, photodynamic therapy; P-C60, porphyrin-C60 dyad; PpIX, Protoporphyrin IX; s.c., subcutaneous; Und-ALA; ALAUndecanoyl ester; ZnPc 2, Zn(II) tetramethyltetrapyridinoporphyrazinium salt; ZnPc 1, Zn(II) tetrapyridinoporphyrazine ∗ Correspondence to: Viamonte 1881 10A, 1056 Buenos Aires, Argentina. Tel.: +54 11 4812 3357; fax: +54 11 4811 7447. E-mail address: [email protected] (A. Batlle).

established by Alcira Batlle in the late sixties, to carry our research on the metabolism of porphyrins, the porphyrias, their diagnosis and treatments. The CIPYP is the Reference Center in Latinamerica. Since its beginning, a great part of the staff was fully devoted to investigate the haem pathway in several organisms. Porphyrin synthesis from 5-aminolevulinic acid (ALA) in a highly dividing plant tissue, considered as a vegetable tumour was already reported from our laboratory in 1975 [1]. Increased porphyrin synthesis in tumour explants [2,3], cells [4] and whole animals [5,6] was described later, showing our early involvement in photodynamic therapy (PDT).

1572-1000/$ — see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2006.06.003

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ALA-based photodynamic therapy Photodynamic therapy of cancer is based on the administration of a photosensitising compound with tumour-localising properties, and subsequent irradiation with light of an appropriate wavelength leading to selective damage to the treated tissue [7]. The use of endogenous Protoporphyrin IX (PpIX) generated through the heme biosynthetic pathway after administration of ALA has led to many applications in PDT. ALA is frequently administered topically or systemically for PDT of several tumour types. ALA-induced PpIX accumulation has been shown to be preferentially greater in certain tumour cells primarily due to the reduced activity of ferrochelatase (Ferro-chel) the enzyme responsible for the conversion of PpIX into heme [8] and a relative enhancement of porphobilinogen deaminase (PBG-D) activity [8,9], which constitutes the biological rationale for the clinical use of ALA based PDT (ALA-PDT).

Porphyrin synthesis from ALA in highly dividing plant tissues and in mice organ explants In the soybean callus system, addition of ALA to the culture medium was found to stimulate porphyrin accumulation and to prevent growth. It was also found to inhibit ALA synthetase (ALA-S) and to enhance ALA dehydratase (ALA-D) and PBG-D activities [1]. As early as in 1988 [10] we demonstrated that using ALA, porphyrin synthesis was increased 20fold in human adenocarcinoma as compared with normal tissue. We then showed that, in both human malignant cells and implanted tumours, the activities of ALA-D, PBG-D, deaminase, uroporphyrinogen decarboxylase and heme oxygenase were increased, while ALA-S was very low and Cyt P450 levels undetectable [8,11—17]. In 1989, our group had already reported our studies on porphyrin synthesis from ALA on explants of mice tissues such as mammary tumour, liver, skin and brain. Explants were cultured for 6, 12 and 22 h in the presence of 0.06, 0.1 and 0.2 mM ALA. It was found that in all tissues, synthesis of porphyrins increased with time and ALA concentration and tumour porphyrins synthesis was of the same order as that of liver, and nearly twice compared with skin and brain. The tissue/tumour porphyrin concentration ratios were highest at short incubation times and at low ALA concentrations. Chromatographic analysis of porphyrins showed that the whole heme

A. Casas, A. Batlle pathway was functional in all organs studied, including tumour. Porphyrin synthesis from ALA was compared with that from liposomal ALA. After 22 h of incubation with 0.4 mM ALA, porphyrin formation was higher when liposomal ALA was used [2]. Employing the same explant tissue culture system we later developed a model for assaying the effectiveness of ALA-PDT photodamage. Tumour tissue explants were incubated in medium containing ALA and afterwards irradiated with a low power He—Ne laser. Immediately after irradiation, a piece of exactly 1 mm3 of the irradiated and nonirradiated control tissue was subcutaneously injected under the right and left flanks of the same animal, respectively. The rate of tumour growth was followed after implantation. Results obtained showed that incubation for 1 h with 0.6 mM ALA and irradiation, produced a reduction of 50—70% of tumour growth whereas no tumour growth was observed in explants exposed 2 h to 0.6 mM ALA [3]. Employing the same model we demonstrated that the cytostatics doxorubicyn [18] and cyclophosphamide enhanced the antitumour effect of ALAPDT whereas the antimetabolite 5-fluorouracil did not have any effect at all [19].

Cell photosensitisation with ALA-PDT More recently we reported photosensitisation in the murine mammary carcinoma LM2 cells. After 3 h incubation with 0.6 mM ALA, light treatment was performed with an Oxford Lasers copper-dye laser set at 630 nm. Applying a light dose of 0.6 J/cm2 cell viability decreased to 50% whereas 1.5 J/cm2 light dose induced more than 80% of cell death, and higher light doses produced total cell killing. Cells treated with ALA-PDT at low light doses exhibited features of apoptosis whereas at high doses necrosis was the mechanism involved [4].

Porphyrin synthesis after ALA administration to mice and tumour growth delay We have also studied for the first time the kinetics of porphyrin accumulation in cultured epithelial cells [20] and in different mice organs after intraperitoneal (i.p.), subcutaneous (s.c.) and intratumour (i.t.) administration of ALA to tumourbearing mice [21]. High porphyrin levels were obtained at short times in tumour, liver, spleen and kidney independently on the administration route, and porphyrin synthesis returned to basal values at 24 h after administration. In addition, the i.p. route was the best for tumour porphyrin synthesis. Low

Photodynamic therapy in Argentina synthesis occurred in muscle, lung, brain, erythrocytes and skin [5]. Topical and i.t. administration of ALA to the LM3 subcutaneously implanted mammary carcinoma produced a significant synthesis of porphyrins and subsequent sensitisation with laser light produced tumour growth delay. Porphyrin accumulation was greater when ALA was administered i.t. and tumour/normal skin porphyrin concentration ratios were higher compared with topical application. However, tumour response to ALA-PDT treatment was similar following either route of drug administration. Both necrosis and apoptosis were the mechanisms involved in cell killing, as it was evaluated by light microscopy. One, two or three PDT sessions were applied and no resistance to the treatment was acquired [12]. ALA-PDT has been used in multiple sessions for different tumour types in several clinical studies; however, there were no studies attempting to elucidate if PDT with ALA could induce resistance. To this end, we have therefore isolated and characterised cell lines resistant to ALA-PDT derived from a murine adenocarcinoma and investigated cross resistance with other injuries. The most resistant clones were identified with numbers 4 and 8, exhibiting 6.7- and 4.2-fold increase in resistance, respectively. Several properties were altered in these clones. A twofold increase in cell volume, higher cell spreading, and a more fibroblastic, dendritic pattern, were the morphology features which led us to think they could show different adhesive, invasive or metastatic phenotypes. Porphyrins formed per cell in the resistant clones were similar to the parenteral line; however, when expressed per mg of protein, there was a twofold decrease, with a higher proportion of hydrophilic porphyrins. These clones were not cross-resistant to photosensitization with benzoporphyrin and merocyanine, but showed some resistance to exogenous PPIX treatment. Also both clones displayed higher protein content, increased number of mitochondria and higher oxygen consumption. These distinctive features made us to think as to how to exploit the changes induced by PDT treatment to target surviving cells. The hypoxic cells could be a preferential target of bioreductive drugs and hypoxia-directed gene therapy, and they could also be sensitive to treatment with other photosensitisers [22].

Topical application of ALA: the role of the vehicles Maximal accumulation was found in the LM3 murine subcutaneously implanted tumour 3 h after ALA

207 application in both cream and lotion preparations. The latter induced higher accumulation of tumour porphyrins, and lotion applied on normal skin was also the most efficient upon inducing total body porphyrins, thus showing the influence of the formulation of ALA vehicle on penetration through the skin [23]. The role of different vehicles on PpIX synthesis from ALA was further studied [24]. We employed ALA in saline lotion with and without dimethylsulphoxide (DMSO), cream, liposomes and vaseline and they were topically applied on the LM3 subcutaneous adenocarcinoma. ALA in saline lotion, with or without 10% DMSO, proved to be the most efficient vehicles for tumour porphyrin accumulation whereas cream and liposomes induced lower levels. Employing ALA + DMSO saline lotion, a higher porphyrin accumulation was found in skin overlying the tumour tissue and in the first 2 mm of tumour, probably due to increased ALA penetration, or greater interconversion to porphyrins, or greater retention of ALA and/or porphyrins.

ALA derivatives The hydrophilic nature of ALA may limit its ability to penetrate through skin or cell membranes and thereby restricts the use of ALA-PDT to the treatment of superficial diseases. As a result, derivatives of ALA, which are less hydrophilic than the parental compound, are under investigation as possible alternatives to ALA [25—28]. The set of ALA derivatives synthesised and tested in our laboratory is shown in Fig. 1.

In vitro studies In the LM2 mammary adenocarcinoma cells, maximal porphyrin synthesis was induced by exposure to 0.6 mM ALA and the same amount was formed by a concentration 60-fold lower of ALA hexyl ester (HeALA) and twofold higher of ALA methyl ester (MeALA). Comparing PpIX production and its induced phototoxicity, the more the amount of porphyrins, the greater the rate of cell death. PpIX formed from either ALA or ALA-esters equally sensitises the cells to photoinactivation. Thus we demonstrated that ALA-PDT can be improved with the use of ALA derivatives, reducing the amount of ALA necessary to induce efficient photosensitisation [4]. We examined in collaboration with Dr MacRobert’s group from the National Medical Laser Centre, University College London, a range of hydrophobic derivatives of ALA. We employed samples of normal human and rat skin in short-term

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A. Casas, A. Batlle

Figure 1 New synthesised ALA derivatives.

organ culture. Using this floating explant cultures, the drugs are absorbed through the dermis, resembling the systemic pathway of drug delivery. Our data suggested that in normal tissues ALA derivatives are less rapidly taken up and/or converted to free ALA. The exception was the two-sided derivative carbobenzoyloxy-D-phenyl-alanyl-5-ALAethyl ester, which was as active as ALA at inducing PpIX fluorescence [29].

In vivo studies We analysed porphyrin formation in chemically induced squamous papillomas, after topical application of ALA and He-ALA in cream and lotion formulations. By chemical extraction of porphyrins from these highly keratinised papillomas, we found porphyrin synthesis from He-ALA and also por-

phyrin accumulation in internal organs. However, He-ALA remained much more confined to the site of application compared with ALA. After application of ALA lotion to a single papilloma, porphyrins were uniformly distributed to all distant papillomas, whereas He-ALA induced a higher concentration in the treated papilloma, and porphyrin accumulation in the nontreated tumours decreased with the distance from the He-ALA applied papilloma. Topical applied He-ALA may be retained in the stratum corneum, which may act as a reservoir, and diffuse superficially, leading to a pattern of skin porphyrin accumulation dependant on the distance of application. In addition, internal organ porphyrin accumulation shows that He-ALA reaches the dermis in this tumour model [30]. We studied the effect on porphyrin synthesis of systemic administration of He-ALA to mice bearing

Photodynamic therapy in Argentina the LM3 implanted mammary adenocarcinoma. In most normal tissues and in tumour, He-ALA induced less porphyrins synthesis than ALA after i.p. or i.v. administration, although explant organ cultures exposed to either ALA or He-ALA revealed equally active esterases. The only tissue that accumulated higher porphyrin levels from He-ALA (seven times more than ALA) was the brain, and this correlated well with a rapid increase in ALA or He-ALA accumulation after administration of He-ALA. We ascribed such behaviour to a differential permeability to lipophilic substances controlled by the blood-brain barrier, which could be further exploited to treat brain tumours [31]. To find a more efficient pro-photosensitiser, we synthesised two ALA esters: R, S-ALA-2(hydroxymethyl)tetrahydropyranyl ester (THP-ALA) and ALA-Undecanoyl ester (Und-ALA). In mice bearing the LM3 subcutaneous tumour, we studied the tissue distribution of the porphyrins formed from these esters after systemic administration. The aim was to establish if these esters are retained in any specific tissue, which could potentially be targeted for photodynamic treatment with ALA derivatives. We also investigated the topical use of these esters. After systemic administration, tumour and skin overlying tumour porphyrin levels were lower from the ALA esters than from ALA. Other tissues such as liver, colon, kidney, skin and spleen also accumulated less porphyrins from the esters, showing that there is no specific retention of the esters in these tissues. However, the brain was the only organ that synthesised more porphyrins from THP-ALA than from ALA. The kinetics of porphyrin synthesis from ALA esters are comparable to those from ALA in almost all tissues, showing that esterases activities are not limiting the availability of the hydrolysed ALA. Both THP-ALA and Und-ALA, applied topically on the skin over the tumour, exhibited higher selectivity than ALA for the site of application, whereas the amount of tumour porphyrin was the same from ALA and THP-ALA but lower from UndALA [32].

Regulation of porphyrin synthesis from ALA and ALA derivatives We tested in vitro the efficacy of the derivatives HeALA, Und-ALA and THP-ALA as pro-photosensitising agents. In the LM3 cells Und-ALA and THP-ALA did not improve ALA efficacy in terms of porphyrin synthesis. On the other hand, half of the amount of HeALA was required to obtain the same plateau porphyrins compared with ALA. However, this plateau value could not be surpassed in spite of the 4-times

209 higher accumulation of ALA or He-ALA from the ALA derivative. This shows that He-ALA conversion to porphyrins but not entry to the cells is limiting. Employing ionic exchange chromatography, we found that 80% of total uptake was He-ALA whereas only 20% was ALA. This suggests that the esterases, probably themselves regulated by the heme pathway, are limiting ALA derivatives conversion into porphyrins. Similar results were found for THP-ALA [33]. We studied later in the same adenocarcinoma cells the porphyrin synthesis regulation from ALA and their derivatives. We found that He-ALA is incorporated into the cells at a higher rate, followed by THP-ALA and ALA, whereas ALA and ALA esters efflux at the same rate mediated by passive diffusion. ALA entrance to the cells might be regulatory at low concentrations, whereas ALA derivative uptake is not a limiting factor. At high concentrations, the regulation of ALA conversion into porphyrins is driven by the enzyme PBG-D, whereas ALA esters hydrolysis is regulated by esterases [34].

ALA transport systems It was demonstrated in our laboratory in 1996 that in Saccharomyces cerevisiae yeasts, ALA shares its transport system with ␥-aminobutyric acid (GABA) [35]. We then characterised the ALA transport system in the LM3 cell line using radiolabelled 14 C-ALA. We found that ALA was incorporated into these cells by two different mechanisms, passive diffusion which is important at the beginning of the incubation, and active transport. Specificity assays suggested that the transporter involved in ALA incorporation is a BETA transporter, probably GAT-2 [36]. Later on, we investigated the ALA derivative transport systems through inhibition of radiolabelled ALA uptake in the LM3 cells. We also performed inhibition studies of GABA uptake. We showed that the more lipohilic ALA derivatives HeALA and Und-ALA inhibit ALA uptake, whereas MeALA, THP-ALA and the dendron aminomethane tris methyl 5-ALA, do not inhibit ALA uptake. A similar pattern was found for GABA, except that the dendron inhibited GABA uptake. However, He-ALA and Und-ALA are not taken up by BETA transporters, but by simple diffusion although they still inhibit ALA uptake by binding to the cell membrane. These results show that different modifications to the ALA molecule lead to different uptake mechanisms. Whereas ALA is taken up by BETA transporters, none of the ALA derivatives shares the same mechanism [37].

210 Since different cell types have different mechanisms for ALA uptake, we also employed Pichia pastoris yeasts expressing the intestinal peptide transporter PEPT1 and the renal PEPT2. In PEPT2 yeasts, the derivatives He-ALA, Und-ALA; THP-ALA and 3mALA competed for ALA transport. In PEPT1 yeasts, only Und-ALA did. The derivatives THP-ALA and HeALA could improve ALA-PDT outcomes through more efficient delivery when applied to tissues expressing PEPT2 such as kidney, mammary gland, brain and lung, but not in tissues like intestine, expressing PEPT1 [38].

Use of liposomes in the delivery of ALA or ALA derivatives Liposomes have been exploited as delivery vehicles for systemic administration of drugs. We have used ALA or ALA derivatives entrapment as an approach to increase ALA delivery to the cell. In our laboratory it was demonstrated that i.t. and i.p. administration of liposome-entrapped ALA to tumour bearing mice, resulted in both a major porphyrin biosynthesis and a higher tumour/normal tissue porphyrin ratio [5]. In addition, we found that previous administration of empty liposomes, enhanced the levels of porphyrins accumulated in tumour, due to blockage of liver uptake [6]. The strategy in designing topical liposomes is to exploit adsorption through the interface of both stratum corneum layers. The tissue distribution and kinetics of porphyrin synthesis after topical application of ALA entrapped in large multilamellar liposomes was determined after application on the LM3 subcutaneous adenocarcinoma model. In contrast to the kinetics of porphyrins formed from free ALA, tumour and skin overlying the tumour produced maximal amount of porphyrins 24 h after liposomal ALA application. However, other vehicles such as saline lotion alone or with DMSO, proved to be more efficient than liposomes in terms of maximal tetrapyrrole accumulation [24]. We also compared ALA with He-ALA in their free and liposomal formulations in explant tumour cultures. We found that neither the use of He-ALA nor the entrapment of either ALA or He-ALA in liposomes increased the rate of tumour porphyrin synthesis. By light and electronic microscopy we demonstrated that exposure of tumour explants to either free or liposomal ALA and subsequent illumination induced the same type of subcellullar damage [39]. However, a great disadvantage in the use of entrapped ALA is the low incorporation efficiency, around 6—10%. In addition, the fact that ALA is a

A. Casas, A. Batlle small, hydrophilic molecule, probably leaking out of the vesicles, determines that the final liposome preparation may be a mixture of free and liposomal fractions, thus making the results difficult to understand. Due to this major drawback, we are trying to entrap ALA derivatives into more stable liposomes.

ALA-containing dendrimers Polymeric drug delivery systems present an attractive method for drug targeting, particularly in cancer chemotherapy [40]. Recent advances in polymer chemistry allow the synthesis of hyperbranched polymers, or dendrimers, which can be conjugated with drug molecules. The molecules can be incorporated into the structure of the dendrimer during synthesis, giving rise to a molecule with a known size and drug loading. Battah et al. [41] synthesised for the first time several ALA-containing dendrimers, where the ALA residues are attached to the periphery by ester linkages, with amide bonds connecting the dendrons. We tested in vivo and in vitro in the LM3 cell line, the dendron aminomethane tris methyl ALA TFA salt (3m-ALA) containing 3 ALA residues, synthesised by Battah et al. [41]. In the LM3 cells, the dendron-induced similar porphyrin levels compared to equimolar concentrations of ALA. Although the dendron was taken up with comparable efficiency to ALA, we found that there was only partial intracellular liberation of ALA residues. Both systemic and topical administration of the dendron to tumour-bearing mice induced higher porphyrin levels than He-ALA in most tissues studied, although it was not possible to surpass the levels induced by ALA [42].

Interactions between ALA-PDT and nitric oxide PDT interactions with nitric oxide are not yet well understood. We have studied the in vitro interactions between ALA-PDT and nitric oxide, as well as the interactions between ALA, porphyrins and some donors and precursors. To this end we have used three murine adenocarcinoma cell lines, LM2 which does not produce NO; LM3 which does produce NO and LM3-SNP, a variant of LM3 resistant to NO, but producing the same amount of NO as the parenteral. We did not find any cross resistance between NO-induced cytotoxicity and ALAPDT. In spite of the lower porphyrin synthesis, LM2 cells show the highest sensitivity to ALA-PDT. Unexpectedly ALA induced in both cells and medium

Photodynamic therapy in Argentina enhancement of NO production from the donor SNP and ALA inhibited NO production from arginine [43]. We have also attempted to elucidate whether NO cytotoxicity and PDT from ALA have independent cell damage mechanisms. We have used again the murine mammary adenocarcinoma cell line LM3 and its NO-resistant variant LM3-SNP obtained after successive exposures to the donor SNP. No cross resistance was found between NO cytotoxicity and ALA-PDT; LM3-SNP cells were not more resistant to ALA-PDT, instead they were more sensitive. We have also induced resistance to ALA-PDT in LM3-SNP cells after multiple cycles of PDT. Then we have isolated two resistant clones identified as Clon 1 and Clon 3, which were 9.2- and 12.5-fold more resistant to ALA-PDT than the parenteral lines, showing that resistance to NO did not interfere in the development of PDT resistance [44]

Porphyrin-derived photosensitisers synthesised in R´ıo Cuarto, C´ ordoba In the National University of R´ıo Cuarto, C´ ordoba, a group is devoted to the synthesis of new porphyrinderived photosensitisers. They study the effects of new porphyrins on photoinactivation of bacterial cells as well as mammalian cell death by photodynamic therapy. They synthesised meso-substituted cationic porphyrins, and the most effective was the tetracationic porphyrin 5-(4-trifluorophenyl)-10, 15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide, which inactivated >99% of Escherichia coli, typical Gram-negative bacterium [47]. The photodynamic activities of a porphyrinC60 dyad (P-C60) which can form a photoinduced charge-separated state, and its metal complex with Zn(II) were compared with 5-(4-acetamidophenyl)10,15,20-tris(4-methoxyphenyl)porphyrin, both in homogeneous medium-bearing photooxidisable substrates and in vitro on the Hep-2-human-larynxcarcinoma cell line. A higher photocytotoxic effect was observed for P-C60, which inactivates 80% of cells after 15 min of irradiation. Moreover, both dyads kept a high photoactivity even under argon atmosphere. Thus, depending on the microenvironment where the sensitiser is localised, these compounds could produce biological photodamage through either an O2 (1delta(g))-mediated photoreaction process or a free-radicals mechanism under low oxygen concentration [48]. The photodynamic activity of a cationic Zn(II) tetramethyltetrapyridinoporphyrazinium salt (ZnPc 2) was compared with that of a noncharged Zn(II) tetrapyridinoporphyrazine (ZnPc 1), both in vitro

211 using human red blood cells and E. coli bacteria. Both phthalocyanines produced similar photohemolysis of red blood cells, reaching values >90% of lysis after 5 min of irradiation with visible light. The photodynamic effect was accompanied by an increase in the membrane fluidity of red blood cells. However, studies on E. coli cells showed that the cationic ZnPc 2 produced a higher photoinactivation of Gram-negative bacteria than ZnPc 1 [49]. Novel asymmetrically meso-substituted cationic porphyrins were also synthesised and it was found that the amphiphilic tricationic porphyrin 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide and its metal complex with Pd(II) were effective in inactivation of E. coli by PDT [50]. A novel 5-[4-(trimethylammonium)phenyl]-10, 15,20-tris(2,4,6-trimethoxyphenyl)porphyrin iodide (2) was synthesised by the same group. A positive charge was incorporated at a peripheral position to increase the amphiphilic character of the structure. The photodynamic effect of the cationic porphyrin was compared with that of noncharged 5-(4-aminophenyl)-10,15,20tris(2,4,6-trimethoxyphenyl)porphyrin (1), on the Hep-2 human larynx carcinoma cell line. It was found a higher photocytotoxic effect for porphyrin 2 in comparison to porphyrin 1 [51]. The photokilling activity of 5-[4-N-(2 ,6 -dinitro 4 -trifluoromethylphenyl) aminophenyl]-10,15,20tris(2,4,6-trimethoxy phenyl) porphyrin was evaluated on a Hep-2 human larynx-carcinoma cell line. They found an apoptotic mechanism of cell death at low irradiation doses, but the mechanism of cell death can be induced depending on the irradiation doses [52].

Clinical experience in C´ ordoba A prototype of a 630 nm noncoherent light source was designed and constructed at the Centre of Electron Microscopy of The Cordoba National University [53]. The prototype consisted of a 400 W arc lamp filled with a special gas mixture that delivers a cold monochromatic beam at 630 nm after appropriate filtering through a bandpass interference filter. The power delivered by the device is about 20 mW/cm2 , with a working area of 25 cm2 . The light is concentrated with reflectors and focused at the window with properly aligned mirrors. The lamp housing is cooled by forced air distributed through appropriate vents. The light source allowed a successful application of PDT, in nonmelanoma skin tumours, for the first time in Argentina. A topical treatment with 20% ALA

212 in aqueous solution was applied in 100 lesions of actinic keratosis of 27 patients. They obtained 84% of complete remissions, 10% of partial remissions and 0% of no responses [54]. Later, in a period of two years they treated around 300 lesions applying 20% ALA and 0.2% taurocholic acid as a diffuser and irradiating afterwards with the noncoherent prototype. In superficial lesions of basal cell carcinoma and squamous cells carcinomas, the responses (total or partial) were 86% and 100%, respectively. In nodular basal cell tumours and in ulcerative squamous cells carcinomas the responses were slightly lower, around 72%. In Bowen’s disease the response was 75% and 94% in actinic keartoses [55].

A. Casas, A. Batlle

[5]

[6]

[7] [8]

[9]

[10]

Conclusions [11]

New ester derivatives of ALA as well as new porphyrin-derived photosensitisers are currently being synthesised and tested in vivo and in vitro models in Argentina. Some clinical studies are also being carried out in nonmelanoma skin cancers employing PpIX synthesised from the precursor ALA. However, none of these compounds have been approved by the national health authorities for their massive use yet. The only photosensitiser approved up to date is Verteporphyrin for the treatment age-related macular degeneration. Cost of the light source and scarce knowledge of the benefits of PDT by physicians limit the spread of the treatment throughout the country.

[12]

[13]

[14]

[15]

[16]

Acknowledgements A.C. and A.B. are Associate and Superior Researchers at the National Research Council—– CONICET.

References [1] Batlle A, Llambias E, Wider E, Tigier H. Porphyrin biosynthesis in soybean callus system -XV. The effect of growth conditions. Int J Biochem 1975;6:591—606. [2] Fukuda H, Paredes S, Batlle A. Tumour-localizing properties of porphyrins. In vitro studies using the porphyrin precursor, aminolevulinic acid, in free and liposome encapsulated forms. Drug Des Deliv 1989;5:133—9. [3] Fukuda H, Casas A, Chueke F, Paredes S, Batlle A. Photodynamic action of endogenously synthesized porphyrins from aminolevulinic acid, using a new model for assaying the effectiveness of tumoural cell killing. Int J Biochem 1993;25:1395—8. [4] Casas A, Fukuda H, Di Venosa G, Batlle A. Photosensitisation and mechanism of cytotoxicity induced by the use

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