Use of 5-Aminolevulinic Acid Esters to Improve Photodynamic Therapy on Cells in Culture'

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[CANCER RESEARCH57, 1481-1486, April 15, 19971 Use of 5-Aminolevulinic Acid Esters to Improve Photodynamic Therapy on Cells in Culture'

Jean-Michel Gauffier,2 Kristian Berg,3 Qian Peng, Helle Anholt, PM Kristian Selbo, Li-Wei Ma, and Johan Moan

Laboratory of Photobiology, National Museum of Natural History, INSERM U.312, 43 rue Cuvier, 75231 Paris Cedex 05, France fJ-M. G.J, and Institute for Cancer Research, Department ofBiophysics. Montebello, N-0310 Oslo, Norway (K. B., Q. P., H. A., P. K. S., L-W. M., J. M.j

ABSTRACT overruled by treating cells with ALA. Several porphyrinogens are produced, from which photoactive porphyrins can be formed. Fortu Human tumor cells of the lines WiDr (adenocarcinoma of the rectosig nately for the treatment of cancer, the ferrochelatase activity needed mold colon), NIIIK 3025 (carcinoma of the cervix), and V79 Chinese for the incorporation of iron into the PpIX macrocycle is low in hamster fibroblasts were treated with 5-aminolevulinic acid (ALA) and ALA esterifled to C@-C3 and C6-C8 chained aliphatic alcohols (ALA neoplastic cells ( I2). esters). In the human cell lines, esterification of ALA with the long-chain Due to the hydrophilic properties of ALA, ALA-PDT may dm1- (C6-C@)alcohols was found to reduce 30-150-fold the amount of ALA cally be limited by the rate of ALA uptake into the neoplastic cells needed to reach the same level of protoporphyrin IX (PpIX) accumulation and/or its penetration through the tissue (13, 14). The penetration of as with non-esterifled ALA. The long-chained ALA-esters were less effi ALA into skin depends on the type and characteristics of the skin. dent in stimulating PpIX formation in V79 cells, i.e., the same amount of ALA seems to penetrate normal human skin poorly compared to skin PpIX was formed by a 1â€”2.6-foldlower concentration of long-chained above malignant lesions (15). Skin that shows signs of chronic sun ALA-esters than with ALA. Short-chained ALA-esters (C1-C3)induced S damage usually has a better permeability for ALA, as do areas of to 10 times lower PpIX accumulation than ALA in all of the cell lines. actinic keratosis (16). However, the hyperkeratosis associated with High-performance liquid chromatography and fluorescence microscopic Verruca vulgaric or some nodular carcinomas greatly inhibits the studies indicated that esterification of ALA has neither impact on the fluorescing porphyrin species formed nor impact on their intracellular penetration of ALA. Clinical work has shown that more than 90% of localization. The PpIX formed from ALA-esters and ALA was found to be superficial BCCs respond well to the topically applied ALA-based equally efficient in sensitizing cells to photoinactivation. The present PDT. But there is a low complete response rate to this treatment for results indicate that esterifIed ALAS are new and promising drugs for use the nodular BCCs, which comprise 45-60% of all of the BCCs. This in photochemotherapy of cancer. has been confirmed by means of microscopic fluorimetry to be due to a limited penetration of ALA (17, 18). Although the penetration of ALA can be enhanced by chemicals (19) or by physical techniques INTRODUCTION (20, 21), the requirements for such methods are strict, and the methods thus far applied are still not optimal. Worldwide, PD'!â€•is being evaluated as a new and promising On the basis of these observations, we propose the use of esterified treatment modality of neoplastic diseases (1, 2). The treatment is ALA. In the present study, it is shown that the same amount of PpIX based on injection of photosensitizing dyes, followed by exposure of is induced in carcinoma cells for concentrations 30â€”I50-fold lower of the tumor area to high fluences of light at appropriate wavelengths. -hexyl, -heptyl or -octyl ALA-esters than in the presence of non Porphyrins and structurally related compounds are applied for treat esterified ALA. ment of cancers, as well as for nontumoral diseases such as psoriasis (3), bacterial and viral eradication (4, 5), vascular stenosis (6), and for tumor detection (7). Nevertheless, the application of PDT remains MATERIALS AND METHODS limited due to the limited penetration of light in tissues, the photo Chemicals. ALA and ALA-methyl ester, provided by Sigma Chemical Co., sensitization of normal tissues, and the remanent skin photosensitivity were dissolved in Dulbecco's PBS, and the pH was adjusted to 7.0 by the observed for at least 4â€”6weeks after treatment (8, 9). The advantages addition of 5 MNaOH. ALA-ethyl, -propyl, -hexyl, -heptyl, and -octyl esters, of this method, as compared to other conventional cancer treatment kindly provided by Photocure (Oslo, Norway), were dissolved in ethanol modalities, are its low systemic toxicity and its ability to destroy (stock solutions) and further diluted in serum-free culture medium (with final tumors selectively (1, 2). concentration of ethanol less than 1%).The stock solutions (25 mM)were made A somewhat different approach to PDT of cancer is based on the same day as they were used. PpIX, coproporphyrin, and uroporphyrin were endogenous accumulation of PpIX after topical or systemic adminis used as standards for HPLC. These drugs, provided by Porphyrin Products tration of ALA (10). The initial step in the porphyrin and heme (Logan, UT), were dissolved in 30% methanol containing 1.5 mMphosphate synthesis pathway is the ALA synthase-induced formation of ALA and adjusted to pH 7.0 before injection in HPLC. from succinyl-CoA and glutamate, regulated by the feedback inhibi Cell Cultivation. NHIK 3025 cells, derived from a carcinoma in situ of the cervix (22), were subculture-I in E2a medium (23) containing 10% human tion by heme (11). The regulation of this pathway can, therefore, be serum and 10% horse serum. V79 Chinese hamster lung fibroblasts were subcultured in MEM medium (Life Technologies, Inc.) containing 10% FCS. Received 7/18196; accepted 2/14/97. WiDr cells, derived from a primary adenocarcinoma of the rectosigmoid colon The costs of publication of this article were defrayed in part by the payment of page (45), were subcultured in RPM! 1640 (Life Technologies, Inc.) containing 10% charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. FCS. All of the media contained 100 units/ml penicillin, 100 @tg/mlstrepto I This work was supported by the Research Council of Norway (NFR) and by the mycin, and 1% glutamine. Cells were subcultured two limes per week (split National Institute of Health and Medical Research (INSERM), as part of a researchers' ratio, 1:100) and maintained at 37Â°Cand 5% CO2 in a humid environment. exchange program between Norway and France. It has also been supported by the Labeling with ALA/ALA-esters and Irradiation. Four X l0@WiDrcells Commission of the European Communities through the program â€œHumanCapital and Mobility Programmeâ€• (PDT Euronet). were inoculated in 25-cm2 plastic tissue-culture flasks (Nunclon) and left for 2 Visiting scientist in Oslo. 42 h for proper attachment to the substratum. The cells were then washed twice 3 To whom requests for reprints should be addressed. Phone: 47 22 93 42 60; Fax: 47 with RPMI 1640 without serum and incubated for 4 h in RPM! 1640 contain 22 93 42 70; E-mail: [email protected]. ing ALA or ALA-esters, without serum to avoid porphyrin excretion to the 4 The abbreviations used are: PDT, photodynamic therapy; ALA, 5-aminolevulinic acid; ALA-esters, esterified derivatives of 5-aminolevulinic acid; PpIX, protoporphyrin culture medium (25). The cells were exposed to light from a bank of four IX; BCC, basal cell carcinoma; HPLC, high-performance liquid chromatography. fluorescent tubes (model 3026; Applied Photophysics, London, United King 1481

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1997 American Association for Cancer Research. ALA ESTERS AND PDT dom) emitting light mainly around 405 nm (26). The fluence rate of the light a mixture of methanol and water (30:70 by volume) containing 1.5 mM reaching the cells was I5 W m 2 as measured by means of a calibrated UDT phosphate, adjusted to pH 7.0. The end solvent was a mixture of methanol and detector (Ysi-Ketterimg model 65A radiometer). The medium was removed water (95:5 by volume) containing 1.5 mMphosphate, adjusted to pH 7.5. A immediately after illumination and replaced with ALA/ALA-ester-free RPMI 30-mm linear gradient between 30 and 95% of methanol was applied, followed 1640 containing 10% FCS. by 5 rain at 95% of methanol. Porphyrins were detected by fluorescence, with Cell Survival. Twenty-four h after photochemical treatment, the WiDr excitation at 330â€”400 nm. Scattered light was eliminated from the fluores cells were washed with 0.9% NaCI solution, fixed in ethanol, and stained with cence by means of a cutoff filter transmitting only light with wavelengths methylene blue. The cell survival was then determined by measuring the longer than 410 nm. absorption of methylene blue at 600 nm with a Shimadzu UV-160A spectro Fluorescence Microscopy Three X iO@WiDrcells were seeded in 10-cm2 photometer (Tokyo, Japan). The results were expressed in percentage of dishes (Falcon). Forty-two h after seeding, the cells were incubated in serum absorption from unin'adiated cells. Because we worked with high cell density free RPMI 1640for 4 h with ALA or ALA-esters and then washed three times to detect PpIX accumulation, the use of conventional colony-forming assay with ice-cold PBS; finally, a cover glass was gently put on top of the PBS (requiring low cell density) was not performed because of the cell density layer. For comparison, other WiDr cells were treated with 10 ,.tM rhodamine dependent formation of ALA-induced PpIX (27, 28). I23 for 10 mm in serum-free culture medium before preparation for micros Measurements of Cellular PpIX Contents. For all cell lines, 2 X l0@ copy as described above. The cells were subsequently studied by a Zeiss cells were seeded into 10-cm2 dishes (Nunclon) and treated with ALA or Axioplan microscope with epifluorescence equipment. A HBO/lOOW mercury ALA-esters in serum-free culture medium 42 h later, as described above. After lamp was used for excitation. The microscope was connected to a cooled treatment, the cells were washed once in PBS and brought into a solution charge-coupled device camera (TE2; Astromed, Cambridge, United Kingdom). containing I M HCIO4 in 50% methanol by scraping with a Costar cell scraper. A computer controlled the camera operations and was used for digital image After 5 mm of incubation, the cell debris was removed by centrifugation. PpIX processing and storage. The microscope was equipped with a 390â€”440-nm was quantitatively extracted from the cells with this procedure. The PpIX band pass excitation filter, a 470-nm dichroic beam splitter, and a 610-nm content of the samples was detected fluorometrically using a Perkin-Elmer long-pass filter for detection of porphyrin fluorescence. For detection of LS5OB spectrofluorimeter. PpIX was excited at 405 nm, and the emitted rhodamine 123 fluorescence, the microscope was equipped with a 450â€” fluorescence was measured at 605 nm. A long-pass cutoff filter (530 nm) was 490-nm band pass excitation filter, a 5 10-nm dichroic beam splitter, and a used on the emission side. A standard of known concentration was added to the 520-nm long-pass filter. In each case, we used a X40 objective plus a X2.5 samples at a concentration increasing the total fluorescence by 50â€”100%. magnifier. Results were corrected for protein contents as measured by the Lowry method (29). HPLC. The porphyrins were extracted from 2 X 106WiDr cells seeded out RESULTS in 75-cm2 plastic tissue-culture flasks (Nunclon) 42 h before treatment by scraping the cells in acidified methanol (5 Mlof concentrated HC1in 10 ml of Porphyrin Synthesis in WiDr, NHIK 3025, and V79 Cells. methanol). The cell debris was pelleted, and the supernatants were collected. Porphyrin synthesis mediated by a 4-h treatment with ALA and The porphyrins were concentrated by flushing the extracts with N2 until the ALA-methyl, -hexyl, -heptyl, and -octyl esters was compared in volumes were reduced to approximately 150â€”200p3and additionally precip WiDr, NHIK 3025, and Chinese hamster V79 fibroblasts cells. In each itated proteins were pelleted. One hundred p3 of the supernatants were mixed cell line, a concentration-dependent formation of PpIX was observed with 235 pJ of 10 mM Na,P04, pH preadjusted to approximately 10.5 by means (Fig. 1), which was slightly sigmoidal-shaped on linear scales (data of 5 M NaOH, and directly used for HPLC analysis. The porphyrins were quantitatively extracted from the cells by this procedure (30). not shown). The same amount of accumulated porphyrins was ob The HPLC system consisted of a pump (Spectra Physics 8800), a reversed tamed with long-chain ALA-esters at concentrations 30 to 150 times phase column (Supelcosil LC-l8-T; 4.6 X 250-mm; Sulpeco, S.A., Gland, lower than with ALA in the human tumor cells and 1 to 2.6 times Switzerland), a fluorescence detector (LDC fluoromonitor III), and an integra lower in the Chinese hamster fibroblasts, when measured as the tor (Spectra Physics Data-jet) connected to a computer. The start solvent was ALA-ester concentration needed to obtain one-half of the maximum

450 â€”:-! 400350

.@ 300 Dl :@â€˜250

,@ 200 C 0 U 150 â€˜C @-100

Concentration (mM)

Fig. 1. PpIX accumulation in wiDe- (A), NHIK 3025 (B), and V79 cells (C) induced by 4-h treatment with ALA (0), ALA-methyl ester (Lx), ALA-hexyl ester (A), ALA-heptyl ester (U), and ALA-octyl ester (@) in serum-free culture medium as described in â€œMaterialsandMethods.â€•ThePpIX production is corrected for protein content. Bars, SD, from three experiments in A and two experiments in B and C. In all cases, experiments were performed in triplicate. 1482

accumulation was 12â€”20times lower with ALA and ALA-methyl @@ethyl esters than with long-chain ALA-esters. The accumulation of PpIX was in all cases linear the first 8 h of treatment and continued to I - @,4ethyl WN@,.@proPyl increase almost linearly, even beyond that time when the WiDr cells were incubated with 100 @.LMALA/ALA-esters. The accumulation @ @ILA reached a maximum after 8 h of incubation when the cells were treated with 10 @MALA-heptyl and -octyl esters. This may be due to E consumption of a significant fraction of the ALA-esters or degrada C 0 0.1 - tion during the incubation (32). Analysis of Accumulated Porphyrin Intermediates. In our stud ies of cellular porphyrin contents, the fluorescence emission spectra were similar to that of PpIX and slightly red-shifted compared to the two other main intermediates in the porphyrin synthesis pathway, uroporphyrin and coproporphynn, indicating that mainly PpIX was I -hexyl 0.01 . -heptyl formed (33). HPLC analysis of extracts from WiDr cells treated for -octyl 4 h with ALA or ALA-esters also showed that mainly PpIX was formed (data not shown; see Ref. 30). No significant amounts of other porphyrins than PpIX were detected, indicating that esterification of ALA mainly influenced the rate of ALA uptake into the cells. 0 1 2 3 4 5 6 7 8 Number of carbon atoms Fig. 2. Concentration of ALA and ALA-ester derivatives needed to induce one-half of the PpIX maximum accumulation induced by ALA in WiDr cells. Bars, SD, from two sets of experiments performed in triplicate. 500

obtain by ALA. In contrast, ALA-methyl ester was needed in con centration 5â€”10times higher than that of ALA needed to produce the 400 same PpIX concentration. The accumulation of PpIX from ALA-ethyl and -propyl esters was found to be similar to that of ALA-methyl ester in WiDr cells (data not shown). The maximum of PpIX production 300 (expressed in mg/mg protein) induced in WiDr cells was 330 Â±10 by ALA, 150 Â±5 by ALA-methyl ester, and 360â€”420 by ALA-hexyl, -heptyl, and -octyl esters. The maximum PpIX accumulation in each 200 cell line treated with long-chain esters was reached at about 0.05â€”0.1 mM ALA-esters, above which PpIX accumulation became lower. This a Dl 100 reduction in PpIX accumulation was followed by a reduction in total E protein content in each sample, indicating cytotoxic effects of the Dl C treatment with such high concentrations (data not shown). The cyto toxic effects of ALA on cells in culture may be due to different C .@ molecular events induced by ALA, such as release of Ca2@ from C 1200 0 mitochondria, mitochondrial swelling and uncoupling respiration U (31), formation of 8-hydroxy-2'-deoxyguanosine in DNA (24), or â€˜C a 1000 release of iron from femtin (30). 0. Influence of the Esterified Chain Length on PpIX Synthesis. The dependency of PpIX production in WiDr cells with the chain 800 length of esterified ALA derivatives was determined as the ALA-ester concentrations needed to obtain one-half of the maximum PpIX production obtained with ALA (Fig. 2). Thus, in WiDr cells, the same 600 amount of PpIX was induced by 400 @LMALA,800-1500 @LMALA methyl, -ethyl, -propyl esters, and by 3â€”6 @MALA-hexyl, -heptyl, 400 and -octyl esters. These results indicate that esterification by aliphatic alcohols with carbohydrate chains lower than C4 leads to a PpIX accumulation less efficiently than ALA, whereas ALA esterified by 200 aliphatic alcohols with carbohydrate chains equal or longer than C6 leads to a PpIX accumulation superior to that observed with ALA. Pharmacokinetics of PpIX Accumulation. The accumulation of PpIX was studied up to 24 h after the addition of ALA and ALA esters to WiDr cells (Fig. 3). The experiments were performed in the Time (hour) absence of serum. The total content of cell protein in each dish Fig. 3. Pharinacokinetics of PpIX synthesis in WiDr cells treated with ALA (0), increased by less than 15% during the time of the experiments (data ALA-methyl ester (i@),ALA-hexyl ester (A), ALA-heptyl ester (U). ALA-octyl ester (s), and control cells treated with serum-free medium only (0). The cells were treated in not shown). At 10 @LM(Fig.3A), the PpIX accumulation was induced serum-free RPM! 1640 as described in â€œMaterialsand Methodsâ€•with 10 @M(A)and 100 only by long-chain ALA-esters. At 100 @M(Fig. 3B), the rate of PpIX @LM(B) of ALA/ALA-esters. Bars, SD, from experiments performed in triplicate. 1483

Fig. 4. Fluorescence micrographs of WiDr cells treated with 1 mM ALA (A) and 0.1 inst ALA-hexyl ester (B) for 4 h in serum-free medium as described in â€œMaterialsandMethods.â€• For comparison, one micrograph of rhodamine 123-treated WiDr cells is given (C).

Intracellular Localization of PpIX. The intracellular localization DISCUSSION of PpIX in WiDr cells after 4 h incubation with ALA and ALA-esters The initial step in the ALA-induced synthesis of porphyrins is the was studied by fluorescence microscopy (Fig. 4). The comparison of cells treated with ALA (Fig. 4A) and with ALA-hexyl ester (Fig. 4B) penetration of ALA through the plasma membrane. ALA is a hydro clearly indicated that there was no difference in the distribution of the philic molecule, and its hydrophilicity may be a clinical limitation for fluorescence emitted by PpIX. The same distribution of porphyrin the use of ALA in PDT because of the low rate of hydrophilic drug induced fluorescence was observed with all of the ALA-esters (data uptake in cells and/or the poor penetration of such drugs through the not shown). The fluorescence was emitted from well-defined spots in tissue (13â€”15).In the present study, esterification of ALA with the cytoplasm. Additionally, a diffuse fluorescence was seen in the aliphatic alcohols was found to reduce 30â€”150-fold the amount of entire cytoplasm, whereas practically no fluorescence was found in drug needed to reach the same level of PpIX accumulation in two the nuclear region. The fluorescence micrographs suggest association different carcinoma cell lines (WiDr and NHIK 3025 cells). Further of some PpIX with the plasma membrane. Comparative studies with more, the maximum PpIX accumulation reached in WiDr cells was the mitochondrially located rhodamine 123 indicate that PpIX is not higher when induced by long-chain ALA-esters than by ALA, mdi located in mitochondria, or that incubation with ALA perturbed the dating that uptake of ALA and ALA-esters may proceed by different mitochondrial morphology as compared to rhodamine 123-treated mechanisms. The mechanism and rate of ALA-uptake in human cells cells (Fig. 4C). are not known (34). In Saccharomyces cerevisiae (35), it has been Phototoxicity Induced by ALA and ALA-esters. The photocy demonstrated that ALA uptake is reduced by substances harboring a totoxicity induced by ALA and ALA-esters was assessed 24 h after methyl-amine terminus. In eukaryotic cells, ALA may be taken up via light exposure. For comparative reasons, in one set of experiments a transmembrane channel, by active transport or facilitated diffusion cells were incubated with ALA and ALA-methyl ester to produce across the plasma membrane (36). ALA-esters, which are more li 125 Â± 5 ng of PpIX/mg protein in all cases. In another set of pophilic may: (a) diffuse passively across the plasma membrane, and experiments, WiDr cells were treated with ALA and long-chain thereby bypass totally or in part potential transport mechanism of ALA-esters to produce 180 Â± 10 ng of PpIX/mg protein in all ALA; or (b) use another active or facilitated transport mechanism than cases. No significant difference in the photocytotoxicity between ALA. ALA and ALA-esters was observed, i.e., 20% of cell survival was ALA-methyl, -ethyl and -propyl esters have recently been shown to reached after exposure to 0.5 Â±0.04 J/cm2 when the cells were induce more porphyrin fluorescence than ALA in normal mouse skin treated with ALA and ALA-methyl ester and after exposure to (37). The present results indicate that this is not due to enhanced 0.36 Â±0.04 J/cm2 in the other case. The photosensitizing efficacy uptake of these compounds into the cells of the skin but rather to was dependent on the amount of PpIX per cell and the fluence enhanced rate of penetration through the interstitial space ofthe tissue. applied, i.e., the â€œphotochemical doseâ€•(defined as the product of The low formation of PpIX in cells treated with short-chained ALA the PpIX amount in the cells and the fluence to reach 20% of cell esters may be due to low rate of uptake through a transport protein and survival) was 62.5 PpIX/mg protein X J cm2 (125 ng of PpIX/mg slow passive diffusion, or less likely, to a slow rate of intracellular protein X 0.5 J cm2) in the ALA/ALA-methyl ester experiments deesterification. The production of PpIX induced by ALA-esters is and 64.5 PpIX/mg protein X J cm2 (180 ng of PpIX/mg pro related to the aliphatic length of the alcohol used for esterification. tein X 0.36 J cm2) in the ALA/ALA long-chain experiments. The present results indicate that the maximum benefit of esterifying Thus, these experiments indicated that PpIXs formed from ALA ALA for the formation of PpIX in vitro is obtained by esterification and ALA-esters are equally efficient in sensitizing the cells to with hexanol. photoinactivation. The enhancement ratio induced by esterification of ALA on PpIX 1484

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1997 American Association for Cancer Research. ALA ESTERS AND PDT accumulation was found to be cell line dependent. Although long 5. Rywkin, S., Ben-Hur, E., Malik, Z., Prince, A. M., Li, Y. S., Kenney, M. E., Oleinick, chain ALA-esters were 30â€”150-fold more efficient in inducing PpIX N. L., and Horowitz, B. New phthalocyanines for photodynamic virus inactivation in red blood cell concentrates. Photochem. Photobiol., 60: 165â€”170,1994. in WiDr and NFHK 3025 cells, the efficiency was only 1â€”2.6times 6. Dartsch, P. C., Coppenrath, E., Coppenrath, K., and Ischinger, T. Photodynamic better in the fibroblast cell line V79. This could be due to: (a) a low therapy of vascular stenosis: results from cell culture studies on human endothelial esterase activity present in fibroblasts, or (b) the lower levels of ALA cells. Coron. Artery Dis., 4: 207â€”213,1993. 7. Andersson-Engels, S., Johansson, J., Svanberg, K., and Svanberg, S. Fluorescence needed to reach a maximum of PpIX accumulation in V79 cells (â€œ0.1 imaging and point measurements of tissue: applications to the demarcation of malig mM) compared to that in the carcinoma cell lines (1â€”2nmi). nant tumors and atherosclerotic lesions from normal tissue. Photochem. Photobiol., 53: 807â€”814,1991. The use of esterified ALA is highly dependent on intracellular 8. Wooten, R. S., Smith, K. C., Ahlquist, D. A., Muller, S. A., and Balm, R. K. esterase activity. This could be a limitation for the use of ALA-esters Prospective study of cutaneous phototoxicity after systemic hematoporphyrin deriv in PDT, i.e., in some cases the esterase content is higher in tumoral ative. Lasers Surg. Med., 8: 294â€”300, 1988. 9. Dougherty, T. J., Cooper. M. T., and Mang, T. S. Cutaneous phototoxic occurrences cells than in their normal counterparts (38, 39), whereas the opposite in patients receiving Photofrin. Lasers Surg. Med., 10: 485â€”488, 1990. is verified in other cases (40, 41). Nevertheless, preliminary clinical 10. Kennedy, J. C., and Pottier, R. H. Endogenous protoporphyrin IX, a clinically useful studies of human nodular BCCs have shown that the fluorescence photosensitizer for photodynamic therapy. J. Photochem. Photobiol. B Biol., 14: 275â€”292,1992. from ALA ester-induced porphyrins is built up more rapidly, to a 11. Moore, M. R., McColl, K. E. L., Rimigton, C., and Goldberg, A. Disorders of higher intensity, and to a more homogeneous distribution than seen in Porphyrin Metabolism. New York: Plenum Publishing Corp., 1987. 12. Dailey, H. A., and Smith, A. Differential interaction of porphyrins used in photora the case of free ALA-induced porphyrins (37). diation therapy with ferrochelatase. Biochem. J., 223: 441â€”445, 1984. ALA and ALA-esters lead to the same intracellular localization of 13. Caimduff, F., Stringer, M. R., Hudson, E. J., Ash, D. V., and Brown, S. B. Superficial fluorescing porphyrins, and no other intermediates were formed by photodynamic therapy with topical 5-aminolaevulinic acid for superficial primary and secondary skin cancer. Br. J. Cancer, 69: 605â€”608, 1994. esterification of ALA. Thus, the esterification of ALA has no detect 14. Wolf, P., Rieger, E., and Kerl, H. Topical photodynamic therapy with endogenous able effects on the pathway leading to PpIX formation or on the porphyrins after application of 5-aminolevulinic acid. An altemative treatment mo intracellular distribution of PpIX. However, the intracellular localiza dality for solar keratoses, superficial squamous cell carcinomas, and basal cell carcinomas? J. Am. Acad. Dermatol., 28: 17â€”21,1993. tion of fluorescing porphyrins induced by ALA seems to be cell line 15. Warloe, T., Peng, Q., Steen, H. B., and Giercksky, K. Localization of porphyrins in dependent. The partly granular localization found in WiDr cells was human basal cell carcinoma and normal tissue induced by topical application of 5-aminolevulinic acid. In: P. Spinelli, M. Dal Fante, and R. Marchesini (eds.), not observed in V79 cells after identical treatment conditions (30). Photodynamic Therapy and Biomedical Lasers, pp. 454â€”458. Amsterdam: Elsevier The staining of WiDr cells with a specific dye for mitochondria led to Science Publishers B. V., 1992. a different fluorescence pattern than treatment with ALA or ALA 16. Calzavara-Pinton, P. G. Repetitive photodynamic therapy with topical &-amin olaevulinic acid as an appropriate approach to the routine treatment of superficial esters, indicating that PpIX induced by ALA and ALA derivatives is non-melanoma skin tumours. J. Photochem. Photobiol. B Biol., 29: 53â€”57, not present only in mitochondria. Also, the plasma membrane, and 1995. other cytoplasmic compartment(s), such as lysosomes, may accumu 17. Peng. Q., Warloc, T., Moan, J., Heyerdahl, H., Steen, H. B., Nesland, J. M., and Giercksky, K. E. Distribution of 5-aminolevulinic acid-induced porphyrins in nodu late PpIX because it has been shown in the case of keratinocytes loulcerative basal cell carcinoma. Photochem. Photobiol., 62: 906â€”913, 1995. NCTC 2544 (42). In other cases, differences in treatment conditions 18. Warloe, T., Peng. Q.. Moan, J., Qvist, H. L., and Giercksky, K. E. Photochemotherapy may explain the differences in intracellular localization patterns seen of multiple basal cell carcinoma with endogenous porphyrins induced by topical application of 5-aminolevulinic acid. In: P. Spinelli, M. Dal Fante, and R. Marchesini (30, 42). It should be noted that the type of porphyrin formed depends (eds.), Photodynamic Therapy and Biomedical Lasers, pp. 449â€”453. Amsterdam: on the cells used, e.g., in B 16 melanoma cells, 95% of the fluorescing Elsevier Science Publishers B. V., 1992. 19. Orenstein, A., Kostenich, G., Tsur, H., Roitman, L., Ehrenberg, B., and Malik, Z. porphyrins are PpIX after ALA incubation (43), whereas other por Photodynamic therapy of human skin tumors using topical application of 5-aminole phynn intermediates may be formed in other cell lines (44). vulinic acid, DMSO and EDTA. in: D. Brault, G. Jori, J. Moan, and B. Ehrenberg The experiments with the WiDr cells showed that for the same (eds.), Photodynamic Therapy of Cancer II, pp. 100â€”105.Proceedings of the Inter national Society of Optical Engineering, 1994. amount of PpIX produced, the same photocytotoxic efficiency was 20. Levy, D., Kost, J., Meshulam, Y., and Langer, R. Effect of ultrasound on transdennal obtained. Therefore, in agreement with our studies of PpIX formation, drug delivery to rats and guinea pigs. J. Clin. Invest., 83: 2074â€”2078,1989. HPLC analysis, and fluorescence microscopy, even if all ALA-esters 21. Luxenberg. M. N., and Guthrie, T. H., Jr. Chemotherapy of basal cell and squamous cell carcinoma of the eyelids and penorbital tissues. Ophthalmology, 93: 504â€”510, are not completely deesterified, they do not interfere with the photo 1986. chemical treatment. Esterification of ALA by long-chain aliphatic 22. Nordbye, K., and Oftebro, R. Establishment of four new cell strains from uterine cervix. I. Exp. Cell Res., 58: 458, 1969. alcohol may, therefore, offer a promising way to improve ALA-PDT 23. Puck, 1. T., Cieciura, S. J., and Fisher, H. Clonal growth in vitro of human cell with by increasing the intracellular PpIX accumulation, probably due to a fibroblastic morphology. J. Exp. Med., 106: 145â€”165,1957. better cellular uptake. ALA-esters may penetrate deeper into the 24. Fraga, C. G., Onuki, J., Lucesoli, F., Bechara, E. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1997 American Association for Cancer Research. Use of 5-Aminolevulinic Acid Esters to Improve Photodynamic Therapy on Cells in Culture

Jean-Michel Gaullier, Kristian Berg, Qian Peng, et al.

Cancer Res 1997;57:1481-1486.

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