zyxwvuts zyxwvu zyx Photochemistry and Photobiology, 1998,68(3): 382-387 Clinical Evaluation of the Cutaneous Phototoxicity of 5,10,15,20=Tetra (m-hydroxypheny1)chlorin Georges Wagnieres*l, Christophe Hadjur’, Pierre Grosjeanz, Daniel Braichottel, Jean-FranGois Savary2, Philippe Monnier2and Hubert van den Berghi f ~2 Institute of Environmental Engineering, Swiss Federal Institute of Technology, Lausanne, Switzerland and Department of Otolaryngology, Head and Neck Surgery, CHUV Hospital, Lausanne, Switzerland Received 28 January 1998; accepted 2 June 1998 ABSTRACT intravenous (iv) injection. The region where a cancer is located is then irradiated with a controlled light dose at a The cutaneous phototoxic reaction induced by intravewavelength that corresponds to an absorption maximum of nous injection of 5,-10,-15,-20-tetra(m-hydroxyphen- the PS. Light at 630 nm is the most frequently used waveg1)chlorin (mTHPC) has been clinically evaluated in palength for PDT based on the above-mentioned porphyrins tients undergoing photodynamic therapy. These tests due to the relatively large penetration depth of red light in were performed on the backs of 23 patients with a solar most of the soft tissues. For very superficial lesions such as simulator at various times after drug administration early stage squamous cell carcinomas (SCC) the treatment ranging from 5 h to 57 days. The mTHPC doses ranged is sometimes performed in the green, at 514 nm, with the from 0.1 to 0.3 mgkg, and the illuminations lasted from same efficacy as with red light (10-15). Photodynamic ther30 s up to 8 min. These tests have shown that the duraapy using HPD or Photofrin has the potential of curing early tion of the skin photosensitizationinduced after a typical cancers. In particular, it has been successfully used as an therapeutic dose of mTHPC (0.15 mgkg) is less imporendoscopic treatment for early carcinomas in the bronchi and tant than with Photofrinn (2 mgkg). The level of mTHPC the gastrointestinal tract (10-14,16-20). However, HPD and in the skin was also assessed in vivo and at times correPhotofrin suffer from a number of significant drawbacks: (1) sponding to the irradiations using an optical fiber-based HPD consists of poorly defined and variable mixture of mulspectrofluorometer. This study indicates that the lighttiple active components (21), (2) they have a moderate phoinduced fluorescence spectroscopy of mTHPC enables totoxicity requiring relatively high drug and light doses, ( 3 ) prediction of the degree of photosensitivity of the skin. their fluorescence quantum yield is low (22), (4) they induce long-term skin photosensitization (23,24), and ( 5 ) the wavelength (630 nm) usually used for HPD- or Photofrin-PDT is INTRODUCTION frequently not optimal in terms of light penetration in the tissues. Photodynamic therapy (PDT)? has been studied clinically by 5 , 10, 15, 20-Tetra-(m-hydroxyphenyl)chlorin(mTHPC) is numerous investigators for treating both early and advanced a second-generation PS that has been investigated in both cancer stagings (1-9). This method is potentially low in cost, preclinical and preliminary clinical studies with promising with few side effects and it may be applied repetitively for results (10,11,13,14,25-30). It is superior to HPD and Phothe selective treatment of malignant tumors after administratofrin in many respects: (1) it is a single component of 98% tion of a phototoxic drug, mostly hematoporphyrin derivative (HPD), a mixture of porphyrins or its somewhat purified purity, (2) it has an absorption coefficient in the red, at 652 form Photofrin” (generic name: porfimer sodium). The latter nm, which is larger than that of HPD and Photofrin by about is the first photosensitizer (PS) that achieved formal governone order of magnitude, (3) it is much more phototoxic than mental recognition for the treatment of various cancers by HPD and Photofrin for a given light dose absorbed by the PDT. Such porphyrins tend to localize somewhat selectively PS (14) and (4) it has a large fluorescence quantum yield in tumors as compared to the surrounding normal tissue after (20%) (31). Thus, mTHPC seems to be an appropriate candidate for further clinical testing of PDT. A major drawback of most PS used today systemically for PDT is the skin *To whom correspondence should be addressed at: Institute of Enphotosensitization. Hence, it is necessary to evaluate this vironmental Engineering, BLtiment de chimie, EPFL, CH-1015 side effect. In fact, the skin reactions observed in 12 out of Lausanne, Switzerland. Fax: +41 (0) 21 693 36 26; e-mail: georges.wagnieres @ epfl .ch the 66 patients of the CHUV Hospital in Lausanne, Swit?Abbreviations: HPD, hematoporphyrin derivative; iv, intravenous; zerland, treated with mTHPC-PDT (10,11,13,14), are the LIF, light-induced fluorescence; mTHPC, tetra(rn-hydroxyphenreason for the systematic cutaneous phototoxicity study rey1)chlorin; PDT, photodynamic therapy: PS, photosensitizer; SCC, ported here. These undesirable skin reactions, never obsquamous cell carcinomas. $5.00+0.00 B ) 1998 American Society for Photobiology 0031-8655/98 served later than the first week after injection of mTHPC, zyxwvutsrq zyxwvutsr zyxwvu zyxwvutsrqpon 382 Photochemistry and Photobiology, 1998,68(3) 383 included redness, edema and occasionally cutaneous blistering of the hands and face. Systematic skin photosensitivity tests were carried out with a filtered xenon light source that simulates the solar spectral irradiance at noon on a clear summer day at sea level at our latitude (45" northern latitude). Light-induced fluorescence (LIF) spectroscopy of the skin was also used to assess the level of rnTHPC in this organ. 'This information was used to predict the degree of photosensitivity of the skin. MATERIALS AND METHODS zyxwvu zyxwvutsr zyxw i i 0 Chemicals. The mTHPC (C,,H,,N,O,) (molecular weight: 680.76) was provided as a lyophilized powder by Scotia QuantaNova Ltd., Guildford, UK, in 20 mg vials. This moderately hydrophobic product is dissolved in the contents of a 5 mL vial containing ethanol 96% (I g). polyethylene glycol 400 (1.5 g) and water, prior to iv injection. Less than 30 min after its preparation, it was administered over a period of 5-10 min through a bacterial filter. Clinical skin irradiation tests. The protocol presented below was accepted by the Ethics Committee of the Medical Department of the University of Lausanne, Switzerland. Enrollment was voluntary and each patient gave written informed consent prior to mTHPC administration. All patients involved in this study had at least one early SCC of the bronchi or the esophagus. The mTHPC was administered to treat these lesions by PDT. Patients were cautioned to strictly avoid direct sunlight for 2 weeks after the drug administration and, for safety, to test their skin photosensitivity by brief exposure to sunlight at regular intervals during the next 2 4 weeks. The skin photosensitization was systematically evaluated with a solar simulator on the backs of 23 patients every week up to 7 weeks after iv injection of 0.1 (2 patients), 0.15 (17 patients) and 0.3 mg (4 patients) of mTHPC per kg of body weight. The irradiations took place during 0.5, 1, 2, 4 and 8 min, which correspond to 3, 6, 12, 24 and 48 J/cm2 of white light (between 380 and 700 nm), respectively. The light power was measured with a Scientech (type 36000 1, Boulder, CO) surface-absorbing calorimeter, the spectral response of which is flat and calibrated with an accuracy of 3% between 250 nm and 35 pm. The skin was irradiated with a 300 W xenon (ILC Technology Inc., Sunnyvale, CA) white light source equipped with a color filter (Schott FG16) to simulate the shape of the solar spectrum in the visible range (380-700 nm) at noon on a clear day in the summer at sea level at our latitude. The light intensity in this spectral window was 100 mW/cm2 at the distal end of a 120 cm long (diameter: 5 mm) flexible liquid lightguide (series 700, Lumatec, Munich, Germany), which is about two times larger than the solar intensity in the same spectral window. This photometric configuration was chosen in order to reduce the time of exposure while avoiding thermal effects. The spot diameters were large enough ( > I 5 mm) to avoid significant edge effects (32). The spot of homogenous light intensity was generated by a lens (Spindler & Hoyer; f = 16 mm) that imaged the lightguide output on the skin of the patient. The skin reaction was evaluated 48 h after irradiation according to the following semiquantitative classification: 0, no reaction; 1, light erythema; 2, moderate erythema; 3, pronounced erythema; 4, papular erythema. The delay of 2 days between the irradiation and the assessment of the skin reaction corresponds to the highest degree of tissue reaction. More importa.nt tissue reactions such as blister formation and necrosis have never been generated deliberately for ethical reasons and are therefore not included in this scale. LIF spectroscopy of mTHPC. The inTHPC fluorescence spectroscopy has also been measured on the skin of 11 patients injected with 0.1 mg/kg (1 patient), 0.15 mglkg (7 patients) and 0.3 m g k g (3 patients), just before the irradiation tests. Such measurements were performed on the back, in areas that were not used for the irradiation tests. The configuration of the optical fiber-based spectrofluorometric apparatus, used to make these fluorescence measurements, has been described in detail elsewhere (33). Briefly, the fluorescence excitation wavelength at 420 nm (full width at half maximum: 10 nm) was selected by passing the light of a short arc xenon lamp through a monochromator. This excitation light was transmit- 1 2 3 4 5 6 WEEKS AFTER INJECTION 7 Figure 1. The skin reaction level observed with 17 patients as a function of the time after injection of 0. 15 mglkg of mTHPC per kg of body weight. The different periods of illumination (0.5, 1, 2, 4 and 8 min) correspond to sunlight between 380 and 700 nrn at fluences of 3, 6, 12, 24 and 48 J/cm2. Each point represents the mean result with error bars corresponding to the 67% confidence interval. zyxwvutsrqpon ted through a dichroic mirror prior to injection via a microscope objective in a 0.6 mm core-diameter quartz optical fiber. The distal end of the fiber was positioned in direct contact with the skin and the power of the excitation light at this distal end was 5 pW. The fluorescence spectrum induced in the tissue by the light at 420 nm was collected by the same optical fiber, reflected by the dichroic mirror, filtered to remove the excitation light and focused onto the entrance slit of a spectrograph that was coupled to an intensified diode array. The acquisition time was about 1 s. No photobleaching can be detected at such low power levels and short acquisition times. Each mTHPC LIF spectra was at least the average of four measurements. The signal obtained was displayed on an optical multichannel analyzer and can be further processed by a microcomputer. The detail of the signal processing for extracting the mTHPC LlF signals at 652 nm from the background generated by the tissue autofluorescence is described elsewhere (34). In order to compare the mTHPC fluorescence between different measurements and between different patients, the power of the excitation light and the intensity of the fluorescence spectrum induced by this light in a fluorescent reference solution were measured. This allows us, for instance, to correct for the variations of the excitation light intensity and slight changes in the optical alignment. Stutisticul analysis. Each point in Figs. 1 and 2 represents the mean tissue reaction obtained with 17 and 23 patients, respectively. Each point in Fig. 3 represents the mean tissue fluorescence averaged over four measurements on each of 11 patients. The error bars correspond to one standard deviation in these three figures. The significance of the different skin reaction observed between various solar light doses and drug doses was determined using the nonparametric Mann-Whitney U-test (a 5 0.05). zyxwvutsrq zyxwvuts zyxwvutsrq RESULTS The results of the skin photosensitivity tests obtained after the administration of 0.15 m g k g of mTHPC are presented in Fig. 1. Such a drug dose induces a measurable skin photosensitization up to about 6 weeks after injection in the 8 min irradiations. As expected, a reduction of the light dose reduces significantly the tissue reaction, no matter what the injection-irradiation delay. This indicates that no measurable saturation of skin phototoxicity exists over the range of tissue reaction investigated here. The drug dose effect is presented in Fig. 2 for an irradiation duration of 4 min. A significant decrease of skin photosensitization is observed if a reduced amount of mTHPC is administered. It is noticeable that, in our conditions, the 384 Georges Wagnieres zyxwvutsrqpo zyxwvutsr zyxwvuts et a/. zyxwvutsrqponm zyxwvutsrqponmlk zyxwvutsrqp zyxwvutsrqpo 0 1 2 3 4 5 6 7 WEEKS AFTER INJECTION 8 9 Figure 2. Skin reaction level as a function of the delay after injection of 0.3, 0.15, 0.10 m g k g of mTHPC per kg of body weight for an irradiation time of 4 min (24 J/cm2). Illumination procedures were carried out as described in the Materials and Methods. Each point represents the mean result with error bars corresponding to the 67% confidence interval. duration of skin photosensitization is roughly proportional to the drug dose, no matter what the tissue reaction. This observation can be used as a rule of thumb to predict approximately the duration of the cutaneous photosensitization induced by a given injected dose of mTHPC. Important interpatient variations have been observed in the context of this study and these variations have to be taken into account in the prediction of the duration of this phototoxicity after the administration of a given drug dose. Figure 2 indicates that the influence of these variations on the prediction of this duration results in an error of typically 1 week. It should also be noted that these tests were performed on the backs of patients on an area not frequently exposed to direct sunlight. Results on areas of the human body exposed more rt:gularly (face, hands) are different as will be seen later. We have carried out the same skin tests with three noninjected patients, demonstrating no reaction after irradiations as long as 15 min (90 J/cm2). This indicates that the skin reactions observed in this study are essentially due to the presence of mTHPC and not to conventional sunburn. Figure 3 presents the fluorescence intensity of mTHPC measured on the skin with the optical fiber-based spectrofluorometer. This curve resembles the data of Fig. 1. Indeed, all these curves show a maximum near or slightly before 1 week after injection, and the phototoxicity vanishes over a time range on the order of several weeks. Surprisingly, even though the scale describing the skin reaction is not completely quantitative nor linear, the mTHPC fluorescence signal from the skin seems to correlate well with the skin reaction. This can be seen in Fig. 4 in which the results obtained with 10 patients injected with 0.1 (1 patient), 0.15 (7 patients) and 0.3 ( 2 patients) mg/kg are reported. This figure concerns patients irradiated at drug-light intervals longer than 4 days, so as to avoid as much as possible artifacts induced by the redistribution of mTHPC in the various cutaneous layers. These results suggests that fluorescence spectroscopy may be used to predict the degree of photosensitivity of the skin. 20 10 10 30 40 50 60 TIME AFTER INJECTION [days] Figure 3. Kinetics of the mTHPC fluorescence measured at 650 nm on the skin of 11 patients. Fluorescence intensities have been normalized for an injected dose of 0.15 mgkg. Fluorescence excitation wavelength: 420 nm. of the goals of this study was to define the limitations imposed by skin photosensitization on the treatment conditions based on the use of mTHPC. This was achieved by varying most of the parameters that are thought to play a significant role in the tissue reaction. Among these parameters are the drug dose, the light dose and the drug-light interval. The fluence rate was kept constant at 100 mW/cm2 between 380 and 700 nm. Such a fluence rate is a typical value used in clinical PDT and, according to the study reported by van Gemert and Welch (35), no significant thermal effect is induced. This statement is in agreement with the comments given by the patients on the thermal effect sensed during the irradiation. Moreover, the use of a solar spectral irradiance that is twice larger than the one observed at noon on a clear summer day at sea level at our latitude is also justified, as a separate study performed on animals indicated that no observable fluence rate effect can be generated under our photometric conditions with mTHPC (36). It should be noted that no UV light was delivered by our solar simulator. This is justified by the fact that the mTHPC main absorption peaks are located at 420 nm and 652 nm (plus some minor peaks located between 500 and 600 nm). Moreover, to our knowledge, it is not likely that the simultaneous irradiation of the skin with UV light during the short (several minutes) zyxwvutsrqpo DISCUSSION In the present paper we present an approach for the assessment of the skin photosensitization induced by mTHPC. One 0 0 -/ , 20 40 60 80 100 FLUORESCENCE INTENSITY [a.u.] Figure 4. Correlation between the mTHPC fluorescence intensity in the skin of 10 patients and the tissue reaction. The light dose was 24 J/cm* (4 min irradiation) and the drug light interval was longer than 4 days. zyxw zy zyxwv zyxwvutsr Photochemistry and Photobiology, 1998,68(3) 385 illumination times considered in this study would have modified the tissue reaction. This statement is supported by measurements we did on one patient. His skin was exposed to direct sunlight on a cloudless day of mid-April at 3:30 P.M., 15 days after injection of 0.15 m g k g of mTHPC. The irradiance of the sunlight in the spectral domain corresponding to the emission of our solar simulator (380-700 nm) was 50 mW/cm2 (half the irradiance of our solar simulator in this spectral domain). The duration of the skin exposure was, respectively, 2, 4, 8 and 16 min (corresponding to 6, 12, 24 and 48 J/cm2 in the spectral domain described above) and the resulting skin reactions were classed as 0, 1, 2 and 3, respectively. These tissue reactions are in good agreement with the results obtained with the solar simulator (see Fig. 1), suggesting that the UV and IR light of the sunlight does not play an important role in the skin reaction, at least for the short exposure times considered in this study. The skin photosensitization study reported here indicates that a reduced drug dose (less than 0.3 mgkg) should be used for routine clinical use of mTHPC-PDT so as to avoid unacceptable skin photosensitization and other possible side effects. Too low drug doses should also be avoided as they may lead to ineffective PDT or to excessive irradiation times. It should be noted that, due to the mTHPC photobleaching, increasing the light dose has to be more than inversely proportional to the reduced injected drug dose to lead to a curative treatment (37). Moreover, high light doses, which imply long irradiation times, are not convenient for endoscopic PDT and necessitate the use of powerful and expensive light sources. Hence, an intermediate drug dose of about 0.15 mgkg, or possibly slightly less, seems to be optimal for the clinical treatment of precancerous and cancerous lesions by mTHPC-PDT. These skin tests indicate that, for a given drug dose, mTHPC is also much more efficient in inducing skin photosensitization with sunlight than Photofrin (23,24). This difference is probably due to the intrinsically stronger mTHPC phototoxicity (14) but also to its absorption spectrum, which is different between 600 and 700 nm than that of Photofrin. The mTHPC absorption spectrum is somewhat similar at shorter wavelengths to that of the Photofrin with a large Soret band close to 420 nm and three low absorption bands between 500 and 600 nm. However, the strong absorption in the red at 652 nm (E = 33 500 M-I crn-') exceeds that of Photofrin around 630 nm by an order of magnitude. This difference could lead to a more efficient photosensitization of the deeper-seated skin layers with mTHPC as compared to Photofrin. The strong phototoxicity of mTHPC also has to be considered for patients who may be subjected to a surgical treatment within several weeks after drug injection. An exposure of 3 h to the lamps of the operating theater during a surgical treatment 2 weeks after mTHPC injection (injected dose = 0.3 mgkg) can induce important tissue necrosis as reported by Savary et al. (26). The mTHPC skin photosensitization seems to remain effective at the applied conditions during 6 weeks after an injected dose of 0.15 mgkg (8 min irradiation). A scatterplot presenting the results of all irradiation tests (results not shown here) indicates that the maximal skin photosensitization is observed between 2 and 6 days after injection. Nev- z P !- 0 2K 3 u) zyxwvu 0 2 4 6 8 10 WEEKS AFTER INJECTION 12 14 Figure 5. Skin reaction level as a function of the time after injection of 0.15 m g k g of mTHPC and 2 m g k g of Photofrin in the same patient for an irradiation time of 4 min (24 J/cm2). The photosensitizer administrations were separated by 3 years. ertheless, the duration of the critical period, i.e. the time during which direct sunlight has to be strictly avoided, is about 2 weeks after the administration of mTHPC (0.15 mg/ kg). The interpatient fluctuations observed in Figs. 1 and 2 indicate that the confidence interval for the duration of this critical period is 2 1 week. The duration of significant skin phototoxicity of 2 weeks is confirmed by the 12 undesirable skin reactions (redness, edema and occasionally cutaneous blistering of the hands and face) observed in the patients of the CHUV Hospital in Lausanne, Switzerland. Solar-induced skin photosensitizations have never been observed later than the first week after injection of mTHPC (10,ll). The kinetics of its decay appear to be faster than that of Photofrin according to comparable studies reported by Dougherty et al. (23) and Mullooly et al. (24) and according to the skin tests performed by our group on two patients injected with Photofrin (2 mgkg) (19). An interesting and unique case investigated in Lausanne is a patient who received an iv dose of both PS (Photofrin 2 mg/kg; mTHPC 0.15 mgkg) 3 years apart. The results of the skin photosensitization tests performed on the back of this patient with the same protocol (4 min irradiation, both with the solar simulator described above in the Materials and Methods) are presented in Fig. 5 . This figure suggests that mTHPC, at least in this single case, is more rapidly cleared from the skin than Photofrin. Indeed, the latter induced a skin photosensitization up to more than 13 weeks, whereas no effect was observed with mTHPC at 6 weeks. It should be noted that the skin tests have been performed at three delays only with mTHPC, and the last one induced no reaction. Considering the data in Fig. 1, it is likely that no reaction would have been observed with this PS if the last irradiation would have been performed 4 weeks after the injection. From this point of view, this case suggests that the duration of the skin photosensitization induced by mTHPC is about three times shorter than that induced by Photofrin. This conclusion has to be considered with caution as it is drawn from one patient only. Nevertheless, this case report is of interest as this comparative test has been performed with the same patient, at the same location and with the same photometric protocol for both drugs. A sort of tissue reaction scale had to be defined in the context of this study in order to describe the tissue damage zyxwvutsrq zyxwvutsrqpon zyxwvu zyxwvutsrqp zyxwvutsrq zyxwvutsrq 386 Georges Wagnieres eta/. induced in the skin. The scale described above is unfortunately not completely quantitative nor linear. Moreover, the assessment of identical tissue reaction may, to some extent, be different if this evaluation is performed by two independent physicians. Nevertheless, our results indicate that the metabolic interpatient variations play a more important role in the fluctuations presented in Fig. 1, for instance. It should be noted that interpatient variations of the dye concentration in tissues at the time of PDT have been observed with other PS, such as Photofrin (3840). These variations are probably due to different pharmacokinetics in different patients as is the case for most anticancer drugs used in conventional chemotherapy (41). Another parameter that could be of importance in the context of this tissue reaction assessment is the time interval separating the irradiation from the evaluation of the phototoxic effect. In fact, we have performed a preliminary study in human skin that showed that the results of repeated evaluations of the tissue reaction performed at different times have reached their maxima and are basically the same between 2 and 4 days after tissue illumination. The skin photosensitization tests reported above were performed on the backs of patients, i.e. on an area not frequently exposed to direct sunlight. Results obtained on the upper side of the hands (skin exposed more regularly to the sunlight) are different as was observed with only one patient. Both the hand and the back of this patient were exposed during 4 min between 2 and 18 days after the administration of 0.15 m g k g of mTHPC. As expected, the skin reaction obtained on the hand was less important than the one observed on the back, i.e. a tissue reaction of 1 (light erythema) was observed on the hand when the reaction 3 (pronounced erythema) was obtained on the back. This case indicates that the type of skin (tissue architecture, content of melanin, etc.) play an important role in the tissue response. Another possible explanation for this reduced skin reaction on the hand could be that the degree of mTHPC photobleaching is greater on the hands versus the back due to ambient light. This study also suggests that the fluorescence spectroscopy of mTHPC can help to predict the degree of photosensitivity of the skin as illustrated by the good correlation between the mTHPC fluorescence intensity and the skin reaction (see Fig. 4). Hence, this correlation can be used to define the time lag during which sunlight has to be avoided for each individual patient. This is of major interest considering the important interpatient variations in the duration of skin photosensitivities observed in this study. The use of convenient spectrofluorometers calibrated for this specific application is therefore of high clinical relevance. The design and optimization of such a device should be carried out in terms of excitation wavelength, size and locations of the probed areas. It is likely that the error bars of Fig. 4 can be significantly reduced if larger (several millimeters in diameter) areas are probed with fluorescence excitation wavelengths that are less absorbed by tissues inhomogeneities. In conclusion, the study reported here indicates that the duration of the skin photosensitization induced after a typical therapeutic dose of mTHPC is shorter than with Photofrin, i.e. the time during which direct sunlight has to be strictly avoided is about 2 weeks after the administration of mTHPC (0.15 mgkg). It is nevertheless highly recommended to test the patient’s skin photosensitivity by brief (several minutes) exposure of a small area (10 cm2) of the skin to sunlight at regular intervals between 2 and 4 weeks after drug administration. This study indicates also that the drug dose of 0.15 m g k g seems to be close to the optimum in terms of skin photosensitization. Finally, we have shown that LIF spectroscopy of mTHPC enables prediction of the degree and duration of photosensitivity of the skin. Acknowledgements-The authors are grateful to Scotia QuantaNova Ltd, Guildford, UK for kindly providing the mTHPC. We are also grateful to Ciba-Geigy, the Fonds National Suisse de la Recherche Scientifique (grant 2100-043507.95/1), the Swiss National Priority Program in Optics, the Fonds CHUV-EPFL-UNIL and the Swiss Commission for Technology and Innovation for technical and financial support. zyxwvu zyxwvu zyxwvutsrqp zyxwvutsr REFERENCES 1. Kessel, D. (1990) Photodynamic Therapy of Neoplastic Disease, Vols. I and 11. CRC Press, Boca Raton, FL. 2. Spinelli, P., M. Dal Fante and R. Marchesini (1992) Photodynamic Therapy and Biomedical Lasers. Excerpta Medica, Amsterdam, The Netherlands. 3. Pass, H. I. 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