Multiple Large-Surface Photodynamic Therapy Sessions with Topical or Systemic Aminolevulinic Acid and Blue Light in UV-Exposed Hairless

Mice
Younan Liu, Gilles Viau, and Robert Bissonnette

DOI: 10.1007/s10227-004-0117-5 J Cutan Med Surg 2004; 131139

Abstract
Background: The use of photodynamic therapy (PDT) with topical aminolevulinic acid (ALA) on large skin surfaces has recently been reported for patients with multiple actinic keratoses. Objective: The current study compared the ability of topical and systemic ALAPDT as well as topical ALAPDT with blue light to delay the appearance of UV-induced skin cancer using the hairless mouse as a model. Methods: Groups of hairless mice were exposed daily to UV radiation and weekly to ALAPDT. Tumor-free survival was compared for mice exposed to UV and treated weekly with ALAPDT and mice exposed only to UV radiation. Results: Weekly topical or systemic ALAPDT was able to delay the induction of skin tumors. A signicant difference in tumor-free survival was also observed for both actinic keratoses and invasive squamous cell carcinoma in mice treated weekly with topical ALAPDT performed with blue light. This was observed even when weekly ALAPDT was started after 8 weeks of UV exposure. Conclusion: Large-surface topical ALAPDT with blue light can delay the appearance of UV-induced actinic keratoses and squamous cell carcinoma in hairless mice.

Sommaire
rapie photodynamique (TPD) combine e a ` lapplication topique de Ante ce dents: La the vulinique (AAL) sur une large surface de la peau a e te utilise e sur des lacide aminole ratoses actiniques multiples. patients souffrant de ke sente e tude utilise les souris sans poils an de comparer la capacite de Objectif: La pre mique et de lAALTPD topique avec irradiation a ` la lulAALTPD topique et syste ` re bleue a ` retarder la manifestation du cancer de la peau cause par les rayons UV. mie te expose s a ` des radiations quotidiMe thode: Des groupes de souris sans poils ont e ` des traitements hebdomadaires a ` lAALTPD. La survie sans ennes de rayons UV et a es et traite es a ` lAALTPD a e te compare e a ` la survie sans tumeurs des souris irradie es mais non traite es. tumeurs des souris irradie mique de lAAL-TPD a pu Re sultats: Lapplication hebdomadaire topique ou syste galement, une diffe rence signicative a e te note e dans retarder Iinduction des tumeurs. E ratose actinique et de carcinome squala survie sans tumeurs des souris atteintes de ke es chaque semaine a ` lAALTPD topique avec meux envahissant lorsquelles sont traite ` la lumie ` re bleue. Cela est vrai me me lorsque le traitement est commence irradiation a ` s lexposition aux rayons UV. ConclusionLapplication de lAAL huit semaines apre

Division of Dermatology, University of Montreal Hospital Centre, Montreal, Quebec, Canada Online publication: 4 May 2004 Correspondence to: Robert Bissonnette, Division of Dermatology, University of Montreal Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Rm K-5201, Montreal, Quebec, Canada H2L 4M1. E-mail: rbissonn@globetrotter.net

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` la lumie `re bleue peut retarder lapparition TPD sur une large surface avec irradiation a ratoses actiniques et des carcinomes squameux cause s par les rayons UV chez la des ke souris sans poils.

hotodynamic therapy (PDT) with topical aminolevulinic acid (ALA) has been approved in the US and Canada for the treatment of actinic keratoses. Investigator-initiated studies have also suggested efcacy in the treatment of other skin diseases such as basal cell carcinoma, acne, Bowens disease, warts, and Pagets disease.1 5 The FDA-approved protocol for the treatment of actinic keratoses involves the application of ALA on individual actinic keratoses followed by blue light exposure one day later. Recently, physicians have started to use ALA on larger skin surfaces to treat multiple AKs, skin aging, and acne.68 This was reported to be well tolerated and resulted in a good cosmetic outcome. Large-surface PDT has the potential to become an alternative to 5-FU for patients with extensive photodamage and multiple actinic keratoses. Weekly large-surface topical ALAPDT performed on mice exposed chronically to ultraviolet (UV) radiation has been shown to delay the appearance of skin tumors.9 However, in these experiments, a higher incidence of invasive squamous cell carcinoma was reported for mice treated weekly with ALAPDT.9 With the increasing use of large-surface ALAPDT in the clinic, we decided to further investigate the effects of large-surface topical ALA-PDT on UV-exposed hairless mice. The specic aims of the current experiments were to study the incidence of squamous cell carcinoma in UV-exposed mice treated weekly with ALAPDT, to study the ability of PDT with blue light to delay the appearance of skin tumors when ALAPDT is started after, as well as at the same time as, UV exposure, and to study the safety of weekly large-surface topical ALAPDT.

skin tumors. The size and location of tumors were recorded and any tumor larger than 1 mm that was present for at least two consecutive weeks was included in the analysis. Euthanasia was performed when the tumor load (combined volume of large and small tumors) reached 1 cm3, when erosions occurred on larger tumors, or at 22 weeks in the experiment where topical and systemic ALAPDT was compared and 32 weeks in the experiment where blue light was used, whichever came rst. All tumors 4 mm or larger that were present at the time of euthanasia were biopsied for routing histopathology for the experiment comparing the ability of topical and systemic ALAPDT to delay the appearance of UV-induced skin tumors.

Materials and Methods

Chemical and Reagents 5-Aminolevulinic acid (Sigma, St Louis, MO) was prepared immediately before administration either in normal saline (40 mg/kg) for intraperitoneal injection or in hydroalcoholic solution (DUSA Pharmaceuticals, Valhalla, NY) at 20% for topical application (10 mg/cm2). Animals Female SKH-1 hairless mice (48 weeks old) were purchased from Charles River Laboratories (St-Constant, Canada). Mice were fed a standard rodent diet and kept on a 12-h on/off ambient light cycle. Mice were kept in the dark for 24 h on days when ALA was administered. All experiments were approved by the University of Montreal Hospital Center Animal Care committee. Mice were allowed to move freely during all exposures. They were examined weekly for the presence of

Comparison of the Ability of Topical and Systemic ALAPDT to Delay the Appearance of UV-Induced Skin Tumors These experiments were conducted in order to understand why weekly topical ALAPDT has been reported to induce a higher incidence of invasive squamous cell carcinoma9 whereas systemic ALA-PDT did not.10 The rst step was to reproduce as best as possible the protocol used by Stender et al.9 with topical ALA as well as the protocol used by Sharfaei et al.10 with systemic ALA. These two experiments used different UV systems. For systemic ALAPDT, the UV system used was identical to the source used by Sharfaei et al.10 and was made of six FS 20 tubes (Westinghouse, Bloomeld, NJ). The irradiance of this system was 1.6 mW/cm2 for UVB and 0.82 mW/cm2 for UVA. A second UV system was built to reproduce the Stender et al. UV source.9 This source was made of 5 Bellariums SA1-12-100 W (Wolff System, Drummondville, Quebec, Canada) and 1 Philips TLK10/ 10R lamp (Philips, Eindhoven, Holland). The irradiance was measured weekly with an International Light IL 1700 radiometer (Newburyport, MA, USA) equipped with an SED 033 detector, a UVA lter, and a W quartz diffuser to measure UVA. To measure UVB, a SED 240 detector, a UVB-1 lters and a W quartz diffuser were used. The irradiance of the combination of Bellariums and the Philips lamp system was 0.25 mW/cm2 for UVB and 4.4 mW/cm2 for UVA. To reproduce Sharfaeis systemic ALAPDT protocol, two groups of 20 mice were exposed 5 days per week to 91 mJ/cm2 of UVB (70% minimal erythematous dose, MED) and to 52 mJ/cm2 of UVA from the FS 20 source. Mice from one group were exposed once a week to 980 mJ/cm2 of visible light from a slide projector (Kodak Carousel 650 H, Toronto, ON, Canada) at 5.2 mW/cm2 (70% minimal phototoxic dose, MPD) 3 h after intraperitoneal injection of ALA. The spectral output of the slide projector peaks at 590 nm and has a full-width

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TABLE I
Experimental groups for comparison of topical and systemic ALA-PDT GROUP A B C D E UV exposure Monday Monday Monday Monday Monday to to to to to Thursday Thursday Thursday Thursday Thursday Weekly ALAPDT On a UV exposure day (Wednesday) No PDT On a UV exposure day (Wednesday) On a UV exposure day (Tuesday) On a day with no UV exposure (Friday) % MPD 200% No PDT 70% 70% 70% Time of PDT after ALA application 6h No PDT 6h 3h 3h

half-maximal bandwidth of 220 nm. The uence was decreased to 35% MPD at week 9 and 20% MPD at week 13. The other group served as a no-PDT control and was exposed to only UV radiation. To study topical ALAPDT, ve groups of 20 mice were used (Table I). Groups A, B, C, D, and E were exposed 4 days per week (MondayThursday) to 13,200 mJ/cm2 of UVA and 840 mJ/cm2 of UVB (70% MED) from the Bellariums SA and Philips TLK10/10R system. The UV uence for all groups was decreased to 50% MED at week 5, 40% MED at week 10, and 30% MED at week 11. The PDT source, built to reproduce as best as possible the Stender et al. Results, consisted of a set of halogen lamps (Philips, AL, 12 V50 W24, Germany) with an irradiance of 9 mW/cm2. The irradiance was measured at each treatment day with the IL 1700 radiometer equipped with a SED 033 detector, a SCS 425 lter, a WBS 465 lter, and a W quartz diffuser. For groups A, C, D, and E, ALA was applied to the back of mice (about 10% of body surface area). To reproduce Stenders protocol as best as possible, ALAPDT was performed on a day when mice were exposed to UV radiation (group A). Group A was also treated 6 h after ALA application with a visible light uence of twice the minimal light dose that induced erythema on mouse skin (200% MPD) as Stender et al.9 describe that mice were erythematous and edematous following ALAPDT. Additional groups were included to compare tumor-free survival for mice exposed at 70% MPD (group C), the MPD that was used in systemic ALAPDT experiments, for mice exposed 3 h after ALA application with 70% MPD (group D), and for mice treated 3 h after ALA application with 70% MPD on a day when mice did not receive UV treatment (group E). Experimental conditions for mice in group E were similar to the ones used by Sharfaei et al.,10 except that topical ALA was used instead of systemic administration. The visible light uence for group A was decreased to 100% MPD at week 10, whereas the visible light uence for groups C, D, and E was decreased to 35% MPD at week 9 and 20% MPD at week 13.

TABLE II
Experimental groups for ALA-PDT with blue light Group 1 2 3 4 5 6 UV exposure From week 0 to 8 From week 0 to 8 Topical ALA From week 0 From week 0 From week 0 From week 0 to 8 From week 9 Blue light exposure From week 0 From week 0 From week 0 From week 9

exposed to UV for a total 8 weeks [10,000 mJ/cm2 of UVA and 645 mJ/cm2 of UVB (50% MED)]. The UV system used was identical to the Bellariums and Philips system previously described for experiments with topical ALA and white light with the addition of a Kodacel lter (cellulose triacetate, Eastman Kodak, Rochester, NY) to lter out UVC. The UVB and UVA irradiance was 0.16 mW/cm2 and 2.52 mW/cm2, respectively. Mice were exposed 5 days per week (from Monday to Friday). Groups 1, 3, 5, and 6 were exposed weekly to 113 mJ/cm2 (60% MPD) at 7.2 mW/cm2 of light from a panel of blue light tubes (Blue-U, DUSA Pharmaceuticals). The BlueU has a peak at 417 nm and a full-width half-maximal bandwidth of 30 nm. ALAPDT was started at the same time as UV exposure for group 1. However, ALAPDT was started 1 week after the end of the 8-week course of UV exposure for group 6. Visible light uence was decreased to 30% MPD at week 16. Groups 1, 3, 4, and 6 were treated weekly with 20% topical (10 mg/cm2) ALA.

Use of Topical ALAPDT with Blue Light to Delay the Appearance of UV-Induced Skin Cancer Six groups of 20 mice for a total of 120 mice were studied (Table II). Mice from groups 1, 2, and 6 were

In Vivo Fluorescence Spectroscopy For groups A, C, and D (Table I), skin PpIX levels were measured before visible light exposure as well as 24 h later, before and after daily UV exposure for groups A and C, and 48 h after PDT, before and after daily UV exposure for group D. PpIX levels were measured with in vivo uorescence spectroscopy using a Skin Skan spectrouorometer (SPEX model, Instruments SA, Edison, NJ) equipped with a xenon lamp and two monochromators. The excitation was set at 401 nm and the emission was recorded from 600 to 700 nm. PpIX uorescence intensity was evaluated by measuring the area under the emission spectra between 616 and 653 nm

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after subtraction of the autouorescence background. A single measurement was performed on 20 mice for each group studied.

Statistical Analysis Tumor-free survival was expressed with KaplanMeier curves and the log rank test was used to study differences between groups. Students t-test was used to compare PpIX intensity before and after irradiation using in vivo uorescence spectroscopy. Data for all tumors include any mice that presented at least one tumor.

PpIX intensity was high in all groups studied. Twentyfour hours after PDT, PpIX levels were still detectable in the skin at 26% on average of what was measured on the day of PDT. PpIX levels present 24 h after PDT were decreased signicantly (p < 0.001) by 87% on average following UV exposure (Table III). In group D PpIX levels were still detectable 48 h after PDT and were also signicantly decreased (p < 0.001) following UV exposure 48 h after PDT.

Results
Comparison of the Ability of Typical and Systemic ALAPDT to Delay the Appearance of UV-Induced Skin Tumors After 5 weeks of daily UV exposure, erythema was noted on the back of mice from all groups which prompted a decrease in UV uence to 50% of the original MED. At week 9 some mice from the PDT groups showed erythema following visible light exposure which prompted an equal decrease in visible light uence to 35% of the MPD in all PDT groups except group C where the uence was reduced to 100% of MPD at week 10. There was a signicant (p < 0.0001) difference in tumor-free survival for mice treated weekly with systemic ALAPDT compared with mice exposed to only UV (Fig. 1A). There was no statistically signicant difference in tumorfree survival for tumors equal or larger than 4 mm for mice treated weekly with systemic ALAPDT (Fig. 1B). There was also a signicant (p < 0.0001) difference in tumor-free survival for mice treated with topical ALA PDT when the analysis was performed on all tumors (Fig. 2A). However, there was no signicant difference in tumor-free survival for tumors equal or larger than 4 mm with topical ALAPDT (Fig. 2B). Comparison of the Ability of Different Tppical ALAPDT Regimes to Delay the Appearance of UV-Induced Skin Tumors The ability of topical ALAPDT performed at 70% (group C) and 200% MPD (group A) 6 h after ALA application was compared with ALAPDT performed at 70% MPD but with light exposure 3 h after ALA application (group D) and with ALAPDT performed at 70% MPD 3 h after ALA application but on a day when UV exposure does not occur (group E) (Fig. 2B). Tumor-free survival curves of all these regimes were statistically different from mice exposed to only UV. The tumor-free survival of group D was also signicantly less than mice from groups A (p = 0.018), C (p = 0.0002), and E (p = 0.0006). In Vivo In vivo mouse (Table Fluorescence Spectroscopy uorescence spectroscopy was also performed on skin 24 and 48 h after PDT in hairless mice III). Immediately before visible light exposure,

Use of Topical ALAPDT with Blue Light to Delay the Appearance of UV Induced Skin Cancer Mild erythema after visible light exposure was noted in 6 mice from the group where PDT was started at the same time as UV exposure and from 4 mice from the group where PDT was started after 8 weeks of UV exposure which prompted an equal decrease in blue light uence as described in the Materials and Methods section. Tumorfree survival of mice treated weekly with topical ALA PDT and blue light was signicantly different (p < 0.0001) compared with mice exposed to only UV (Fig. 3A). This was signicant for mice where ALAPDT was started at the time of UV exposure (group 1; p < 0.0001) as well as for mice where ALAPDT was started one week after the end of UV exposure (group 6; p < 0.001). There was no statistical difference (p = 0.33) in tumor-free survival between mice for which ALA-PDT was started after UV exposure and mice for which ALA PDT was started at the same time as UV exposure. Weekly topical ALAPDT also induced a delay in large tumor appearance compared with mice exposed to only UV (Fig. 3B). Large tumor-free survival was signicantly different for mice where ALAPDT was started at the end of UV exposure (group 6; p = 0.0003) as well as at the same time as UV exposure (group 1; p < 0.0001).

Discussion
Since the original report of Slender et al.,9 which demonstrated that weekly topical ALAPDT can delay the appearance of UV-induced skin tumors, our group has shown that weekly PDT with systemic ALA or topical methylaminolevulinate can also delay the appearance of UV-induced skin tumors.10,11 The current experiments were performed to explore additional questions raised by these studies in view of the growing clinical use of largesurface ALAPDT. The rst question raised was related to the increased incidence of large tumors observed by Stender et al. in mice treated weekly with ALAPDT despite a lower overall incidence of small tumors. This observation is crucial for the development of large-surface ALAPDT as small (<4 mm) tumors are mostly actinic keratoses and large tumors are mostly invasive squamous cell carcinoma.12 If PDT can delay only actinic keratoses but not invasive skin cancer, there is little interest in using multiple large-surface ALAPDT sessions in patients with multiple AKs. It was therefore decided to

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FIGURE 1 Tumor-free survival for all tumors (A) and tumors equal or larger than 4 mm (B) in mice treated with systemic ALAPDT. PDT was performed with a slide projector.

reproduce Sharfaeis10 and Stenders9 experiments as best as possible. Using in vivo uorescence spectroscopy, PpIX levels were similar in mouse skin after topical ap-

plication of 20% ALA at 10 mg/cm2 (FDA-approved concentration for the treatment of actinic keratoses and intraperitoneal ALA at 40 mg/kg (not shown). In

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FIGURE 2 Tumor-free survival for all tumors (A) and tumors equal or larger than 4 mm (B) in mice treated with topical ALAPDT. PDT was performed with a Halogen lamp. Refer to Table I for differences between groups.

this study as well as in all other studies using PDT as a modality to delay skin tumor appearance that our group performed, there was no increase in incidence of large tumors in mice treated with PDT. In the experiment using blue light 3 h after topical ALA application (groups 1 and 6), we even observed a decrease in the incidence of large tumors. Histological analysis of large tumors in the

current experiments showed that 93% were invasive squamous cell carcinoma which conrms ndings from previous studies that tumors larger than 4 mm are mostly squamous cell carcinoma.12 Stender et al.9 reported their large-tumor data by giving the number of mice that had to be withdrawn because of large tumors. They showed only a Kaplan

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FIGURE 3 Tumor-free survival for tumors smaller than 4 mm (A) and equal or larger than 4 mm (B) in mice treated with topical ALAPDT. PDT was performed with blue uorescence tubes. Refer to Table II for differences between groups.

Meir graph to report tumor-free survival for small tumors. They withdrew 5 out of 20 mice for large tumors in the vehiclePDT group, 10 out of 20 mice in mice exposed to only and UV, and 13 or 14 out of 20 in groups of mice exposed to ALAPDT. We have also analyzed the number of mice withdrawn because of large tumors in our experiments and found no difference between mice

exposed to only UV radiation and mice exposed to UV and treated with ALAPDT (not shown). In the experiment that reproduced Stenders conditions (unltered UV source), we were able to conrm a delay in the appearance of small tumors but a difference in the appearance of large tumors was not observed. However, when blue light was used to activate PpIX (Kodacel-

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TABLE III
Protoporphyrin IX intensity before and after UV exposure PpIX intensity (a.u.) SD Group A Time of measurement Before UV exposure After UV exposure Before PDT Before UV exposure After UV exposure Before PDT Before UV exposure After UV exposure Before PDT Monday 0.002 0.000 na 0.001 0.000 na 0.003 0.000 na 0.001 0.000 0.001 0.000 0.002 0.000 Tuesday 0.003 0.000 na 0.002 0.000 na 0.007 0.000 4.690 0.003 0.000 0.003 0.000 0.002 0.000 0.250 Wednesday 0.012 0.000 4.200 0.011 0.000 4.750 1.096 0.159 na 0.004 0.000 0.200 0.004 0.000 0.310 0.094 0.029 Thursday 1.048 0.165 na 1.397 0.126 na 0.185 0.021 na 0.078 0.021 0.078 0.021 0.038 0.009 Friday na na na na na na na na na

na = not applicable.a.u. = arbitrary units.

ltered UV source), a delay in large-tumor appearance was observed (groups 1 and 6). The reason for this phenomenon is unknown. The absence of UVC in the source used in experiments with blue light might explain this observation as UVC is highly carcinogenic and contributes signicantly to the immunosuppressive effects of unltered FS 20 sources.1315 This could have contributed to the progression of actinic keratoses to invasive squamous cell carcinoma in the experiments where topical and systemic ALA were compared since a Kodacel paper to lter out UVC was not used in these experiments. In the experiment where blue light was used, it was decided to add Kodacel paper to lter out UVC as this waveband does not reach the surface of the earth. Another question that was addressed with the current experiments was the ability of ALAPDT to delay the induction of skin cancer when PDT is started at the end of UV exposure (group 6). In all previous studies published on the use of PDT to prevent skin tumors, UV exposure and PDT were started at the same time. This is clearly different from the clinical situation where patients get most of their sun exposure before they develop actinic keratoses. We therefore designed an experiment where mice were exposed to UV radiation for 8 weeks followed by ALAPDT with blue light (group 6) and compared the tumor-free survival to an experiment where UV and ALA PDT were started at the same time (group 1). In both cases we observed a delay in the induction of actinic keratoses as well as invasive squamous cell carcinoma. The rst tumor was seen earlier in mice treated with ALAPDT at the end of UV exposure compared with mice where ALAPDT was started at the beginning of UV exposure but the overall tumor-free survival was not statistically different. Stender et al.9 also observed an increase in mortality in mice treated with weekly ALAPDT and exposed daily to UV compared with mice exposed to only UV. We did not observe an increase in mortality in mice treated with ALAPDT in all experiments conducted so far including previous experiments with systemic ALA.10 A new nding reported in this study is the presence of detectable PpIX in mouse skin 24 h after PDT under the

current conditions (groups A, C, and D). It was shown that this PpIX can be photobleached by UV exposure suggesting that additional PDT effects are created by exposing mice to UV radiation 24 and 48 h post-PDT. This could inuence the results and eventually the design of this type of experiment as mice treated with PDT on Wednesdays and exposed to UV from Monday to Friday may benet from additional PDT effects compared with mice treated with PDT on Friday and exposed to UV from Monday to Friday as the latter group is exposed to only UV 72 h after ALA-PDT. The presence of additional PDT effects is suggested by the signicant decrease in tumor-free survival for mice treated on Friday (group E) compared with all other groups in the current study. To our knowledge this phenomenon has never been studied in patients. If PpIX is present in the skin of patients after ALAPDT, sun or ambient lighting exposure could induce additional PDT effects. In conclusion, large-surface topical ALAPDT with blue light can delay induction of actinic keratoses as well as squamous cell carcinoma in hairless mice. This phenomenon is observed even when ALAPDT is started after the end of UV exposure.

Acknowledgments
This study was funded in part by DUSA Pharmaceuticals and in part by the Canadian Dermatology Foundation.

References
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10. Sharfaei S, Viau G, Lui H, et al. Systemic photodynamic therapy with aminolevulinic acid delays the appearance of ultraviolet-induced skin tumours in mice Br J Dermatol 2001; 1446:12071214. 11. Sharfaei S, Juzenas P, Moan J, et al. Weekly topical application of methyl aminolevulinate followed by light exposure delays the appearance of UV-induced skin tumours in mice Arch Dermatol Res 2002; 294:237242. 12. Gruijl FR de , Leun JC van der . Development of skin tumours in hairless mice after discontinuation of ultraviolet irradiation Cancer Res 1991; 513:979984. 13. Hurks HM, OutLuiting C, Vermeer BJ, et al. In situ action spectra suggest that DNA damage is involved in ultraviolet radiation-induced immunosuppression in humans Photochem Photobiol 1997; 661:7681. 14. Roberts LK, Beasley DG, Learn DB, et al. Ultraviolet spectral energy differences affect the ability of sunscreen lotions to prevent ultraviolet-radiation-induced immunosuppression Photochem Photobiol 1996; 636:874884. 15. Learn DB, Beasley DG, Giddens LD, et al. Minimum doses of ultraviolet radiation required to induce murine skin edema and immunosuppression are different and depend on the ultraviolet emission spectrum of the source Photochem Photobiol 1995; 626:10661075.