ORIGINAL ARTICLE

A time course investigation of the fluorescence induced by topical application of 5-aminolevulinic acid and methyl aminolevulinate on normal human skin
Andrea Lesar, James Ferguson & Harry Moseley
Department of Photobiology, Ninewells Hospital, Dundee, UK

Summary
Key words:
5-aminolevulinic acid (ALA); methyl aminolevulinate (MAL); photodynamic therapy (PDT); protoporphyrin IX (PpIX).

Correspondence:
Harry Moseley, Department of Photobiology, Ninewells Hospital, Dundee DD1 9SY, UK. e-mail: h.moseley@dundee.ac.uk

Accepted for publication:

25 February 2009

Conicts of interest:
None declared.

Background: Treatment of non-melanoma skin cancers (NMSC) with topical photodynamic therapy (PDT) is a treatment of choice for many clinicians. The two most commonly used PDT photosensitizer precursors are 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL). Current PDT treatment regimes advise longer (46 h) application times for ALA and shorter times (3 h) for MAL. Aims: To establish the time course characteristics of protoporphyrin IX (PpIX) uorescence following the application of ALA and MAL in normal skin. Methods: A total of 17 healthy volunteers were recruited, and both ALA and MAL were applied to the inner forearm for varying times (16 h). PpIX uorescence was detected using a non-invasive spectroscopy system. Results and conclusion: PpIX uorescence (following the application of either ALA or MAL) is dependent on duration of application. Following the application of ALA for 13 h peak uorescence was noted at 7 h. Longer duration times (46 h) resulted in sustained uorescence, which peaked at 24 h. MAL-induced uorescence peaked at 7 h and was signicantly decreased by 24 h for all application times. ALA induced uorescence was shown to be signicantly greater than MAL. The ndings from this study have shown that potentially it would be more benecial to apply ALA for shorter periods of time and MAL for longer than current practice.

he therapeutic benets of photodynamic therapy (PDT), following the application of either 5-aminolevulinic acid (ALA) or methyl aminolevulinate (MAL), for the treatment of non-melanoma skin cancers (NMSC) are well documented (15). Tumor selectivity is important to ensure that during treatment the surrounding normal tissue is conserved, and it has been shown that ALA ester derivatives show more tumor selectivity than ALA (68). The application of either ALA or MAL results in the formation of the photosensitizer protoporphyrin IX (PpIX). Illumination of PpIX with blue/ violet light (approximate wavelength 400410 nm) results in the excitation of the molecules and subsequent release of this absorbed energy is mainly by emission of a characteristic uorescence which has two peaks at 635 and 700 nm. PpIX is an extremely photolabile molecule, and has been shown to photobleach rapidly when exposed to light (9, 10). Work conducted by Peng et al. (6) demonstrated that in nude mice, induced PpIX uorescence following application of MAL

was restricted to the application site. However, following the application of ALA, PpIX uorescence was detected out with the application site. It has been documented that peak ALA induced PpIX tumor uorescence occurs after 46 h (11). However, following the application of MAL, peak uorescence was observed after only 3 h (12). Studies conducted by Peng et al. substantiated this work, demonstrating that optimal selectivity of MAL-induced PpIX uorescence was observed in basal cell carcinomas 3 h after application. Comparison of ALA and MAL in patients with actinic keratoses (AK), showed that, although MAL was shown to be more selective than ALA, higher PpIX uorescence was observed following the application of ALA (7). To date there are no comparative studies investigating the time course characteristics of ALA and MAL-induced PpIX uorescence in normal skin. This study aims to investigate these characteristics, to provide a better understanding of the uorescence kinetics in normal tissue, which could contribute to optimization of future PDT treatment regimes.
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Lesar et al.

Materials and methods

The study protocol was reviewed and approved by the Tayside Committee on Medical Research Ethics and written informed consent was obtained from each volunteer. A total of 17 normal healthy volunteers were recruited. Exclusion criteria were pregnancy, volunteers younger than 18 years of age and volunteers with known photosensitivity. A range of skin types, mainly types IIII (13) participated in the studies. The median age of the volunteers was 40 (range 2261 years); there were six males and 11 females. Topical ALA (20% w/w in Unguentum M; Crawfords Pharmaceuticals, Milton Keynes, UK) and MAL (Metxivs 160 mg/g of MAL as hydrochloride; Galderma [UK] Ltd, Herts, UK) were applied at different times on aluminum FINN Chambers (Epitest Ltd Oy, Tuusula, Finland) under occlusion to the inner forearms. (A FINN chamber is a patch test device, which provides good occlusion because of the chamber design. The 8 mm inner diameter provides a 50 mm2 area.) A total of six different application times were studied (16 h). Previous work carried out by the authors showed that ALA/MAL-induced uorescence was not dose dependent when applied at quantities of 0.010.09 ml, therefore a standardized quantity of 0.03 ml was used (14). The ALA patches were applied to the right inner forearm with the shortest application time (1 h) being placed at the distal end, and the MAL patches in the left inner forearm. All patches were evenly spaced, approximately 3 cm apart. The FINN Chambers were removed at hourly intervals in a darkened room, and any excess cream was carefully wiped off. After removal of the creams, uorescence measurements were taken at each site hourly up to 7 h then at 24 and 28 h. A point spectroscopy system was used to measure uorescence as it has been demonstrated to be a reliable system for the detection and monitoring of PpIX uorescence (1517). The system comprises of a 5 mW, 405 nm gallium-nitride diode laser (corresponding to the excitation wavelength of PpIX), bifurcated ber (allowing both the delivery of excitation light and collection of uorescent signal via a single ber), probe, compact grating spectrometer and a laptop computer. To operate the system a computer controlled shutter is activated, allowing laser light to pass along one arm of the bifurcated ber, through a glass lter, before reaching the target tissue. The acquired uorescence passes along the other arm of the bifurcated ber, through another lter into the spectrometer. These components are housed in a box to prevent any stray light from entering the system. The spectrometer is connected to a laptop computer and with the use of specialized LabVIEW programme software visual displays of the acquired spectra can be viewed. Before taking uorescence measurements the system requires a dark reference spectrum to correct for any stray light and possible uorescence of the probe and optical bers. This was done in a darkened room by inserting the end of the probe into a black rubberized cushion. Computer software allowed the spectra to be saved automatically and any subsequent readings were corrected by subtracting the dark reference. The system allows the shutter integration time to vary from 100 to 2000 ms.
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Fig. 1. Protoporphyrin IX uorescence at various times up to 28 h after 5-aminolevulinic acid (ALA) application. ALA applied at varying times (16 h) to the inner forearm. Values are represented as the means SD (n = 17).

Throughout this study an integration time for the shutter was set at 500 ms. This time was chosen as it provides enough signal to minimize the effects of background noise while ensuring unnecessary overexposure therefore reducing the possibility of photobleaching. To further reduce the possibility of photobleaching the test sites after each reading the probe was carefully repositioned. Five readings were taken at each site. Between readings all volunteers were asked to keep the sites covered and on completion of the study were advised to keep the sites out of direct sunlight for a further 24 h.

Results
The mean and standard deviations (SD) of all 17 data sets were determined. Statistical comparisons of results were performed using paired Students t-test analysis and statistical signicance was taken as P 0.05. PpIX uorescence following the application of ALA The cumulative results of PpIX uorescence are graphically represented in Fig. 1. This shows that at all time points PpIX uorescence was detected. It was noted that for the application times of 13 h peak uorescence was achieved by the 7 h time point. However, the longer application times (46 h) resulted in peak values at the 24 h time point. Comparison between 1 and 6 h ALA application times showed similar uorescence at the 6 h time point, with sustained uorescence at the 7, 24 and 28 h time points. Although no statistical differences were seen when comparing the 7 h time point, at the 24 h time point the uorescence following the 6 h application was signicantly higher. The uorescence values at 7 h were 182 113 (1 h) and 220 88 (6 h) (P = 0.11) and at 24 h were 52 63 (1 h) and 312 88 (6 h) (P = 0.0001). PpIX uorescence following the application of MAL The accumulative results of MAL-induced PpIX uorescence are graphically represented in Fig. 2. This showed that PpIX

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PpIX uorescene time course

Fig. 2. Protoporphyrin IX uorescence at various times up to 28 h after methyl aminolevulinate (MAL) application. MAL applied at varying times (16 h) to the inner forearm. Values are represented as the means SD (n = 17).

uorescence was detectable at all time points. It was noted that for all application times peak uorescence was achieved by the 7 h time point. Comparison between 1 and 6 h MAL application times showed increased uorescence at all time points following the 6 h application. This was statistically different at both the 7 and 24 h time points. The uorescence values at 7 h were 97 112 (1 h) and 206 99 (6 h) (P = 0.0009) and at 24 h were 18 14 (1 h) and 68 64 (6 h) (P = 0.005). ALA/MAL comparison At all time points, ALA-induced uorescence was greater than MAL. This was shown to be statistically signicant at all time points when comparing the uorescence intensities following a 1 h application (Fig. 3 and Table 1). Following the 6 h application, although a greater uorescence was noted at the ALA sites, there was no statistical difference detected at the 7 h time point. However, at 24 and 28 h there was signicantly more uorescence at the ALA sites (Fig. 4 and Table 1).

Fig. 3. Comparison of protoporphyrin IX uorescence following a 1 h application of 5-aminolevulinic acid/methyl aminolevulinate (ALA/ MAL) to the inner forearm. Values are represented as means SD; P 0.05 (n = 17).

Discussion
There is currently little agreement as to the optimum quantity of ALA or MAL required for successful treatment, and this is rarely reported in clinical studies. However, where it has been reported there is little consistency with a range of doses from 10 to 200 mg/cm2 being used (18). The two creams used throughout this study were acquired and used in their prepared states of ALA 20% w/w in Unguentum M, supplied by Crawfords Pharmaceuticals and MAL 160 mg/g of MAL as hydrochloride, supplied by Galderma (UK) Ltd. In previously published data (14) it was shown that PpIX-induced uorescence following the topical application (within the range of 0.010.09 ml) of both ALA and MAL was not dose dependent. Therefore it was decided that a standard quantity (0.03 ml) should be applied in the present study. This study examined the time course characteristics of ALA and MAL-induced PpIX uorescence in normal human skin. Results have shown that the duration of application time does

inuence PpIX uorescence following the application of ALA and MAL. Examination of the ALA data showed that PpIX uorescence was detectable even after only a 1 h application time (Fig. 1). The shorter application times of 13 h resulted in peak uorescence being achieved by the 7 h time point. The uorescence at the later 24 and 28 h time points was greatly reduced by approximately 75%. This rapid reduction may be due to availability of ALA being the limiting factor. Longer application times (46 h) resulted in more sustained uorescence, which peaked at 24 h. The uorescence was noted to have reduced by 28 h, a reduction of approximately 30%. It is possible that at these longer application times the heme biosynthetic pathway may be the limiting factor, thus supporting the work done by Di Venosa et al. (19). The longer the application time the more ALA will accumulate within the cell, this surplus ALA would remain within the cell until enzymatically converted to PpIX. It was also noted that PpIX uorescence was detectable following a 1 h application of MAL (Fig. 2). Peak uorescence for all application times was achieved at 7 h, and had decreased by approximately 75% at 24 h. This substantial decrease is consistent with previous work (12, 14, 20). Although the exact mechanisms for this rapid reduction are unclear, low intracellular ALA ester levels may be achieved by an increased rate of ALA ester efux from the cell, a hypothesis presented by Di Venosa et al. (19). Statistical analysis comparing the 1 and 4 h ALA application times showed no difference in uorescence at the 47 h time points. However, at 24 and 28 h there was signicantly more uorescence noted following the 4 h application time. Similar results were noted when comparing the 1 and 6 h ALA application times. It would be of interest to investigate if the same were true in tumor tissue. If so, then shorter application times (e.g. 12 h) would not only provide enough PpIX to be therapeutically effective, but would also reduce any unwanted phototoxic reactions 2428 h post PDT treatment. Comparison between the 4 and 6 h application times did not show any difference in uorescence at any of the time points. Currently application
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Lesar et al. Table 1. Mean uorescence and standard deviation (SD) following the application of ALA/MAL to the inner forearm (n = 17) Application time (h) 1 Time (h) 1 2 3 4 5 6 7 24 28 6 7 24 28 Mean ALA induced uorescence SD 27 21 78 46 121 70 131 79 173 102 198 123 182 113 52 63 35 41 195 75 220 88 312 88 224 105 Mean MAL induced uorescence SD 22 16 42 36 70 63 87 88 86 98 99 107 97 112 18 14 18 9 178 95 206 99 68 64 49 43

0.05) between ALA- and MAL-induced uorescence. ALA. 5-aminolevulinic acid; MAL, methyl aminolevulinate.

Statistically signicance (P

Fig. 4. Comparison of protoporphyrin IX uorescence following a 6 h application of 5-aminolevulinic acid/methyl aminolevulinate (ALA/ MAL) to the inner forearm. Values are represented as means SD; P 0.05 (n = 17).

times of 46 h are used when treating NMSC (18, 21). It would be benecial to study the uorescence characteristics in tumor tissue using 4 and 6 h application times, as these results (although in normal skin) would suggest that there is no additional benet using the longer 6 h application time. Statistical analysis comparing the 1 and 4 h MAL application times showed no difference in uorescence at the 4 and 5 h time points. However, at 628 h there was signicantly more uorescence noted following the 4 h application time. Similar results were noted when comparing the 1 and 6 h MAL application times; signicant differences were noted at all time points (628 h). Comparison between the 4 and 6 h application times did not show any difference in uorescence at the 6 and 7 h time points. However, the uorescence following a 6 h application was signicantly 4 4 h application, suggesting that longer application times result in a more sustained uorescence. Again these investigations should be repeated in tumor tissue. Comparison of ALA and MAL highlighted striking differences. When comparing the shorter application time (13 h) at all time
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points (except for 28 h time point following a 1 h application) ALA-induced PpIX uorescence was signicantly greater than MAL-induced uorescence (Fig. 3 and Table 1). This contradicts previous work in both cell line and animal models, which have shown that ALA esters increase PpIX production (22, 23). However, the results obtained in this study support the ndings by Fritsch et al. (7), who showed that PpIX production in AKs was lower following the application of MAL when compared with ALA. When comparing the 6 h application time, although ALA induced increased levels of uorescence this was only signicant at the 24 and 28 h time points (Fig. 4 and Table 1). These ndings would suggest elevated sustained PpIX uorescence could only be achieved by longer MAL application times. It would be of interest to repeat this work in tumor tissue because potentially by dening these time course characteristics PDT treatments could be optimized. Given the reported selectivity of MAL-induced uorescence to the application site in nude mice (6), in contrast to the spread of ALA-induced uorescence, it would be interesting to study the spread of MAL- and ALA-induced uorescence in human subjects. This was beyond the scope of the present investigation. However, it should be noted that in an earlier study in which we applied ALA to one half of a lesion only, the other half showed no uorescence over a 6 h time period, suggesting that there was no signicant lateral diffusion of ALA during this time (24). The results obtained in this study have shown ALA and MAL induced PpIX uorescence in normal skin is dependent on application time. Therefore emphasizing the importance of dening the time course characteristics of photosensitizers in tumor tissue. Currently PDT treatment regimes for NMSC advise longer ALA application times (46 h) and shorter MAL time (3 h). However, these results (although in normal skin) would suggest that shorter ALA application times might not only achieve the same production of PpIX, but also reduce the delayed photosensitivity. These results would also suggest that longer MAL application times result in increased PpIX production, which could be of therapeutic benet.

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PpIX uorescene time course

References
1. Soler AM, Warloe T, Tausjo J, Giercksky KE. Photodynamic therapy of residual or recurrent basal cell carcinoma after radiotherapy using topical 5-aminolevulinic acid or methylester aminolevulinic acid. Acta Oncol 2000; 39: 6059. 2. Soler AM, Warloe T, Berner A, Giercksky KE. A follow-up study of recurrence and cosmesis in completely responding supercial and nodular basal cell carcinomas treated with methyl 5-aminolaevulinate-based photodynamic therapy alone and with prior curettage. Br J Dermatol 2001; 145: 46771. 3. Horn M, Wolf P, Wulf HC, et al. Topical methyl aminolaevulinate photodynamic therapy in patients with basal cell carcinoma prone to complications and poor cosmetic outcome with conventional treatment. Br J Dermatol 2003; 149: 12429. 4. Kormeili T, Yamauchi PS, Lowe NJ. Topical photodynamic therapy in clinical dermatology. Br J Dermatol 2004; 150: 10619. 5. Rhodes LE, De RM, Enstrom Y, et al. Photodynamic therapy using topical methyl aminolevulinate vs surgery for nodular basal cell carcinoma: results of a multicenter randomized prospective trial. Arch Dermatol 2004; 140: 1723. 6. Peng Q, Moan J, Warloe T, et al. Build-up of esteried aminolevulinic-acid-derivative-induced porphyrin uorescence in normal mouse skin. J Photochem Photobio B 1996; 34: 956. 7. Fritsch C, Homey B, Stahl W, Lehmann P, Ruzicka T, Sies H. Preferential relative porphyrin enrichment in solar keratoses upon topical application of delta-aminolevulinic acid methylester. Photochem Photobiol 1998; 68: 21821. 8. Ninomiya Y, Itoh Y, Tajima S, Ishibashi A. In vitro and in vivo expression of protoporphyrin IX induced by lipophilic 5-aminolevulinic acid derivatives. J Dermatol Sci 2001; 27: 11420. 9. Schneckenburger H, Konig K, Kunzi-Rapp K, Westphal-Frosch C, Ruck A. Time-resolved in-vivo uorescence of photosensitizing porphyrins. J Photochem Photobiol B 1993; 21: 1437. 10. Moan J, Streckyte G, Bagdonas S, Bech O, Berg K. Photobleaching of protoporphyrin IX in cells incubated with 5-aminolevulinic acid. Int J Cancer 1997; 70: 907. 11. Fritsch C, Lehmann P, Schulte KW, et al. Optimum porphyrin accumulation in epithelial skin tumours and psoriatic lesions after topical application of 5-aminolaevulinic acid. Br J Cancer 1999; 79: 16038. 12. Juzenas P, Sharfaei S, Moan J, Bissonnette R. Protoporphyrin IX uorescence kinetics in UV-induced tumours and normal skin of hairless mice after topical application of 5-aminolevulinic acid methyl ester. J Photochem Photobiol B 2002; 67: 117.

13. Fitzpatrick TB. The validity and practicality of sun-reactive skin types I through VI. Arch Dermatol 1988; 124: 86971. 14. Lesar AE, Padgett M, ODwyer M, Ferguson J, Moseley H. An investigation of the uorescence induced by topical application of different quantities of 5-aminolevulinic acid and methyl aminolevulinate on normal human skin. Photodiagn Photodyn Ther 2007; 4: 2249. 15. Nadeau V, Hamdan K, Hewett J, et al. Endoscopic uorescence imaging and point spectroscopy system for the detection of gastro-intestinal cancers. J Mod Optic 2002; 49: 73141. 16. ODwyer M, Nadeau V, Padgett M. A spectroscopic tool based on an interference lter and birefringent prisms: demonstration of detection of 5-aminolaevulinic acid-induced protoporphyrin IX uorescence. J Phys D Appl Phys 2003; 36: 17036. 17. Nadeau V, ODwyer M, Hamdan K, Tait I, Padgett M. In vivo measurement of 5-aminolaevulinic acid-induced protoporphyrin IX photobleaching: a comparison of red and blue light of various intensities. Photodermatol Photoimmunol Photomed 2004; 20: 1704. 18. Morton CA, Brown SB, Collins S, et al. Guidelines for topical photodynamic therapy: report of a workshop of the British Photodermatology Group. Br J Dermatol 2002; 146: 55267. 19. Di Venosa GM, Fukuda H, Batlle A, Macrobert AJ, Casas A. Photodynamic therapy: regulation of porphyrin synthesis and hydrolysis from ALA esters. Photochem Photobiol 2006; 5: 12936. 20. Bugaj A, Iani V, Juzeniene A, Juzenas P, Li-Wei M, Moan J. The effect of dimethylsulfoxide,1-[2-(decylthio)ethyl]azacyclopentan-2-one and Labrafacs CC on porphyrin formation in normal mouse skin during topical application of methyl 5-aminolevulinate: a uorescence and extraction study. Photodiagn Photodyn Ther 2006; 1: 2733. 21. Ibbotson SH. How we treat a supercial basal cell carcinoma with topical photodynamic therapy in Dundee. Photodiagn Photodyn Ther 2006; 3: 12831. 22. Brunner H, Hausmann F, Knuechel R. New 5-aminolevulinic acid esters efcient protoporphyrin precursors for photodetection and photodynamic therapy. Photochem Photobiol 2003; 78: 4816. 23. Ogasawara T, Miyoshi N, Fukuda M, et al. Fluorescent analysis of 5-aminolevulinic acid-induced protoporphyrin-IX in mouse transplanted tumour tissues. Int Congr Ser 2003; 1248: 4058. 24. Moseley H, Brancaleon L, Lesar AE, Ferguson J, Ibbotson SH. Does surface preparation alter ALA uptake in supercial nonmelanoma skin cancer in vivo? Photodermatol Photoimmunol Photomed 2008; 74: 725.

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