Fluorescence induction of protoporphyrin IX by a new 5-aminolevulinic acid nanoemulsion used for photodynamic therapy in a full-thickness ex vivo skin model

Authors


Tim Maisch, Department of Dermatology, University Hospital of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany, Tel: +49(0)941-944-8944, Fax: +49(0)941-944-8943, e-mail: tim.maisch@klinik.uni-regensburg.de

Abstract

Please cite this paper as: Fluorescence induction of protoporphyrin IX by a new 5-aminolevulinic acid nanoemulsion used for photodynamic therapy in a full-thickness ex vivo skin model. Experimental Dermatology 2010; 19: e302–e305.

Abstract:  An ex vivo porcine skin model was utilized to analyse the penetration of 5-aminolevulinic acid (5-ALA) contained in a nanoemulsion-based formulation BF-200 ALA (10% 5-ALA-hydrochloride) versus 16% aminolevulinate methyl ester-hydrochloride in a commercially cream (MAL cream) by fluorescence microscopy of their common metabolite protoporphyrin IX (PpIX) after 3, 5, 8 and 12 h. Fluorescence signals of PpIX in pig skin treated with BF-200 ALA were stronger than those for MAL cream. At 8 and 12 h, the PpIX fluorescence signals were 4.8- and 5.0-fold higher than those measured after MAL cream application. Fluorescence signals of PpIX after application of BF-200 ALA were detected in deeper tissue layers of the epidermis than after application of MAL cream (97.2 ± 5.7 μm for BF-200 ALA vs 42.0 ± 4.2 μm for MAL cream). These data implicate that BF-200 ALA in photodynamic therapy might lead to a superior therapeutically effect of intraepidermal (in situ) squamous cell carcinomas.

Background

Meanwhile, topical photodynamic therapy (PDT) with 5-aminolevulinic acid (5-ALA) reached approval status for actinic keratosis in US and Canada, whereas methylaminolevulinate (MAL) is approved worldwide for actinic keratosis, for Bowen’s disease and Morbus bowen in Europe and Australia (1). In the last years, little attention has been paid to the penetration properties of 5-ALA or MAL, assuming that these small molecules penetrate easily into the epidermal tissue. The structure of the stratum corneum has a great influence on the transepidermal penetration of topically applied photosensitizers (2–4). In order to enhance skin penetration of 5-ALA, either the vehicle composition or the skin permeability can be altered in order to increase the penetration of 5-ALA (2,4,5). Recently, a soybean lecithin microemulsion gel demonstrated high affinities to epidermal tissue and higher in vitro skin penetration rates (6,7). The preparation of such a lecithin microemulsion gel containing the antitumor agent tetra-benzamidine demonstrated antitumor activity using an in vivo mice model (8).

Questions addressed

The aim of this study was to determine the kinetics of protoporphyrin IX (PpIX) generation and distribution of PpIX after topical application of 5-ALA delivered in a new nanoemulsion or MAL in the conventional formulation using an ex vivo full-thickness porcine skin model.

Experimental design

Sixteen per cent aminolevulinate methyl ester hydrochloride (methyl-5-amino-4-oxopentanoate 160 mg/g; MAL) were used in a cream containing sterile water, glycerol, white petrolatum, cholesterol, isopropylmyristate, almond oil, peanut oil and other stabilizers, preservatives and emulgators (Galderma SA, Paris, France) and a lecithin-containing nanoemulsion (particle diameter 30 nm) containing 10% 5-ALA hydrochloride (Biofrontera AG, Leverkusen, Germany). Details of the used ex vivo skin model are described elsewhere (9,10). The excised skin pieces were classified to a skin typ I/II; washed with distilled water, carefully depilated using an electric razor. For time-response kinetic experiments, the same amount of active ingredient per square centimetre was applied in each case (0.012 g/cm2). At different time points, fluorescence signals were used as an individual marker to determine induction and distribution of PpIX (11). Greyscale images with a bit depth of 16, which provides 65.536 levels of grey, were used to measure the fluorescence intensities of PpIX (Image Pro Plus 5.0, Media Cybernetics, Silver Spring, MD, USA). A thresholding was used to segment the images in order to extract the grey intensity levels of the fluorescence above which the values were stated as positive signals. The greyscale distribution of the fluorescent images was represented by red-coloured areas on the micrographs. The maximal depth of the epidermis to the basal membrane was determined as describe elsewhere (11). The viability of each porcine skin sample was tested using the Fluorescein FragEL DNA Fragmentation Detection Kit (Calbiochem, Darmstadt, Germany) (10). The data were evaluated by ANOVA. Pairs of data were compared using Bonferroni’s multiple comparison test (differences were significant when P < 0.05).

Results

Figure 1a shows the increase in the PpIX fluorescence signal over time after incubation of the skin with the BF-200 ALA. PpIX fluorescence signals could be observed 3 h after application. The intensity of the fluorescent signal continued to increase during the remaining observation period up to 12 h. Fluorescence signals of PpIX were also detected in the epidermis after MAL cream application for 3 h (Fig. 1b). Figure 1c represents the relationship between the fluorescence intensity of PpIX and time after application of BF-200 ALA or MAL cream. Following 3 and 5 h incubation with BF-200 ALA or MAL cream, the fluorescence intensity of PpIX was slightly higher after application of BF-200 ALA but differences were not statistically significant. The PpIX fluorescence signals measured more than 5 h following the application of BF-200 ALA were much stronger than the corresponding results for MAL cream. The PpIX fluorescence signals measured 8 and 12 h after application of BF-200 ALA were 4.8- and 5-fold higher than those measured after MAL cream application. The fluorescence signal intensity measured after 12 h of incubation with MAL cream was not higher in value than that induced by a 3- or 5-h incubation with BF-200 ALA. Application of the BF-200 ALA placebo or MAL cream control did never induce any fluorescence signals in the epidermis above threshold value (Fig. 1c and data not shown). Viability of the ex vivo porcine skin was not influenced during the entire observation period. A short incubation time (3 h) resulted in the induction of PpIX in the upper layers of the epidermis when BF-200 ALA or MAL cream was applied (Fig. 2a). Fluorescent signals were detected in deeper layers (97.2 ± 5.7 vs 42.0 ± 4.2 μm) of the epidermis after 12 h of incubation with BF-200 ALA and these signals were significantly stronger than the fluorescence induced by MAL cream (Fig. 2b). No penetration was detected beyond the basal cell membrane into the dermis for either formulation.

Figure 1.

 Induction of PpIX fluorescence in an ex vivo porcine skin model by BF-200 ALA. PpIX fluorescence intensity was measured after application of the BF-200 ALA formulation (a) or MAL cream (b) at different time points. (c) Mean fluorescence intensity of 42 individual values obtained from six independent experiments following application of BF-200 ALA or MAL cream are plotted against time of incubation (h). Grey dotted line shows the limit of detection of a positive fluorescence signal. The grey horizontal line describes the median value; the vertical grey line shows the interquartile range. Control: BF-200 ALA placebo. (Control: BF-200 ALA Placebo).inline image, P < 0.001 vs 3, 5, 8 and 12 h MAL; inline image, P < 0.001 vs 3, 5, 8 and 12 h MAL.

Figure 2.

 PpIX distribution after application of BF-200 ALA or MAL cream. (a) Porcine skin was incubated with BF-200 ALA (Left column) or MAL cream (right column). Samples were removed at the time points indicated and assessed for the induction of PpIX. The blue line indicates the border between epidermis and dermis (the basal membrane). (b) The depth of penetration was determined by analysing the fluorescence intensity of PpIX (red) along several vertical virtual lines. The virtual line was drawn at various points on each of the skin sections and the maximal depth of the epidermis (border = basal membrane) was also determined at the same points. (Grey bars; n = 7 median ± standard deviation). The black line with diamond-shaped symbols represents the maximal vertical extent of the epidermis (n = 7, median ± standard deviation). inline image, P < 0.05 vs 3 h MAL; inline image, P < 0.001 vs 3 and 5 h MAL; inline image, P < 0.001 vs 3, 5 and 8 h MAL; inline image, P < 0.001 vs 3, 5, 8 and 12 h MAL.

Conclusion

A prerequisite for a successful PDT of epidermal lesions is the effective penetration of a photosensitizer or a precursor across the stratum corneum into the deeper layers of the epidermis. It is possible that the different phases of the oil/water emulsion of the MAL cream result in a poorer penetration of the active ingredient and a correspondingly low conversion MAL to PpIX. 5-ALA or methyl-ALA are both water-soluble and will therefore be mostly found in the aqueous phase of a water/oil emulsion. Thus, the distribution and localization of hydrophilic and/or lipophilic agents can be affected by the nature of the formulation and also by the agent itself (4,12). Therefore, the lipophilic phase of an oil/water emulsion may inhibit the contact of the hydrophilic phase with the lipophilic surface of skin and thereby prevent a satisfactory penetration of the hydrophilic 5-ALA/methyl-ALA phase. It may be necessary for 5-ALA to leave the aqueous phase and to pass through the lipophilic phase before it can penetrate the stratum corneum. This may not exist for the BF-200 ALA formulation so that 5-ALA is available for direct penetration of the stratum corneum. The comparably effective transport of 5-ALA down to the basal membrane after BF-200 ALA application cannot be entirely explained by a better efficacy. It is conceivable that the fluorescence development in deeper layers of the epidermis is observed because the 5-ALA transport is supported by components of the BF-200 ALA formulation, which (i) stabilize 5-ALA by preventing dimerization and oxidation to pyryazin and (ii) supports uptake of 5-ALA by the cells. Comparable supportive effects by the MAL cream on the active ingredient MAL may not exist. The stratum corneum barrier is believed to be the primary barrier to the transdermal permeation of drugs. Transdermal delivery systems that make the skin surface locally more permeable, thereby facilitating the penetration of drugs are of great interest. One possibility to enhance permeation of 5-ALA is the use of a lecithin microemulsion transdermal delivery system (7,13,14). Phospholipids can change the skin lipid fluidity leading to enhanced percutaneous absorption of anti-inflammatory drugs, like indomethacin or diclofenac (15). A full-thickness porcine skin model was used to study the effect of BF-200 ALA, a new nanoemulsion containing 10% 5-ALA. BF-200 ALA facilitates the transport of 5-ALA through the stratum corneum and leads to a uniform distribution of PpIX within the epidermis. This emphasizes the potential benefit of BF-200 ALA for the treatment of more deeply seated superficial basal cell cancers.

Acknowledgement

The authors gratefully acknowledge financial support for this study from Biofrontera AG, Leverkusen, Germany.

Conflict of interest

Dr Szeimies received financial support for performing preclinical and clinical trials in the field of epithelial skin cancer from Biofrontera AG, 3M, meda Pharma, Photocure, photonamic and received travel support and speakers honoraria from Almirall Hermal, Galderma International, Intendis, Photocure and photonamic. The other authors declare no conflict of interest.

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