Fluorescence contrast and threshold limit: implications for photodynamic diagnosis of basal cell carcinoma

This study was designed to evaluate what application time of delta-5-aminolaevulinic acid (ALA) results in highest contrast between tumour and normal skin, in the interval 1-4 h, when using photodynamic diagnosis (PDD) of basal cell carcinomas (BCC) located on the face. Moreover, a value of the demarcation limit has been derived based on the fluorescence variation in normal skin adjacent to the tumour. Forty patients were included in the study, randomly allocated to four different groups with varying ALA application time in the range 1-4 h. The contrast, defined as the ratio between the fluorescence intensity in ALA-treated tumour tissue and normal skin, was calculated for each patient, and the mean values in each group were evaluated as a function of ALA application time. In addition, the fluorescence intensity variation in ALA-treated normal skin adjacent to the tumour was assessed. The results from this study show a peak of the mean contrast values after 3 h ALA application, but due to large interpatient variation, the mean contrast did not differ significantly in the interval 2-4 h. After 2 h ALA application, the fluorescence intensity variation in the normal ALA-treated skin was found to be at a maximum, which suggests that 2 h ALA application is not preferable when using PDD. Based on data of the fluorescence variation in ALA-treated normal skin after 3 and 4 h ALA application, a tolerance interval was calculated implying that values above 1.4 times the mean normal fluorescence indicate an abnormal condition. This tolerance limit agrees well with results obtained in a former study.     

Comparison of ALA- and ALA hexyl-ester-induced PpIX depth distribution in human skin carcinoma

Photodynamic therapy (PDT) based on the use of photoactivable porphyrins, such as protoporphyrin IX (PpIX), induced by the topical application of amino-levulinic acid (ALA) or its derivatives, ALA methyl-ester (m-ALA), is a treatment for superficial basal cell carcinoma (BCC), with complete response rates of over 80%. However, in the case of deep, nodular-ulcerative lesions, the complete response rates are lower, possibly related to a lower bioavailability of PpIX. Previous in vitro skin permeation studies demonstrated an increased penetration of amino-levulinic acid hexyl-ester (h-ALA) over ALA. In this study, we tested the validity of this approach in vivo on human BCCs. An emulsion containing 20% ALA (w/w) and preparations of h-ALA at different concentrations were applied topically to the normal skin of Caucasian volunteers to compare the PpIX fluorescence intensities with an optical fiber-based spectrofluorometer. In addition, the PpIX depth distribution and fluorescence intensity in 26 BCCs were investigated by fluorescence microscopy following topical application of 20% ALA and 1% h-ALA. We found that, for application times up to 24h, h-ALA is identical to ALA as a PpIX precursor with respect to PpIX fluorescence intensity, depth of penetration, and distribution in basal cell carcinoma, but has the added advantage that much smaller h-ALA concentrations can be used (up to a factor 13). We observed a non-homogenous distribution in BCCs with both precursors, independent of the histological type and depth of invasion in the dermis.     

Topical application of 5-aminolevulinic acid and its methylester,hexylester and octylester derivatives: considerations for dosimetry in mouse skin model

Ester derivatives of 5-aminolevulinic acid (ALA-esters) have been proposed as alternative drugs for ALA in photodynamic therapy. After topical application of creams containing ALA, ALA methylester (ALA-Me), ALA hexylester (ALA-Hex) and ALA octylester (ALA-Oct) on mouse skin, typical fluorescence excitation and emission spectra of protoporphyrin IX (PpIX) were recorded, exhibiting a similar spectral shape for all the drugs in the range of concentrations (0.5-20%) studied. The accumulation kinetics of PpIX followed nearly a similar profile for all the drug formulations. The fluorescence of PpIX peaked at around 6-12 h of continuous cream application. Nevertheless, some differences in pharmacokinetics were noticed. For ALA cream, the highest PpIX fluorescence was achieved using 20% of ALA in an ointment. Conversely, 10% of ALA-Me and ALA-Hex, but not of ALA-Oct, in the cream was more efficient (P < 0.05) than was 20%. The cream becomes rather fluid when 20% of any of these ALA-esters is used in ointment, whereas 10% and lower concentrations of ALA-esters do not significantly increase fluidity of the cream. The dependence of PpIX accumulation on the concentration of ALA and ALA-ester in the applied cream followed (P < 0.002) kinetics as described by a mathematical model based on the Michaelis-Menten equation for enzymatic processes. Under the present conditions, the PpIX amount in the skin increased by around 50% by the application of ALA-Me, ALA-Hex or ALA-Oct for 4-12 h as compared with ALA for the same period. Observations of the mice under exposure to blue light showed that after 8-24 h of continuous application of ALA, the whole mouse was fluorescent, whereas in the case of ALA-Me, ALA-Hex and ALA-Oct the fluorescence of PpIX was located only at the area of initial cream application. The amount of the active compound in the applied cream necessary to induce 90% of the maximal amount of PpIX was determined for normal mouse skin. Optimal PpIX fluorescence can be attained using around 5% ALA, 10% ALA-Me and 5% ALA-Hex creams during short application times (2-4 h). Topical application of ALA-Oct may not gain optimal PpIX accumulation for short applications (<5 h). For long application times (8-12 h), it seems that around 1% ALA, 4% ALA-Me, 6% ALA-Hex and 16% ALA-Oct can give optimal PpIX fluorescence. But for long application times and high concentrations, systemic effect of ALA applied topically on relatively large areas should be considered.     

In vivo pharmacokinetics of protoporphyrin IX accumulation following intracutaneous injection of 5-aminolevulinic acid

Photodynamic therapy with 5-aminolevulinic acid (ALA) derived protoporphyrin IX (PpIX) as photosensitizer is a promising treatment for basal cell carcinomas. Until now ALA has been administered topically as an oil-in-water cream in most investigations. The disadvantage of this administration route is insuffici?nt penetration in deeper, nodular tumours. Therefore we investigated intracutaneous injection of ALA as an alternative administration route. ALA was administered in 6-fold in the normal skin of three 6-week-old female Dutch pigs by intracutaneous injection of an aqueous solution of ALA (pH 5.0) in volumes of 0.1-0.5 ml and concentrations of 0.5-2% and by topical administration of a 20% ALA cream. During 8 h fluorescence of ALA derived PpIX was measured under 405 nm excitation. For the injection the measured fluorescence was shown to be dose dependent. All injected doses of 3 mg ALA or more lead to a faster initial increase rate of PpIX synthesis and significantly greater fluorescence than that measured after topical administration of ALA. Irradiation (60 Jcm(-2) for 10 min) of the spots was performed at 3.5 h after ALA administration. After 48 and 96 h visual damage scores were evaluated and biopsies were taken for histopathological examination. After injection of 2 mg ALA or more the PDT damage after illumination was shown to be significantly greater than after topical application of 20% ALA. An injected dose of 10 mg ALA (0.5 ml of a 2% solution) resulted in significantly more tissue damage after illumination than all other injected doses.     

Kinetic fluorescence studies of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation in basal cell carcinomas.

Laser-induced fluorescence (LIF) investigations have been performed in connection with photodynamic therapy (PDT) of basal cell carcinomas and adjacent normal skin following topical application of 5-aminolaevulinic acid (ALA) in order to study the kinetics of the protoporphyrin IX (PpIX) build-up. Five superficial and 10 nodular lesions in 15 patients are included in the study. Fluorescence measurements are performed prior to the application of ALA, 2, 4 and 6 h post ALA application, immediately post PDT (60 J cm-2 at 635 nm), and 2 h after the treatment. Hence, the build-up, photobleaching and re-accumulation of PpIX can be followed. Superficial lesions show a maximum PpIX fluorescence 6 h post ALA application, whereas the intensity is already the highest 2-4 h after the application in nodular lesions. Immediately post PDT, the fluorescence contribution at 670 nm from the photoproducts is about 2% of the pre-PDT PpIX fluorescence at 635 nm. Two hours after the treatment, a uniform distribution of PpIX is found in the lesion and surrounding normal tissue. During the whole procedure, the autofluorescence of the lesions and the normal skin does not vary significantly from the values recorded before the application of ALA.     

Hyperpigmentation induced by topical 5-aminolaevulinic acid plus visible light

Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) is an alternative tool for the treatment of superficial non-melanoma skin cancers. Recently ALA-PDT has been employed with encouraging results also for warts, condylomata and psoriasis. In this study the effects of topical ALA plus irradiation with visible light on intact human skin have been evaluated. Five skin areas (A, B, C, D, and E) on the inner upper part of the arms of five healthy volunteers (skin types III and IV) were treated with (A) ALA 20% in base cream without irradiation, (B) only the vehicle (base cream) without ALA, (C, D and E) ALA cream at the concentrations of 5, 10 and 20%, respectively; all treatments were applied with an occlusive dressing. Four hours after ALA or vehicle application areas B, C, D and E were irradiated with a fixed dose of 40 J/cm(2). ALA penetration through the intact skin was evaluated by in vivo fluorescence determination. The effects on healthy skin were evaluated by clinical and chromometric examinations, light microscopy, immunohistochemistry and electron microscopy. RESULTS: (1) in vivo fluorescence demonstrated that ALA is able to penetrate through the intact skin, when applied with occlusive dressing and induces a classical fluorescence peak due to Protoporphyrin IX (PpIX) formation, which is the active photosensitiser. (2) Skin areas receiving ALA plus irradiation showed erythema and swelling just after the irradiative session and hyperpigmentation 48-72 h later. (3) Colourimetric data confirmed significant skin colour changes: values a* (representing the erythematous changes) increased only on the skin areas where ALA+irradiation were applied and during the 48 h after irradiation, thereafter a* began to decrease; values L* (pigmentation) increased during the 2 weeks following treatment. (4) Histopathological, immunohistochemical (S100, HMB-45) and electron microscopic findings showed an absolute increment of the number of melanocytes, which appeared clearly activated. In conclusion the application of ALA cream followed by irradiation is able to induce a pigmentation response in healthy human skin, at least in skin types III and IV. This melanocytic activation could have a potential for the treatment of skin disorders characterised by hypopigmentation.     

Kinetics of Photobleaching of Protoporphyrin IX in the Skin of Nude Mice Exposed to Different Fluence Rates of Red Light

Abstract— The purpose of the present study was to determine the kinetics and the fluence rate dependency of the photo-bleaching of protoporphyrin IX (PpIX) in normal skin of Balb/c nude mice after systemic and topical application of 5-aminolevulinic acid (ALA). ALA was administered systemically (200 mg/kg body weight, i.p.) and topically (20% w/w ALA cream) to the mice. Fluences of up to 40 J/cm² were delivered by a dye laser (636 nm) at fluence rates of 37.5, 75, 150, 300 and 500 mW/cm². The photo-bleaching rate was constant within this range of fluence rates. This result suggests that there is no oxygen effect for PpIX photobleaching in this region for the skin of Balb/c nude mice. During light exposure the fluorescence decay followed neither first- nor second-order kinetics. The decay rate was slightly faster after systemic application than after topical application of ALA, but did not depend on the time (1–8 h) between application and analysis.     

Oral aminolevulinic acid induces protoporphyrin IX fluorescence in psoriatic plaques and peripheral blood cells

Photodynamic therapy (PDT) with topical aminolevulinic acid (ALA) has been shown in previous studies to improve psoriasis. However, topical ALA-PDT may not be practical for the treatment of extensive disease. In order to overcome this limitation we have explored the potential use of oral ALA administration in psoriatic patients. Twelve patients with plaque psoriasis received a single oral ALA dose of 10, 20 or 30 mg/kg followed by measurement of protoporphyrin IX (PpIX) fluorescence in the skin and circulating blood cells. Skin PpIX levels were determined over time after ALA administration by the quantification of the 635 nm PpIX emission peak with in vivo fluorescence spectroscopy under 442 nm laser excitation. Administration of ALA at 20 and 30 mg/kg induced preferential accumulation of PpIX in psoriatic as opposed to adjacent normal skin. Peak fluorescence intensity in psoriatic and normal skin occurred between 3 and 5 h after the administration of 20 and 30 mg/kg, respectively. Ratios of up to 10 for PpIX fluorescence between psoriatic versus normal skin were obtained at the 30 mg/kg dose of ALA. Visible PpIX fluorescence was also observed on normal facial skin, and nonspecific skin photosensitivity occurred only in patients who received the 20 or 30 mg/kg doses. PpIX fluorescence intensity was measured in circulating blood cells by flow cytometry. PpIX fluorescence was higher in monocytes and neutrophils as compared to CD4+ and CD8+ T lymphocytes. PpIX levels in these cells were higher in patients who received higher ALA doses and peaked between 4 and 8 h after administration of ALA. There was only a modest increase in PpIX levels in circulating CD4+ and CD8+ T lymphocytes. In conclusion oral administration of ALA induced preferential accumulation of PpIX in psoriatic plaques as compared to adjacent normal skin suggesting that PDT with oral ALA should be further explored for the treatment of psoriasis.     

Study of protoporphyrin IX (PpIX) pharmacokinetics after topical application of 5-aminolevulinic acid in urethral condylomata acuminata

The pharmacokinetics of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in lesions of urethral condylomata acuminata were investigated. Sixty patients (20 to 60 years old, 48 male and 12 female) were divided randomly into five groups and received topic application of different concentrations of ALA solution (0.5%, 1%, 3%, 5% or 10%). Biopsy was performed between 1 and 7 h and specimens were subjected to histological, PpIX fluorescence and human papillomavirus (HPV) DNA typing analyses. Fluorescence examination confirmed that ALA-induced PpIX fluorescence was dominantly distributed in the HPV-infected epidermis. In contrast, only a minimal amount of PpIX fluorescence was detected in the dermis. The maximal fluorescence intensity was detected at 5 h incubation. Higher ALA concentration (e.g. 5% and 10%) produced a stronger intensity. These results suggest that the topical application of 5-10% ALA solution for 3-5 h is the optimal condition for the photodynamic therapy of urethral condylomata acuminata. The selective damage of the condylomata acuminata lesions in the epidermis without damaging the dermis ensures a better control of recurrence and side effects such as ulceration or scarring. DNA typing showed that all patients were positive for low risk-HPV DNA and among them 18.3% of patients harbored high risk-HPV DNA.     

In vitro fluorescence, toxicity and phototoxicity induced by delta-aminolevulinic acid (ALA) or ALA-esters

Synthesis of delta-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 micrograms/mL), E1 and E2 (10 micrograms/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 micrograms/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm2) applied 6 h after 5 min incubation with 168 micrograms/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.     

Targeting of the tumor microcirculation by photodynamic therapy with a synthetic porphycene

9-acetoxy-2,7,12,17-tetrakis-(β-methoxyethyl)-porphycene (ATMPn) is a chemically pure substance with fast pharmacokinetics and superior photodynamic properties in vitro as compared to Photofrin®. In this study the pharmacokinetics, photodynamic efficacy and tissue localization of ATMPn were investigated in vivo.

Amelanotic melanomas (A-Mel-3) were implanted in dorsal skin fold chambers fitted to Syrian Golden hamsters. Fluorescence kinetics of ATMPn (1.4 μmol kg⁻¹ b.w.i.v; n = 8) were monitored by intravital microscopy. Quantitative measurements of fluorescence intensity were carried out by digital image analysis. For tumor growth studies 1.4 μmol kg⁻¹ was injected 24 h (n = 3), 3 h (n = 3), 1 min (n = 6) and 2.8 μmol kg⁻¹ 1 min (n = 6) before PDT (Laser (630 nm) or lamp (600–750 nm), 100 mW cm⁻², 100 J cm⁻²). Tumor volume was measured for 28 d. Solid tumors (n = 3) were excised 1 min after injection of ATMPn (2.8 μmol kg⁻¹) and cryostat sections (20 mm) were analyzed by confocal laser scanning microscopy (CLSM) for tissue localization of the dye.

Maximal fluorescence (mean ± S.E.) arose in the tumor (94 ± 7%) and surrounding host tissue (67 ± 5%) 30 s post injection followed by a rapid decrease. Hardly any fluorescence was detectable 12 h after administration. Only PDT 1 min after injection of ATMPn was effective yielding 3/6 complete remissions (2.8 mmol kg⁻¹, laser) and 6/6 complete remissions (2.8 μmol kg⁻¹, lamp), respectively. One minute after injection the dye is primarily localized in the vascular wall of normal and tumor vessels as shown by CLSM.

PDT at a time, when the dye is localized primarily in the tumor microcirculation, exhibits the best tumor killing effects showing that vascular targeting is effective in treating solid malignant tumors. ATMPn in liposomes makes administration and light irradiation in one session possible due to its fast pharmacokinetics. Thus, using ATMPn as a photosensitizer may provide more flexibility to perform PDT after surgical exploration and debulking as adjuvant therapy.     

Systemic component of protoporphyrin IX production in nude mouse skin upon topical application of aminolevulinic acid depends on the application conditions

Topical application of 5-aminolevulinic acid (ALA) for protoporphyrin IX (PpIX)-based photodynamic therapy of skin cancer is generally considered not to induce systemic side effects because PpIX is supposed to be formed locally. However, earlier studies with topically applied ALA have revealed that in mice PpIX is not only produced in the application area but also in other organs including skin outside the application area, whereas esterified ALA does not. From these results, it was concluded that it is not redistribution of circulating PpIX that causes the fluorescence distant from the ALA application site, but rather, local PpIX production induced by circulating ALA. In the present study we investigate the effects of the ALA concentration in the cream, the application time, the presence of a penetration enhancer, the presence of the stratum corneum and esterification of ALA on the PpIX production in nude mouse skin outside the area where ALA is applied. For this purpose, ALA and ALA hexyl ester (ALAHE) were applied to one flank, and the PpIX fluorescence was measured in the contralateral flank. During a 24 h application of ALA, PpIX was produced in the contralateral flank. No PpIX could be detected in the contralateral flank after ALA application times ranging from 1 to 60 min. Tape-stripping the skin prior to short-term ALA application, but not the addition of a penetration enhancer, resulted in PpIX production in the contralateral flank. When ALAHE was applied, no PpIX fluorescence was measured in the contralateral flank under any application condition. The results suggest that the systemic component of PpIX production outside the ALA application area plays a minor or no role in relevant clinical situations, when the duration of ALA (ester) application is relatively short and a penetration enhancer is possibly added.     

Uptake of topically applied 5-aminolevulinic acid and production of protoporphyrin IX in normal mouse skin: dependence on skin temperature

The temperature dependence of the uptake phase of 5-aminolevulinic acid (ALA) and the following production phase of protoporphyrin IX (PpIX) in normal mouse skin was investigated. A cream containing 20% ALA was topically applied on the skin for 10 min. The amount of ALA-induced PpIX was evaluated by measuring the fluorescence of PpIX from the treated skin. No measurable amount of PpIX was found in the skin immediately after 10 min application of ALA. The penetration of ALA into the skin was almost temperature independent while the following production of PpIX was found to be a strongly temperature-dependent process. Practically no PpIX was formed in the skin as long as skin temperature was kept low (12 degrees C).     

On the role of iron and one of its chelating agents in the production of protoporphyrin IX generated by 5-aminolevulinic acid and its hexyl ester derivative tested on an epidermal equivalent of human skin

Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) or its derivatives as precursors of protoporphyrin IX (PPIX) is routinely used in dermatology for the treatment of various pathologies. However, this methodology suffers to some extent from a limited efficacy. Therefore, the main goal of this study was to investigate the modulation and pharmacokinetics of PPIX buildup after a 5 h incubation with ALA (1.5 mM) and one of its derivatives, the hexyl ester of ALA (h-ALA) (1.5 mM), on the human epidermal equivalent Epidex. PPIX production was modulated with (L+) ascorbic acid iron (II) salt (LAI) or the iron (II)-specific chelating agent deferoxamine (DFO). PPIX fluorescence from the Epidex layers was measured up to 150 h after the precursor administration using a microspectrofluorometer (lambda(ex): 400 +/- 20 nm; lambda(det): 635 nm). The maximum PPIX fluorescence intensity induced by h-ALA was about 1.7 x larger than that induced by ALA. The addition of DFO resulted in a more than 50% increase in PPIX fluorescence for both precursors. The decay half life measured for PPIX fluorescence is 30 and 42.5 h, respectively, for ALA and h-ALA. These half lives are doubled when the samples contain DFO. In the samples with the highest fluorescence intensity, a modified fluorescence spectrum was observed after 10 h, with the emergence of a peak at 590 nm, which is attributed to zinc protoporphyrin IX (Zn PPIX).     

CELLULAR FLUORESCENCE OF THE ENDOGENOUS PHOTOSENSITIZER PROTOPORPHYRIN IX FOLLOWING EXPOSURE TO 5-AMINOLEVULINIC ACID

Abstract— Supplying 5-aminolevulinic acid (ALA), a precursor in the biosynthetic pathway to heme from an external source leads to an accumulation of the endogenous fluorescent photosensitizer protoporphyrin IX (PPIX). Following instillation of ALA in the urinary bladder neoplastic tissue can be discerned by fluorescence cystoscopy or treated by illumination with light of an appropriate wavelength. In order to provide a biological rationale for the clinical findings, we have analyzed the capacity of three different cell lines to accumulate PPIX by flow cytometry. Three different urothelial cell lines, normal fibroblasts and endothelial cells were exposed to ALA under varying conditions. Urothelial cell lines J82 and RT4, derived from malignancies of the bladder displayed fluorescence intensities 9- and 16-fold, respectively, above the fluorescence level of the normal urothelial cell line HCV29. Human umbilical cord endothelial cells fluoresced moderately while the fibroblast cell line Nl exhibited a fluorescence level comparable to those of the cancer cells. Fluoresence increased with increasing cell density and was also dependent on the growth of cells as monolayers or multicellular spheroids. Increasing ALA concentrations led to saturation of fluorescence after 4 h of incubation at cell type-specific fluorescence levels obtained at different ALA concentrations. Continuous incubation in medium containing serum resulted in a linear rise of fluorescence during the first 4 h, which was followed by a saturation period (8–24 h) and a renewed rise. In the case of serum depletion, fluorescence intensities were significantly higher and increased linearly during the entire 48 h incubation period. By replacing serum with albumin, it could be shown that the emission of PPIX into the medium in the presence of serum is mainly caused by this protein. The ALA-induced fluorescence was predominantly perinuclear after 4 h of incubation and relocated toward the cell membrane after prolonged incubation. This study demonstrated the complexity of factors influencing the ALA-induced fluorescence and should stimulate further research in this field.     

PENETRATION POTENCY OF TOPICAL APPLIED δ-AMINOLEVULINIC ACID FOR PHOTODYNAMIC THERAPY OF BASAL CELL CARCINOMA *

Abstract Penetration potency of δ-aminolevulinic acid (ALA) was studied by examining fluorescence of endogenous protoporphyrin IX in different histological types of basal cell carcinoma. Ten basal cell carcinomas were coated with an ointment containing 10% ALA prior to excision; five served as controls. Tumors were excised either 4 h or 12 h after application of ALA using a modified Mohs’micrographic surgical technique. Horizontal sections were cut from deep dermis to tumor surface and examined under a fluorescence microscope. After 4 h of application, only skin appendages demonstrated fluorescence typical of protoporphyrin IX. After 12 h, fluorescence was detectable in tumor cells in deep dermis. The five controls revealed no fluorescence at any site. These results may confirm the high penetration potential of topically applied ALA and its usefulness in photodynamic therapy. For tumors penetrating to deep dermis, an application time of more than 4 h seems necessary, at least when hydrophilic solvents for ALA are used.     

Protoporphyrin IX fluorescence kinetics and localization after topical application of ALA pentyl ester and ALA on hairless mouse skin with UVB-induced early skin cancer

In order to improve the efficacy of 5-aminolevulinic acid-based (ALA) photodynamic therapy (PDT), different ALA derivatives are presently being investigated. ALA esters are more lipophilic and therefore may have better skin penetration properties than ALA, possibly resulting in enhanced protoporphyrin IX (PpIX) production. In previous studies it was shown that ALA pentyl ester (ALAPE) does considerably enhance the PpIX production in cells in vitro compared with ALA. We investigated the in vivo PpIX fluorescence kinetics after application of ALA and ALAPE to hairless mice with and without UVB-induced early skin cancer. ALA and ALAPE (20% wt/wt) were applied topically to the mouse skin and after 30 min, the solvent was wiped off and PpIX fluorescence was followed in time with in vivo fluorescence spectroscopy and imaging. At 6 and 12 h after the 30 min application, skin samples of visible lesions and adjacent altered skin (UVB-exposed mouse skin) and normal mouse skin were collected for fluorescence microscopy. From each sample, frozen sections were made and phase contrast images and fluorescence images were recorded. The in vivo fluorescence kinetics showed that ALAPE induced more PpIX in visible lesions and altered skin of the UVB-exposed mouse skin, but not in the normal mouse skin. In the microscopic fluorescence images, higher ALAPE-induced PpIX levels were measured in the stratum corneum, but not in the dysplastic layer of the epidermis. In deeper layers of the skin, PpIX levels were the same after ALA and ALAPE application. In conclusion, ALAPE does induce higher PpIX fluorescence levels in vivo in our early skin cancer model, but these higher PpIX levels are not located in the dysplastic layer of the epidermis.     

Protoporphyrin IX Fluorescence Induced in Basal Cell Carcinoma by Oral δ-Aminolevulinic Acid*

Limited depth of penetration significantly limits photodynamic therapy of nodular basal cell carcinoma (BCC) using topical δ(5)-aminolevulinic acid (ALA). To demonstrate safety and efficacy of orally administered ALA in inducing endogenous protoporphyrin IX (PpIX) production in BCC, 13 patients with BCC ingested ALA in a dose-escalation protocol. All dose ranges (10, 20 or 40 mg/kg single doses) resulted in formation of PpIX in human skin and BCC, measurable by in vivo fluorescence spectrophotometry. The PpIX fluorescence peaked in tumors before normal adjacent skin from 1 to 3 h after ALA ingestion. Gross fluorescence imaging of ex vivo specimens revealed greater PpIX fluorescence in tumor than normal skin only at the 40 mg/kg dose. Fluorescence microscopy confirmed this finding by showing distinct, full-thickness PpIX fluorescence in all subtypes of BCC only after ALA given at 40 mg/kg. Side effects were dose dependent and self limited. Photosensitivity lasting less than 24 h and nausea coinciding with peak skin PpIX fluorescence occurred at 20 and 40 mg/kg doses. After 40 mg/kg ALA, serum hepatic enzyme levels rose to a maximum within 24 h, then resolved over 1–3 weeks. Transient bilirubinuria occurred in two patients.     

Effects of photodynamic therapy with topical application of 5-aminolevulinic acid on normal skin of hairless guinea pigs.

Photodynamic therapy (PDT) is a relatively new approach to the treatment of neoplasms which involves the use of photoactivatable compounds to selectively destroy tumors. 5-Aminolevulinic acid (ALA) is an endogenous substance which is converted to protoporphyrin IX (PpIX) in the synthetic pathway to heme. PpIX is a very effective photosensitizer. The goal of this study was to evaluate the effect of PDT using topical ALA on normal guinea pig (g.p.) skin and g.p. skin in which the stratum corneum was removed by being tape-stripped (TS). Evaluation consisted of gross examination, PpIX fluorescence detection, reflectance spectroscopy, and histology. There was no effect from the application of light or ALA alone. Normal non-TS g.p. skin treated with ALA and light was unaffected unless high light and ALA doses were used. Skin from which the stratum corneum was removed was highly sensitive to treatment with ALA and light: 24 h after treatment, the epidermis showed full thickness necrosis, followed by complete repair within 7 d. Time-dependent fluorescence excitation and emission spectra were determined to characterize the chromophore and to demonstrate a build-up of the porphyrin in the skin. These data support the view that PDT with topical ALA is a promising approach for the treatment of epidermal cutaneous disorders.     

Protoporphyrin IX fluorescence kinetics in UV-induced tumours and normal skin of hairless mice after topical application of 5-aminolevulinic acid methyl ester

Accumulation of protoporphyrin IX (PpIX) was investigated in normal skin and UV-induced tumours in hairless mice after topical application of a cream containing 2, 8 or 16% of 5-aminolevulinic acid methyl ester (ALA-Me). Higher levels of PpIX were measured in tumours compared to normal skin. The maximal amount of PpIX was reached at 1.5, 3 and 4 h after 2, 8 and 16% ALA-Me application, respectively. Higher tumour to normal skin PpIX fluorescence ratios were measured after application of 8 and 16% ALA-Me than after application of 2%. After irradiation with a broad spectrum of visible light from a slide projector, more than 90% of PpIX was bleached by fluences of 36 and 48 J/cm2, at fluence rates of 10 and 40 mW/cm2 respectively. At these fluences, the PpIX photobleaching rate was significantly higher (P<0.05) in normal mouse skin than in tumours. In addition, for a given fluence, more PpIX was photobleached at the lower fluence rate (10 mW/cm2) than at the higher fluence rate (40 mW/cm2) in normal skin (P<0.001) as well as in tumours (P<0.05) after exposure to 24 J/cm2 of light. In conclusion, the highest tumour to normal skin PpIX ratio was observed 3 h after application of 8% ALA-Me, suggesting that light exposure should be performed at this time in order to achieve an optimal PDT effect in this tumour model.