Relationship of protoporphyrin IX synthesis to photodynamic effects by 5-aminolaevulinic acid and its esters on various cell lines derived from the skin

Background  5-Aminolaevulinic acid (ALA) and its esters act as precursors to the fluorescent photosensitizer protoporphyrin IX (PpIX) in photodynamic therapy (PDT). There is little information about how ALA and its esters induce PpIX synthesis and photodynamic effects in cell lines derived from the skin.
Objectives  We compared the amount of PpIX synthesis induced by ALA and its esters in skin cell lines, and evaluated the relationship of PpIX synthesis to photodynamic effects by ALA and its esters in vitro .
Methods  Four cell lines, including human epidermal keratinocytes (HEK), human dermal fibroblast (hF), A431, and TXM13 were used. Cell survival was evaluated by the MTT assay. Fluorescence spectroscopy was used to measure the amount of PpIX synthesis induced by ALA and its esters. Flow cytometry measured cell death induced by ALA- and its esters-mediated PDT.
Results  ALA and its esters were not toxic at concentrations lower than 2 mmol L⁻¹ in all cell lines. PpIX synthesis was dose-dependent at low doses (0·01–0·1 mmol L⁻¹), and ALA esters were more effective than ALA. Cell death occurred from necrosis rather than apoptosis just after light irradiation illumination on both ALA and its esters-treated cells. Cell death related more to PpIX synthesis than the irradiation light dose.
Conclusions  PpIX production by ALA and its esters was induced on both normal and malignant cell lines derived from the skin, and cell death of PDT responses is closely related to the amount of PpIX synthesis rather than to the irradiation dose.     

Characteristics of 5-aminolaevulinic acid-induced protoporphyrin IX fluorescence in human skin in vivo

BACKGROUND/PURPOSE: Topical photodynamic therapy (PDT) is increasingly being used to treat skin cancers. Knowledge of the detailed characteristics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence in diseased and normal skin is incomplete. Understanding the characteristics of PpIX fluorescence in normal skin may facilitate optimization of PDT regimes while minimizing side effects in the surrounding normal skin. We investigated the characteristics of ALA-induced PpIX fluorescence in normal skin. METHODS: ALA was applied to the arm, back and leg skin of 21 healthy volunteers for 1-6 h, and PpIX fluorescence was measured for up to 24 h after ALA application using a fluorescent spectrometer. The effect of tape stripping on fluorescence was also examined. RESULTS: Considerable inter-subject variation was observed in the time to reach peak PpIX fluorescence. Intra-subject variation in the time to peak fluorescence was dependent on ALA application time. Six hours after ALA application, no significant difference was observed in the degree of fluorescence achieved irrespective of ALA application times ranging between 1 and 6 h. PpIX fluorescence was reduced on the leg and increased by tape stripping. CONCLUSIONS: Marked inter- and intra-subject variation in ALA-induced PpIX fluorescence occurs in normal human skin. ALA application time, body site and the state of the stratum corneum are all determinants of PpIX fluorescence within subjects and these factors need to be taken into account in optimization of PDT regimes.     

A quantitative assessment of protoporphyrin IX metabolism and phototoxicity in human skin following dose-controlled delivery of the prodrugs 5-aminolaevulinic acid and 5-aminolaevulinic acid-n-pentylester

BACKGROUND: Topical 5-aminolaevulinic acid (ALA) is widely used in photodynamic therapy (PDT) to generate protoporphyrin IX (PpIX) in the skin. However, other prodrugs may be more effective. OBJECTIVES: The pharmacokinetics of ALA- and ALA-n-pentylester-induced PpIX, together with the phototoxicity after PDT, were compared in human skin in vivo, using iontophoresis as a quantitative drug delivery system. METHODS: A series of six increasing doses of equimolar prodrug solutions was iontophoresed into normal skin of the upper inner arms of 20 healthy subjects. The kinetics of PpIX metabolism in skin (n = 4) and the response to light exposure, performed at 4.5 h (n = 6) and 6 h (n = 10) after application, were assessed by skin surface PpIX fluorescence and postirradiation erythema. RESULTS: ALA and ALA-n-pentylester showed a linear correlation between logarithm of dose and PpIX fluorescence (P < 0.005), and logarithm of dose and skin phototoxicity with irradiation at 4.5 h (P < 0.001 and P < 0.005, respectively) and 6 h (P < 0.05 and P < 0.0001, respectively) after iontophoresis. Higher phototoxicity was observed with ALA-n-pentylester than with ALA when sites were irradiated at 6 h, as indicated by the significantly lower theoretical threshold dose for erythema (P < 0.05) and the shift of the PpIX fluorescence/phototoxicity curve towards greater skin erythema at equal PpIX fluorescence levels. Depth of PpIX fluorescence in skin, as determined by fluorescence microscopy, was similar for both prodrugs, but a more homogeneous distribution of PpIX was seen with the more lipophilic ALA-n-pentylester. CONCLUSIONS: The observed greater phototoxicity of ALA-n-pentylester relative to ALA may be attributable to a more favourable PpIX localization in tissue and/or greater intrinsic toxicity.     

A placebo-controlled randomized study on the clinical effectiveness, immunohistochemical changes and protoporphyrin IX accumulation in fractionated 5-aminolaevulinic acid-photodynamic therapy in patients with psoriasis

BACKGROUND: Topical 5-aminolaevulinic acid (ALA)-photodynamic therapy (PDT) for the treatment of psoriasis has been evaluated in a few studies. In these studies different treatment parameters were used, there was a variable clinical response, and a nonhomogeneous fluorescence was seen after irradiation with Wood's light. OBJECTIVES: To study the clinical effectiveness, immunohistochemical changes and protoporphyrin IX accumulation in ALA-PDT in patients with psoriasis. Eight patients with stable plaque psoriasis with symmetrical involvement were included in the study. Two symmetrical plaques were randomly allocated to PDT either with 10% ALA or with placebo. Irradiation consisted of 2 and 8 J cm(-2) with a dark interval of 2 h (Waldmann PDT 1200 L, 600-750 nm, 40 mW cm(-2)) once weekly for 4 weeks. Before, during and after irradiation, fluorescence diagnosis was performed. Biopsies were taken at baseline, week 1 and week 6 for immunohistochemical assessment. Psoriatic plaques were clinically assessed using the plaque severity (sum) score. Fluorescence diagnosis was performed and expression of immunohistochemical markers for proliferation, differentiation and T-cell infiltration [Ki67, keratin 10 (K10), CD4, CD8 and CD45RO] was assessed. RESULTS: From week 1 up to week 6, ALA-PDT gave a significant reduction in the number of Ki67+ nuclei, while the K10 expression increased. After 6 weeks significant improvement was observed for CD8 and CD45RO. These changes were absent in the placebo-treated lesions. The sum scores were also significantly lower in the ALA-treated plaques. Heterogeneity of macroscopic fluorescence was observed during treatment despite keratolytic treatment. CONCLUSIONS: The present study shows that clinical improvement during fractionated ALA-PDT in psoriasis parallels histological improvement as seen in normalization of epidermal proliferation, differentiation and infiltration of relevant T-cell subsets. Optimizing the current treatment protocol may increase clinical efficacy further.     

The effect of an iron chelating agent on protoporphyrin IX levels and phototoxicity in topical 5-aminolaevulinic acid photodynamic therapy

BACKGROUND: In 5-aminolaevulinic acid (ALA)-photodynamic therapy (PDT), the prodrug ALA is endogenously converted to the active sensitizer protoporphyrin IX (PpIX), while further conversion of PpIX to haem requires iron. OBJECTIVES: To explore the potential of the iron chelator desferrioxamine (DFO) to enhance PpIX levels and phototoxicity in ALA-PDT. METHODS: A series of six doses of 2% ALA solution was iontophoresed into the healthy skin of each ventral forearm of 10 volunteers. One arm was pretreated with 20% DFO in aqueous cream, while the control arm received aqueous cream alone, for 16 h. At 5 h following iontophoresis, skin-surface PpIX fluorescence was measured, following which the forearms were simultaneously irradiated with 100 J cm-2 broadband red light. The phototoxic reaction was assessed at 24 h postirradiation as the minimal phototoxic dose (MPD) and with quantification of erythema. Next, eight patients with two superficial basal cell carcinomas or two plaques of Bowen's disease of similar appearance received 20% ALA topically to one lesion and 20% ALA with 20% DFO to the other, for 3 h. Skin-surface PpIX fluorescence was measured at 5 h, following which lesions were irradiated with 100 J cm-2 broadband red light. RESULTS: In healthy skin, PpIX fluorescence increased with increasing ALA dose at DFO-treated and untreated sites (P < 0.0005); PpIX fluorescence peak values were consistently higher in DFO-treated compared with control sites (P < 0.02). Erythema also correlated with ALA dose (P < 0.0005), but a significant difference between active and control sites occurred only at low ALA dose (P < 0.05). The median MPD appeared lower at the DFO-treated sites, at 6 mC vs. 12 mC (P = 0.06). In contrast, in lesional skin there was no consistent difference in PpIX fluorescence levels between those treated with and without DFO. CONCLUSIONS: While iron chelation augmented ALA-PDT phototoxicity in normal skin, this occurred only at low ALA dose. Addition of DFO does not appear to confer additional benefit in ALA-PDT of nonmelanoma skin cancers.     

Topical application of 5-aminolaevulinic acid, methyl 5-aminolaevulinate and hexyl 5-aminolaevulinate on normal human skin

BACKGROUND: 5-Aminolaevulinic acid (ALA) and its ester derivatives are used in photodynamic therapy. Despite extensive investigations, the differences in biodistribution and pharmacokinetics of protoporphyrin IX (PpIX) induced by ALA and its derivatives are still not well understood, notably for humans. OBJECTIVES: To study porphyrin accumulation after topical application of ALA and two of its ester derivatives in normal human skin. METHODS: Creams containing 0.2%, 2% and 20% (w/w) of ALA, methyl 5-aminolaevulinate (MAL) and hexyl 5-aminolaevulinate (HAL) were applied on normal human skin of six volunteers. The amount and distribution of porphyrins formed in the skin was investigated noninvasively by means of fluorescence spectroscopy. RESULTS: Fluorescence emission and excitation spectra exhibited similar spectral shapes for the all drugs, indicating that mainly PpIX was formed. Low concentrations (0.2% and 2%) of MAL induced considerably less PpIX in normal human skin than similar concentrations of ALA and HAL. A high concentration (20%) of ALA gave higher PpIX fluorescence in normal human skin than was found for MAL and HAL. CONCLUSIONS: The concentrations inducing half of the maximal PpIX fluorescence are around 2% for ALA, 8% for MAL and 1% for HAL.     

In vitro/in vivo correlations between transdermal delivery of 5-aminolaevulinic acid and cutaneous protoporphyrin IX accumulation and effect of formulation

BACKGROUND: Photodynamic therapy (PDT) using topical application of 5-aminolaevulinic acid (ALA) has been widely reported for the treatment of a variety of neoplastic and non-neoplastic cutaneous diseases. Although different formulations containing variable amounts of ALA have been applied in PDT, the dose-response relationships between transdermal ALA delivery and cutaneous protoporphyrin IX (PpIX) accumulation have not been studied. OBJECTIVES AND METHODS: The objectives of this study were to investigate the effect of permeability barrier function, ALA concentration and formulation on the in vitro penetration of ALA through nude mouse skin and cutaneous PpIX formation at 2 h following a 2-h application of ALA to nude mouse skin in vivo, and to delineate the relationships in between. RESULTS: Results demonstrated that variations in barrier integrity, in addition to ALA concentration, profoundly influenced ALA delivery to generate PpIX. Saturable correlations were found to exist between PpIX concentrations in both the epidermis and dermis in vivo and its transdermal flux in vitro, and the relationships were well described by the Emax model. The established correlations based on pure aqueous solutions were applicable to different formulations containing hydroxypropylmethylcellulose as the gelling agent and ethylenediamine tetraacetic acid as the iron chelator. Moreover, incorporation of desferrioxamine, another iron chelator, in the formulation prolonged cutaneous PpIX accumulation in the skin in comparison with 3% ALA aqueous solution, but the peak PpIX levels were not increased. Application of a liposomal formulation resulted in similar prolongation in ALA-induced PpIX accumulation, as well as better epidermal targeting. CONCLUSIONS: Knowledge of the dose-response relationships and the effect of formulation is important for designing optimal formulations and treatment schedules for topical ALA-PDT.     

Calcitriol treatment improves methyl aminolaevulinate-based photodynamic therapy in human squamous cell carcinoma A431 cells

Background  Photodynamic therapy (PDT) using methyl aminolaevulinate (MAL) provides a new, approved method for treatment of skin cancer and its precursors. However, MAL-based PDT is not very efficient for poorly differentiated skin carcinoma. Thus, novel strategies to enhance the PDT effect are needed.
Objectives  In order to improve the efficacy of MAL-based PDT, we investigated the effect of adding calcitriol, a prodifferentiation hormone, to human squamous cell carcinoma A431 cells in vitro .
Methods  A short course (24 h) of calcitriol pretreatment was applied in A431 cells, and, subsequently, MAL-induced protoporphyrin IX (PpIX) was measured.
Results  Calcitriol pretreatment of the cells elevated their PpIX levels. Furthermore, the cell damage after exposure to blue light was significantly higher in calcitriol-treated cells. Increased photoinactivation correlated with higher levels of PpIX in the calcitriol-pretreated cells.
Conclusions  Calcitriol enhances MAL-based PDT in A431 cells.     

A time course investigation of the fluorescence induced by topical application of 5-aminolevulinic acid and methyl aminolevulinate on normal human skin

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 (4–6 h) application times for ALA and shorter times (3 h) for MAL.
Aims: To establish the time course characteristics of protoporphyrin IX (PpIX) fluorescence 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 (1–6 h). PpIX fluorescence was detected using a non-invasive spectroscopy system.
Results and conclusion: PpIX fluorescence (following the application of either ALA or MAL) is dependent on duration of application. Following the application of ALA for 1–3 h peak fluorescence was noted at 7 h. Longer duration times (4–6 h) resulted in sustained fluorescence, which peaked at 24 h. MAL-induced fluorescence peaked at 7 h and was significantly decreased by 24 h for all application times. ALA induced fluorescence was shown to be significantly greater than MAL. The findings from this study have shown that potentially it would be more beneficial to apply ALA for shorter periods of time and MAL for longer than current practice.     

Randomized comparison of photodynamic therapy with topical 5-fluorouracil in Bowen's disease

BACKGROUND: Bowen's disease (BD; intraepithelial squamous cell carcinoma) is therapeutically challenging because lesions, which may be multiple, are frequently located at sites that heal poorly. There is a small risk of progression to invasive carcinoma. Photodynamic therapy (PDT) is an effective treatment for certain non melanoma skin cancers, but comparison studies with other, better-established therapies are limited. OBJECTIVES: To compare the efficacy and tolerability of PDT and topical 5-fluorouracil (5-FU) in BD. METHODS: Forty patients from two centres were randomized to either topical PDT or 5-FU. The PDT group was treated with 20% 5-aminolaevulinic acid (ALA) applied 4 h before illumination with 100 J cm-2 narrowband red light (630 +/- 15 nm). 5-FU was applied to lesions for 4 weeks. A repeat treatment cycle was performed after 6 weeks if required. Results Twenty-nine of 33 (88%) lesions treated with PDT initially responded completely, compared with 22 of 33 (67%) after 5-FU. After 12 months, two recurrences in the PDT group and six in the 5-FU group reduced complete clinical clearance rates to 82% and 48%, respectively. PDT was significantly more effective (P = 0.006, odds ratio 4.78, 95% confidence interval 1.56-14.62). In the 5-FU group, severe eczematous reactions developed around seven lesions, ulceration in three and erosions in two. No such reactions occurred following PDT. There was no difference in overall pain experienced during each therapy. CONCLUSIONS: Topical ALA-PDT is more effective than topical 5-FU in the treatment of BD, with fewer adverse events. ALA-PDT should be considered one of the first-line therapeutic options for BD.     

The effect of psoralen plus ultraviolet A in vitro in HUT-78 enhances by 5-aminolevulinic acid

BACKGROUND: Sezary syndrome and mycosis fungoides are forms of cutaneous T-cell lymphoma, and in the early stage of these diseases psoralen plus ultraviolet A (PUVA) is one of the treatments of choice. Photodynamic therapy using 5-aminolevulinic acid (ALA-PDT) is an effective, non-invasive, and safe treatment for most superficial skin cancers. In order to obtain greater efficacy of PUVA, we investigated the synergistic anti-tumor effects of ALA-PDT and PUVA using 8-methoxypsoralen (8-MOP) and a UVA lamp. METHODS: The in vitro effects of PUVA and ALA-PDT and their combination in HUT-78 cell line from human SS were determined by MTT assay. RESULTS: In our results, cell proliferation compared with controls was inhibited to 53.2% with UVA alone, 52.3% with 1 microM 8-MOP, 43.8% with 100 microM ALA, and 19.2% with combined 8-MOP and ALA. CONCLUSION: Combined use of ALA and PUVA using 8-MOP and UVA lamps, which are widespread in Japan, had a strong anti-tumor effect in vitro. Combined treatment with ALA-PDT and PUVA using a UVA lamp appears to have a strong treatment effect.     

In vitro and in vivo comparison of two different light sources for topical photodynamic therapy

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) is an effective and safe treatment option for the treatment of actinic keratosis (AK). Incoherent lamps are often used, matching the absorption maxima of ALA. OBJECTIVES: A comparative trial was performed to evaluate the efficacy of recently developed light-emitting diodes (LEDs). METHODS: Human epidermal keratinocytes were incubated for 24 h with ALA (100, 200, 300, 400 or 500 micromol L(-1)) and irradiated consecutively using either an incoherent halogen lamp (lambda(em) = 580-750 nm; 24 J cm(-2); 40 mW cm(-2)) or an LED system (lambda(em) = 633 +/- 3 nm; 3, 6, 12 or 24 J cm(-2); 40 mW cm(-2)). Topical ALA-PDT was performed on 40 patients with AK (n = 584) in a symmetrical distribution suitable for two-sided comparison. After incubation with ALA (20% in cream base) irradiation was performed with the incoherent lamp (100 J cm(-2); 160 mW cm(-2)) on one side and the LED system (40 J cm(-2); 80 mW cm(-2)) on the opposite side followed by re-evaluation up to 6 months. RESULTS: No significant differences between the LED system (3, 6, 12 or 24 J cm(-2)) and the incoherent light source (24 J cm(-2)) regarding cytotoxicity was found in vitro. The complete remission rate yielded in the in vivo investigation was also not significantly different at 6 weeks (P = 0.95), 3 months (P = 0.75) and 6 months (P = 0.61) following therapy. Six weeks following therapy complete remission rates of 84.3% (LED system) and 82.8% (incoherent lamp) were achieved. There was also no significant difference between both light sources regarding pain during light treatment (P = 0.67), patient satisfaction (P = 1.0) or cosmesis (P = 1.0) following therapy. CONCLUSIONS: These results show the efficacy of an LED system for ALA-PDT both in vitro and in vivo. ALA-PDT with the LED system showed a noninferiority regarding the clinical outcome in the treatment of AK compared with the incoherent lamp.     

Induction of protoporphyrin IX by aminolaevulinic acid in actinic keratosis, psoriasis and normal skin: preferential porphyrin enrichment in differentiated cells

Background In photodynamic therapy the endogenous photosensitizer protoporphyrin IX (PpIX) is synthesized following topical application of aminolaevulinic acid (ALA). However, different tissues have distinct PpIX‐accumulating properties, due to differences in penetration of ALA through the stratum corneum and/or alterations in the haem cycle. Preferential PpIX accumulation has been reported in terminally differentiated cell cultures but ex vivo data are lacking. Objectives To study the intrinsic PpIX‐accumulating capabilities of skin explants from lesional and nonlesional skin in psoriasis and actinic keratosis, with normal skin serving as a control, and to study PpIX accumulation in relation to differentiation status in normal skin. Methods Skin explants from patients with psoriasis, actinic keratosis and normal skin were incubated with ALA and PpIX was measured spectrofluorometrically. PpIX was measured in basal (β₁‐integrin‐positive) and suprabasal (β₁‐integrin‐negative) keratinocytes in normal skin. In addition, PpIX accumulation was studied in cell cultures at different levels of confluence and after induction of terminal differentiation. Results No significant differences in PpIX content were found between the different tissues. However, increased PpIX was found in β₁‐integrin‐negative compared with β₁‐integrin‐positive cells. In addition, in subconfluent cell cultures less PpIX was found compared with confluent cell cultures. Induction of terminal differentiation in vitro, however, resulted in less PpIX, which was likely to be related to cell volume. Conclusions As no significant differences in PpIX synthesis could be found between the different tissue types, these data emphasize the importance of the penetration route of ALA through the stratum corneum. Preferential PpIX accumulation observed in suprabasal epidermal keratinocytes and confluent cell cultures points towards a terminal differentiation‐specific effect.     

Phototoxicity of exogenous protoporphyrin IX and delta-aminolevulinic acid in the photo hen's egg test

BACKGROUND: Oxygen, appropriate light sources, and special photosensitizers are necessary to induce photochemical damage in tumor cells via photodynamic therapy (PDT) delta-aminolevulinic acid (ALA) is increasingly used in PDT, because topical or systemic administration of ALA induces accumulation of endogenous porphyrins preferentially in neoplastic tissues. Subsequent radiation with light of approximately 630 nm leads to selective damage of tumor cells. PDT should optimally leave peritumoral tissues unaffected, but only few data are reported on the effects and the time course of ALA-induced porphyrins in tumor-free tissues. METHODS: Therefore, we studied the phototoxic effects of protoporphyrin IX (PP) and ALA-induced porphyrins in a recently established phototoxic model based on tumor-free tissue, the photo hen's egg test (PHET). RESULTS: Employing this test procedure, PP provoked strong phototoxic reactions when irradiated with Ultraviolet A immediately and up to 30 h after substance application. In contrast, ALA induced a significant phototoxic effect only if irradiated 24 h after application. CONCLUSION: Thus, we observed a delayed phototoxic effect of ALA in tumor-free tissue of the yolk sac (YS) blood vessel system. This delayed phototoxic response 24 h after ALA application is probably caused by endogenously synthesized porphyrins. In contrast, epithelial tumors show a maximum porphyrin accumulation 4-8 h after ALA application whereas in healthy human skin porphyrin synthesis is less intensive but prolonged with maximum levels 24-48 h after ALA application. Thus, ALA induced virtually the same delayed phototoxic effect in the tumor-free YS blood vessel tissue as in healthy human skin. These results show that the PHET is a useful model for the predictive preclinical risk assessment of exogenous or endogenous photosensitizers.     

Expression pattern and intensity of protoporphyrin IX induced by liposomal 5-aminolevulinic acid in rat pilosebaceous unit throughout hair cycle

We have developed liposomal formulation of 5-aminolevulinic acid (ALA) to enhance topical delivery and examined ALA-induced protoporpyrin (PpIX) expression in rat pilosebaceous unit throughout hair cycle. Two types of liposomes—glycerol dilaulate (GDL) and phosphatidylcholine (PC)—were formulated and both liposomal ALA increased PpIX expression in rat dorsal skin and pilosebaceous units when compared with free ALA. However, iontophoresis combined with liposomal ALA reduced the expression intensity of PpIX in hair bulbs although it achieved deeper and wider expression of PpIX through transfollicular pathway. After topical application in intact or depilated rat skin, liposomal ALA produced excellent PpIX expression in pilosebaceous units. The expression pattern and intensity of PpIX changed in hair cycle-dependent manner: specific expression only in sebaceous glands was observed at telogen; strong expression in whole pilosebaceous units was shown at anagen with intense expressions in hair bulbs and sebaceous glands; and a pattern similar to anagen but reduced intensity in the hair bulbs was seen at catagen. Throughout hair cycle, the expression pattern and intensity were dramatically changed in hair follicular epithelial cells depending on the cell density and proliferation activity of those cells, whereas those were consistent in sebaceous glands regardless of hair cycle. Little expression was shown in dermis. Photoactivation effect of 20% liposomal ALA-PDT using a red filtered-halogen lamp damaged sebaceous glands, hair follicles and epidermal layers. Formation of a thicker epidermal layer was observed, and hair induction after depilation was inhibited along with damage in sebaceous glands.     

Thioredoxin upregulation by 5-aminolaevulinic acid-based photodynamic therapy in human skin squamous cell carcinoma cell line

Background/purpose: 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) is widely performed in the clinical setting for superficial skin cancers, giving favorable results, but residual tumor and recurrence occur occasionally. Thioredoxin is a common antioxidant that suppresses apoptosis and facilitates cell growth. We investigated the expression of thioredoxin following ALA-PDT in human skin squamous cell carcinoma cell line, HSC-5.
Methods: ALA-PDT was performed in HSC-5 cells using low-dose (5 J/cm², 100 mW/cm²) or high-dose (30 J/cm², 100 mW/cm²) irradiation, and the expression of thioredoxin was measured by Western blotting. An MTT assay was used to assess cell growth following a low dose of multiple irradiations. Cell death was examined by Western blotting for caspase-3 and PARP. Immunofluorescence double staining using annexin V and propidium iodine was also performed.
Results: Expression of thioredoxin was only observed following low-dose exposure ALA-PDT. Multiple low-dose exposure ALA-PDT significantly proliferated cell growth. With high-dose exposure ALA-PDT, caspase-3 and PARP expression were seen, and cell death due to apoptosis and/or necrosis was observed, but thioredoxin was barely detected.
Conclusion: Low-dose exposure ALA-PDT increased the expression of thioredoxin and facilitated the growth of HSC-5 cells.     

Topical 5-aminolaevulinic acid photodynamic therapy for cutaneous lesions: outcome and comparison of light sources

BACKGROUND: Topical 5-aminolaevulinic acid (ALA) photodynamic therapy (PDT) is increasingly used for superficial non-melanoma skin cancers and their precursors. METHODS: We report our 3-year experience of topical ALA-PDT, with a preliminary comparison of the effects of broadband and laser light sources. RESULTS: We performed 688 treatments on 483 lesions in 207 patients. Complete clearance was achieved in 222/239 lesions of Bowen's disease (BD), superficial basal cell carcinoma (sBCC) and actinic keratosis (AK) (93%) - 117/129 BD (91%), 84/87 sBCC (97%) and 21/23 AK (91%), with a median follow up of 48 weeks. Broadband and laser light sources were of similar efficacy. Recurrences have occurred in 10.3% BD, 4.8% sBCC and 4.8% AK. Adverse effects from PDT were uncommon but included pigmentary change (2%) and minor scarring (2%). How-ever, severe pain was experienced in 16-21% of treatments using the high-output broadband and laser sources, but in only 2% with the low-output xenon arc source. CONCLUSION: Topical ALA-PDT is effective for BD, sBCC and AK and has been an invaluable addition to our dermatology service. Efficacy is similar for broadband and laser light sources, although treatment at higher surface irradiance may be painful, and excellent cosmetic results can be achieved.     

5-aminolaevulinic acid and photodynamic therapy reduce HSV-1 replication in HaCat cells through an apoptosis-independent mechanism

Background: Photodynamic therapy (PDT) involves the use of a photosensitizing agent, which may require metabolic synthesis (i.e. a prodrug), followed by light activation. Numerous studies have advanced PDT as a means for treating bacteria, fungi and viruses. In this study, the photoinactivation of Herpes simplex virus type 1 (HSV‐1) in human keratinocytes using 5‐aminolaevulinic acid (5‐ALA) was investigated. Methods: HaCat cells were infected with HSV‐1 and treated with 5‐ALA to verify its antiviral effect during the stages of adsorption and penetration to host cells. Immunoblot analysis was used to estimate the effect of ALA–PDT on the production of viral proteins glycoprotein D (gD), infected cell proteins (ICP) 27 and virion protein (VP) 16. We also investigated whether the effect of ALA–PDT was associated with a cellular apoptotic mechanism through DNA fragmentation and the study of p53, PARP and caspase‐3 protein expression. Results: While the treatment of ALA–PDT after the viral adsorption period reduced HSV‐1 replication by about 70%, it did not act on the virus in the first phase of infection. The viral proteins' expressions were reduced by ALA–PDT treatments. There was no evidence of ALA–PDT‐induced apoptosis. Conclusion: Our data suggest that the target of photoinactivation appears to be viral replication and not a cellular response.     

Enhancement of topical 5-aminolaevulinic acid delivery by erbium:YAG laser and microdermabrasion: a comparison with iontophoresis and electroporation

BACKGROUND: 5-aminolaevulinic acid (ALA) is used as a protoporphyrin IX-precursor for the photodynamic therapy of superficial skin cancer and cutaneous metastases of internal malignancies. However, the permeability of hydrophilic ALA across the skin is very low. OBJECTIVES AND METHODS: The objective of this study was to optimize and enhance the in vitro skin permeation of ALA by two resurfacing techniques: erbium:yttrium-aluminium-garnet (Erb:YAG) laser and microdermabrasion. Light microscopic changes in pig skin caused by these techniques were also compared. The electrically assisted methods, iontophoresis and electroporation, were also used to facilitate ALA permeation across laser- or microdermabrasion-treated skin. RESULTS: Among the modalities tested in this study the Erb:YAG laser showed the greatest enhancement of ALA permeation. The laser fluence was found to play an important role in controlling the drug flux, producing enhancement ratios from 4-fold to 246-fold relative to the control. The skin permeation of ALA across microdermabrasion-treated skin was approximately 5-15-fold higher than that across intact skin. Both the ablated effect of the stratum corneum (SC) and ALA flux were proportional to the treatment duration of microdermabrasion. The application of iontophoresis or electroporation alone also increased the ALA permeation by approximately 15-fold and 2-fold, respectively. The incorporation of iontophoresis or electroporation with the resurfacing techniques caused a profound synergistic effect on ALA permeation. CONCLUSIONS: This basic study has encouraged the further investigation of ALA permeation by laser or microdermabrasion.