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Folate is essential for cell division and growth. Deficiency is linked to birth defects, magaloblastic anaemia, cardiovascular disease, etc. Folic acid is a synthetic form of folate and is used to fortify food and in supplements. In aqueous solutions, in blood and even in human skin, folic acid may be degraded by ultraviolet radiation. Consequently, photodegradation of folic acid in human blood may lead to folate deficiency. However, the degree and the health consequences of such photodegradation are unknown. It is not clear which spectral region plays the most important role in the photodegradation of folic acid. In this study the photodegradation of folic acid in aqueous solution under different wavelengths of ultraviolet radiation (260–400 nm) was investigated by fluorescence spectroscopy. The photodegradation rate of folic acid was dependent on wavelength. Action spectrum for 1 μM folic acid photodegradation was determined. Its action spectrum is not identical to its absorption or fluorescence excitation spectra. The action spectrum demonstrated that UVB and UVA degrade folic acid. Protecting skin against UVB and UVA radiation by sunscreens may help to protect folic acid in human blood under intense solar radiation.

Most studies have demonstrated that sunscreens with lower sun protection factor (SPF) do not prevent the production of vitamin D because much lower amount of sunscreen (SPF30) is applied than recommended (2 mg/cm(2)) indicating that a significant amount of UV radiation can penetrate the skin. Since less sunscreen is applied, higher SPF sunscreens may be used to achieve the desired protection. However, there is little information regarding the application of high-SPF sunscreen and vitamin D formation. The aim of this study was to measure the influence of the amount of two SPF 30 sunscreens on pre-vitamin D formation in a cuvette with 7-dehydrocholesterol.Sunscreen with physical (reflecting) or chemical (absorbing) UV filters exhibits different levels of protection in vitro even if the SPF is the same. The level of photoprotection is differentially reduced when less sunscreen than the recommended application thickness is applied.The usual application of 0.8-1 mg/cm(2) is below the recommended value of 2 mg/cm(2), and pre-vitamin D may be formed when lower amounts of SPF ≤30 sunscreen are applied, showing that a significant amount of UV radiation may enter the skin.

This study investigated photobleaching of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) and ALA-heptyl ester during superficial photodynamic therapy (PDT) in normal skin of the female BALB/c-nu/nu athymic mouse. We examined the effects of two light sources (laser and broadband lamp) and two different illumination schemes (fractionated light and continuous irradiation) on the kinetics of photobleaching. Our results show that light exposure (0-30 minutes, 10 mW/cm2) of wavelengths of approximately 420 nm (blue light) and 635 nm (red light) induced time-dependent PpIX photobleaching for mouse skin of 2% ALA and ALA-heptyl ester. Blue light (10 mW/cm2) caused more rapid PpIX photobleaching than did red light (100 mW/cm2), which is attributed to stronger absorption at 407 nm than at 632 nm for PpIX. In the case of light fractionation, fractionated light induced faster photobleaching compared with continuous light exposure after topical application of 2% ALA and ALA-heptyl ester in vivo. These have been suggested to allow reoxygenation of the irradiated tissue, with a consequent enhancement of singlet oxygen production in the second and subsequent fractions.

Summary Background Measurements of tissue oxygenation and blood supply are of vital importance during photodynamic therapy. The aim of this study was to measure photosensitization responses in healthy mouse skin using non-invasive reflectance spectroscopy and fluorescein angiography. Methods Healthy mouse skin was treated topically with hexylaminolevulinate (HAL) for 3 h and then exposed to red light (632 nm). Results Non-invasive reflectance spectroscopy showed that the fraction of oxygenized haemoglobin decreased considerably after treatment with HAL and red light. This indicates reduced tissue oxygenation. Fluorescein angiography showed no vasoconstriction in the mouse skin after short (3 h) treatment with HAL and light, indicating that the oxygen concentration was high enough to give a photodynamic effect consistent with the skin damage (scar formation) observed for a few days after the treatment. However, longer treatment with HAL (17 h) resulted in complete vasoconstriction with oxygen depletion and no skin damage in the centre, while a circular (rim) wound was seen around the light exposed area. Skin damage was larger for the lowest fluence rate (20 mW/cm2) than for the highest one (100 mW/cm2) applied. Conclusion Skin treatment with topical HAL and red light in fluencies and fluence rates as applied here does not result in complete vasoconstriction. Haemoglobin oxygenation monitored by reflectance spectroscopy can be used as a rough measure of the resulting photodynamic response. However, haemoglobin oxygenation alone is not a complete predictive factor since reflectance spectroscopy did not reveal any differences in the status of haemoglobin oxygenation for different fluence rates.

To determine the impact of skin pretreatment with microneedles (MNs) on ALA- and MAL-induced protoporphyrin IX (PpIX) production, as well as MN impact on pain sensations during light exposure and erythema after PDT.The skin of 14 healthy volunteers was preteated with MNs. Equal amounts of creams containing 2%, 8% and 16% (w/w) ALA and MAL were applied on 1 cm(2) areas for 4 h. Additionally, 16% ALA and MAL creams were applied for 24 h. Afterwards, PpIX fluorescence spectra were measured. Sixteen percent ALA and MAL spots were exposed to red light (632 nm, 77 mW/cm(2)). Time for pain to occur was measured in seconds, and erythemal response was monitored up to 6 h after the end of the light exposure.Use of MNs increased the PpIX fluorescence after 4 h incubation time with 2% and 8% ALA or MAL, but not with 16% ALA or MAL. Pretreatment with MNs did not increase the pain sensations during light exposure, nor did it influence erythema occurrence.MNs are a promising tool for improving the efficiency of topical PDT by improving the cutaneous delivery of ALA and MAL, without increase in side effects.

A novel 5-aminolevulinic acid (ALA)-containing microparticulate system was produced recently, based on incorporation of ALA into particles prepared from a suppository base that maintains drug stability during storage and melts at skin temperature to release its drug payload. The novel particulate system was applied to the skin of living animals, followed by study of protoporphyrin IX (PpIX) production. The effect of formulating the microparticles in different vehicles was investigated and also the phototoxicity of the PpIX produced using a model tumour. Particles formulated in propylene glycol gels (10% w/w ALA loading) generated the highest peak PpIX fluorescence levels in normal mouse skin. Peak PpIX levels induced in skin overlying subcutaneously implanted WiDr tumours were significantly lower than in normal skin for both the 10% w/w ALA microparticles alone and the 10% w/w ALA microparticles in propylene glycol gels during continuous 12h applications. Tumours not treated with photodynamic therapy continued to grow over the 17 days of the anti-tumour study. However, those treated with 12h applications of either the 10% w/w ALA microparticles alone or the 10% w/w ALA microparticles in propylene glycol gel followed by a single laser irradiation showed no growth. The gel formulation performed slightly better once again, reducing the tumour growth rate by approximately 105%, compared with the 89% reduction achieved using particles alone. Following the promising results obtained in this study, work is now going on to prepare particle-loaded gels under GMP conditions with the aim of initiating an exploratory clinical trial. Copyright r 2009 John Wiley & Sons, Ltd.

Hypericin is a promising photosensitizer for photodynamic therapy (PDT) characterized by a high yield of singlet oxygen. Photobleaching of hypericin has been studied by means of absorption and fluorescence spectroscopy in different biological systems: in human serum albumin solution, in cultured human adenocarcinoma WiDr cells and in the skin of nude mice. Prolonged exposure to light (up to 95 min, 100 mW/cm2) of wavelength around 596 nm induced fluence-dependent photobleaching of hypericin in all studied systems. The photobleaching was not oxygen dependent, and singlet oxygen probably played no significant role. Emission bands in the spectral regions 420–560 nm and above 600 nm characterize the photoproducts formed. An emission band at 615–635 nm was observed after irradiation of cells incubated with hypericin or of mouse skin in vivo but not in albumin solution. The excitation spectrum of these products resembled that of hypericin. Hypericin appears to be more photostable than most sensitizers ...

5-Aminolevulinic acid (ALA) is a natural precursor of protoporphyrin IX (PpIX) and heme in cells. Photodynamic therapy (PDT) utilizes a metabolic imbalance in cancer cells, leading to increased PpIX generation from exogenous ALA. Due to chemical instability of ALA in therapeutic concentrations at pH values larger than 5.0 and at high temperatures, it looses its activity by spontaneous dimerization to 2,5-dicarboxyethyl-3,6-dihydropyrazine (DHPY). ALA esters are now supplementing ALA in PDT, but little is known about their stability. We have studied the stability of ALA and its methyl ester (MAL) stored under different conditions (temperatures, pH values) by measuring their ability to generate PpIX. 100mM solutions of both compounds were found to be stable at pH 4 and at 4 degrees C. However, at pH 5.5 they lost almost 10% of the initial activity during 5days of storage at 4 degrees C. The fastest decay of ALA and MAL was seen at pH 7.4 and at 37 degrees C, and followed first order kinetics. At pH 7.4 and at 4 degrees C MAL lost its PpIX producing ability more slowly than at 37 degrees C. Our work shows that solutions should be prepared immediately before use and stored at low temperatures. The pH of stock solutions should not exceed 5.

Significant amounts of protoporphyrin IX (PpIX) are formed after 6 min of topical application of 5-aminolevulinic acid (ALA) and its hexylester derivative, whereas PpIX is formed after 10 min of topical application of ALA-methylester derivative in normal mouse skin at 37 degrees C. Lowering the skin temperature to 28-32 degrees C by the administration of the anesthetic Hypnorm-Dormicum reduces the PpIX fluorescence by a factor of 2-3. Practically no PpIX was formed as long as the skin temperature was kept at 12-18 degrees C. At around 30 degrees C PpIX fluorescence appears later after application of ALA-ester derivatives (14-20 min) than after application of ALA (8 min), indicating differences in their bioavailability (delayed penetration through the stratum corneum, cellular uptake, conversion to ALA, PpIX production) in mouse skin in vivo. The difference in lag time in the PpIX formation after application of ALA and ALA-esters may be partly related to deesterification of the ALA-ester molecules. The temperature dependence of PpIX production may be used for improvement of photodynamic therapy with ALA and ALA-ester derivatives, where accumulation of PpIX can be selectively enhanced by increasing the temperature of the target tissue.

Melanotic melanomas have a poor response to photodynamic therapy (PDT). The reason for this is that melanin absorbs light over the entire wavelength region used for PDT (400-750 nm). Photobleaching of melanin is an approach to overcome this obstacle. In the present work, reflectance spectroscopy was applied to study depigmentation of human and murine skin with different melanin contents, and effects induced by PDT with topical application of methyl 5-aminolevulinate (MAL) on B16F10 melanotic melanomas transplanted to nude mice. Depigmentation and inhibition of tumor growth after violet light (420 nm) exposure, red light (634 nm) exposure, and combinations of both were studied. Reflectance spectroscopy was suitable for evaluation of the pigmentation of both human and murine skin. Skin depigmentation leads to increase in reflectance. PDT with violet light bleached some of the melanin in the skin above the B16F10 melanomas, and possibly also in the upper part of the melanomas. This resulted in a larger growth inhibition of tumors first given PDT with violet light and then with red light compared to treatments using the reverse order of illumination, namely, red light before violet light. It is concluded that violet light PDT can bleach melanin in melanotic tumors and therefore increase their sensitivity to red light PDT. This finding indicates a new PDT modality that can be further developed for treatment of superficial melanotic melanomas and possibly other diseases where pigmentation is a problem.

Hypericin is a potential photosensitizer for photodynamic diagnosis and therapy of cancer. Using a non-invasive fluorescence method we have shown that it is selectively accumulated in WiDr colon carcinoma tumours grown in nude mice after intraperitoneal (i.p.) administration (2–5 mg/kg). The maximal concentration of hypericin in the tumour was recorded 24 h after injection. The fluorescence of the drug recorded from the surface of the tumour was 6.5, 17, 34 or 6 times higher than that measured in skin, brain, liver or muscles, respectively. Photodynamic therapy with high drug doses and light exposures (5 mg/kg hypericin, i.p., 120 J/cm 2 ) at 6 or 24 h after drug administration delayed tumour growth. Hypericin appears to be a suitable drug, mainly for fluorescence diagnosis.

Summary 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.

5-Aminolevulinic acid (ALA) or its derivative methyl 5-aminolevulinate (MAL) combined with folic acid was applied in nude mice bearing human colon adenocarcinoma. The aim of the study is to see whether folic acid may increase biosynthesis of porphyrins in tumor tissue after systemic or topical administration of ALA or MAL. The production of porphyrins was determined by spectrofluorometric measurements with an optical fibre probe. It was found that the porphyrin production after i.p injection of 200 mg kg(-1) ALA or MAL was significantly increased by i.p injection of 100 mg kg(-1) folic acid. However, in the case of topically applied 20% ALA, folic acid had no effect. In the case of topically applied 20% MAL, folic acid (i.p or topically applied) reduced the porphyrin synthesis. This might be used for the protection of normal skin against photosensitization. The effects of folic acid were similar in tumors and normal skin. Two mechanisms may explain the results: enhancement of the efficiency of the rate-limiting enzyme porphobilinogen deaminase by folic acid or interference of folic acid with the transport of ALA and MAL to and into the cells synthesizing porphyrins in the tissues. The present data seem to favour the latter mechanism. Folic acid may have a role as an adjuvant in photodynamic therapy with systemically administered ALA and its derivatives.

This paper illustrates that fluorescence spectroscopy and imaging can be used to measure the extent and distribution of lipid oxidation in meat. Minced turkey thighs and pork semimembranosus muscles were stored for 7 and 12 days at 4°C in high oxygen (O(2)) modified atmosphere packages and vacuum. Turkey meat packed in high O(2) atmosphere was oxidised already after 7 days of storage. The sensory rancid odour score was 4.7 (on a scale from 1 to 9) and the TBARS value was 1.86mg MDA/kg. There was also an increase in fluorescence emission intensity in the 410-550nm region, which arises from lipid oxidation products. The combination of unsaturated fatty acids and access to O(2) resulted in lipid oxidation gradients in the turkey meat samples, and these gradients were clearly visualised by fluorescence images. In comparison, pork meat was more stable against lipid oxidation, with TBARS values0.2mg MDA/kg and no development of fluorescent lipid oxidation products was detected. The fluorescence spectra measured in the present experiment suggest that turkey thighs and pork semimembranosus muscle in addition to protoporphyrin also have a natural content of Zn protoporphyrin. The porphyrin content was higher in pork meat than in turkey meat. It increased during storage time when the meat was packed in vacuum, and it decreased with O(2) availability. The distribution of porphyrins in the meat was visualised by fluorescence imaging.

Formation of protoporphyrin IX (PpIX) after topical application of 5-aminolevulinic acid (ALA) and its hexylester derivative (ALA-Hex) was studied on healthy human skin. Temperature, density of hai ...

The influence of skin permeation enhancers, such as dimethyl sulphoxide (DMSO) and 1-[2-(decylthio)ethyl]azacyclopentan-2-one (HPE-101), LabrafacCC, Labrafil, Labrasol and Transcutol in a concentration of 10% (wt./wt.) on the formation of porphyrins in normal mouse skin from topical application of creams with methyl 5-aminolevulinate (MAL) was studied. The concentration of porphyrins in the mouse skin was determined by direct fluorescence measurements. The results show that studied permeation enhancers increase the formation of porphyrins, and therefore also the skin penetration 2% MAL whereas for 10% and 20% (wt./wt.) MAL concentrations only DMSO, HPE-101 and LabrafacCC increased the porphyrin formation. At all studied MAL concentrations DMSO gave the largest enhancing effect, similarly to that of HPE-101. This suggests that in 2–20% MAL creams HPE-101 may be substituted by LabrafacCC to reduce skin irritation induced by HPE-101 without impairing the porphyrin formation. 2005 Elsevier B.V. All rights reserved.

In this work, the effect of 10% of dimethylsulfoxide (DMSO), 1-[2-(decylthio)ethyl]azacyclopentan-2-one (HPE-101) and Labrafac(®)CC (a mixture of caprylic and capric acid triglycerides) on porphyrin formation in mouse skin during topical application of methyl 5-aminolevulinate (MAL) was studied. The porphyrin level in mouse skin was determined by measuring directly fluorescence and by extraction method. The porphyrin fluorescence kinetics during continuous application of MAL in creams in concentrations 2, 10 and 20% (wt./wt.) for up to 7h showed that in this concentration range the kinetics of porphyrin production in the site of application does not depend significantly on the MAL concentration. After 24h of application of all studied creams the porphyrin fluorescence in the area of treatment was dramatically reduced to be about two-fold higher than the skin autofluorescence, suggesting a significant decrease of the porphyrin concentration in these sites, although in all cases traces of porphyrins were found. It was found by extraction method that a 10% MAL cream with 10% DMSO for 4h increased the concentration of porphyrin about four-fold compared with 10% MAL cream alone. The presence of 10% HPE-101 or 10% Labrafac(®)CC in the 10% MAL cream increased the porphyrin concentration in the area of application about 2.5- and 2-fold, respectively, as compared with MAL cream without enhancers. No statistically significant difference was found between the effects of the creams containing 10% MAL with 10% HPE-101 or 10% Labrafac(®)CC. The results obtained after 24h of mouse skin treatment with the same creams showed a large decrease of porphyrin formation in comparison with results found after 4h. All porphyrin concentrations measured after this time of MAL creams application were similar. Skin erythema was observed using MAL cream with DMSO and HPE-101, but not with Labrafac(®)CC. Our work suggests that the new penetration enhancer Labrafac(®)CC in creams with MAL may be used to increase the penetration of MAL through the mouse skin and at the same time increase production of porphyrin in the skin. This may reduce skin erythema, which is induced by DMSO and HPE-101.

We have evaluated the efficacy of lipid cubic phases, highly ordered self-assembly systems on the nanometer level, as drug delivery vehicles for in vivo topical administration of delta-aminolevulinic acid (ALA) and its methyl ester (m-ALA) on nude mice skin. ALA, a precursor of heme, induces the production of the photosensitizer protoporphyrin IX (PpIX) in living tissue. Measuring the PpIX fluorescence at the skin surface, after topical administration, makes indirect quantification of the penetration of ALA into the tissue possible. Cubic phases were formed of lipid (monoolein or phytantriol), water and drug. In some cases, propylene glycol was included in the cubic phase as well. The drug concentration was 3% (w/w, based on the total sample weight) in all investigated vehicles. When the formulations were applied for 1 h, the monoolein cubic systems and the three-component phytantriol sample showed higher fluorescence compared to the standard ointment during the 10 h of measurement. Both ALA and m-ALA yielded similar results, although the differences between the investigated vehicles were more pronounced when using m-ALA. For the 24-h applications, the monoolein cubic systems with m-ALA showed faster PpIX formation than the standard ointment, implying higher PpIX levels at short application times (less than 4 h). The systemic PpIX fluorescence of ALA was elevated by using the lipid cubic formulations. Notably, a small systemic effect was also observed for the monoolein cubic sample with m-ALA. These results imply improved PpIX formation when using the lipid cubic systems, most probably due to enhanced drug penetration.

This paper suggests that dairy products have natural contents of porphyrins and chlorins, and that these light sensitive compounds play an important role in photo-oxidation of cheese. Samples of the Gouda-like Norvegia cheese were exposed to light through plastic films of different colors. In addition, some samples were stored under transparent films and some in the dark. Sensory evaluation showed that cheese stored under transparent, violet and blue films were severely oxidized, while those stored under green, yellow, orange and red were less, but still significantly oxidized. Fluorescence analysis indicated that the photo degradation of porphyrins and chlorins correlated closely (R>0.90) with the sensory attributes oxidized odor, sun flavor and acidic flavor. The corresponding degradation of riboflavin correlated well with oxidized odor, but significantly less with the two other attributes ( R = 0.65 – 0.69 ). Fluorescence analysis indicated that the naturally present compounds in the cheese are protoporphyrin, hematoporphyrin, chlorophyll a and chlorophyll b. It is suggested that photo-induced breakdown of these compounds provides a significant contribution to photo-oxidation in cheese, and other dairy products.

The kinetics of photobleaching and formation of photoproducts upon irradiation (735 nm) of 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin (m-THPBC) in phosphate buffer saline (PBS) supplemented with human serum albumin (HSA) were studied by means of absorption and steady-state fluorescence spectroscopy. Measurements were performed either immediately after the dye was dissolved in the HSA solution (0 h) or after six hours incubation in the HSA solution (6 h). Spectroscopic studies indicated that the dye was mainly present as aggregates in freshly prepared solutions, whereas incubation favored monomerisation. Irrespective to incubation time, the rates of photobleaching obtained by fluorescence measurements were higher than those obtained from absorbance measurements. Photobleaching of freshly prepared m-THPBC can be described by a single exponential decay, while the absorbance and fluorescence decays of the incubated dye solutions better fit a bi-exponential decay. Two photobleaching rates probably reflect differences in the photosensitivity of monomer (bound to proteins) and aggregated (non-bound) forms. Irradiation of the freshly prepared m-THPBC solution led to phototransformation of 50% of the bleached m-THPBC into 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin (m-THPC), a clinically used second generation photosensitizer. For irradiation 6 h after dissolving m-THPBC, different kinetics of m-THPC formation were found. A rapid decrease in concentration of m-THPBC was accompanied by a slow formation of m-THPC. The quantum yield of this process was small since only 5% of m-THPBC was transformed to m-THPC. The kinetics characteristics of m-THPBC photobleaching reported in the present study, together with the different kinetics of photoproduct formation during m-THPBC photobleaching, may provide important indications in the m-THPBC-based PDT dosimetry.

Aminolevulinic acid (ALA), ALA methylester (ALA-Me) and ALA hexylester (ALA-Hex) were topically applied for 5 and 20 hr, respectively, on normal skin of mice. The distribution of protoporphyrin IX (PpIX) induced in 7 different tissues by these drugs was determined either by spectrofluorometric measurements with an optical fibre probe or by chemical extraction of PpIX from the tissues. The results from these 2 types of measurements were compared. Both methods showed that ALA and the esters induced similar amounts of PpIX at the skin spot where they were applied and that the esters produced much less PpIX at remote skin spots (i.e., spots outside the location where the drugs were applied) than ALA did, notably after 20 hr application. After 20 hr of drug application ALA produced much more PpIX in liver, intestine and lungs than the esters did. In contrast with the direct fluorescence measurements, the extraction method showed detectable amounts of PpIX in liver, intestine and lung after application of the esters, notably of ALA-Me. The discrepancy is probably related to the fact that the pigmented tissues absorb light and, therefore, the direct fluorescence readings are misleading. Notably in the liver, which contains high concentration of light-absorbing pigments, very weak direct fluorescence was seen. In no case there was any accumulation of PpIX in muscle tissue nor in brain. The esters seem to penetrate less into the circulation than ALA, and PpIX formed by them in the skin is faster cleared than PpIX formed from ALA. This is also true after oral and i.p. administration of the drugs. © 2002 Wiley-Liss, Inc.

The fluorescence of PpIX induced by topical application of 5-aminolevulinic acid (ALA) in normal mouse skin was studied noninvasively by means of a fibre optic probe. The fluorescence excitation spectrum of PpIX exhibits five distinct peaks at around 408, 510, 543, 583 and 633 nm under fluorescence monitoring at the second emission peak of PpIX (705 nm). The transmission of the excitation light is wavelength dependent: the long wavelength light (>600 nm) penetrates deeper into the tissues by a factor of 6 compared with the short wavelength light (

No fluorescence of protoporphyrin IX (PpIX) was measured using a fiber optic probe in pigmented B16F10 melanoma in mice after topical application of 5-aminolevulinic acid methylester (ALA-Me). However, chemical extraction of tissues excised from mice after intratumoral administration of ALA-Me or its parent compound ALA revealed that this tumor had the capability to produce PpIX. Small amounts of endogenous porphyrins, mainly PpIX, were found in the melanoma not treated with these drugs. Topical application of ALA-Me followed by exposure with laser light (633nm) delayed the growth of the tumors slightly. Light alone also had a significant effect on the tumor growth.

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.

Hypericin, a natural polycyclic quinone extracted from Hypericum perforatum, has been recently shown to be a powerful sensitiser for photodynamic therapy (PDT). However, its intracellular localisation remains unclear and contradictory. In the present work we compared the intracellular localisation of hypericin in three cultured cell lines (adenocarcinoma cells WiDr, carcinoma cells NHIK 3025 and glioblastoma cells D54Mg) with the distribution of fluorescent probes specific to lysosomes (LysoTracker Blue DND-22), mitochondria (MitoTracker Green FM) and endoplasmic reticulum (ERTracker Blue-White DPX). It was shown that the hypericin staining pattern was different compared to the intracellular distribution of mitochondria or lysosomes. Hypericin was concentrated in the perinucleolar cytoplasmic area mainly on one side of the nucleus--the region rich in endoplasmic reticulum and Golgi. Sometimes nuclear envelope was also stained. Plasma membrane was not stained but the dye was often accumulated in the intercellular space between the tightly contacting WiDr cells in colonies. Hypericin concentrations of 10 microM or less were not toxic for WiDr cells in the dark. Orange light (lambda max approximately 600 nm; 6 mW/cm2) killed the cells stained with 1 microM hypericin with LD50 approximately 1 J/cm2.

When 5-aminolevulinic acid ( ALA ) is administered topically, intravenously or intraperitoneally to tumor-bearing animals or humans, tumor selective production of protoporphyrin IX ( PpIX ) is frequently observed. Several biophysical, biochemical and physiological factors explain this tumor selectivity, among which the most important ones seem to be: (1) a relatively high concentration of the rate-limiting enzyme porphobilinogen deaminase (PBGD) in tumors; (2) relatively low concentrations of ferrochelatase (FC) and iron in many tumors; (3) a slightly elevated tumor temperature, related to inflammation; and (4) a compromised barrier of the skin overlying tumors, leading to an increased penetration of ALA into tumors. All these factors are discussed in the present work and new data concerning the significance of the skin barrier and the tumor temperature are presented. It is shown that compromising the skin of mice by tape-stripping the stratum corneum leads to a strongly enhanced ALA penetration into the skin and even into the circulation. The skin overlying tumors (WiDr colon carcinoma tumors transplanted subcutaneously to mice) is also much more permeable to ALA than normal skin, even through no physical damage of the tumor skin can be seen. PpIX formation from ALA can be increased by increasing the temperature.

5-aminolevulinic acid heptyl ester was investigated in human adenocarcinoma WiDr cells and in healthy skin of athymic nude mice in comparison with 5-aminolevulinic acid (ALA). Incubation of WiDr cells with ALA and ALA heptyl ester resulted in production of protoporphyrin IX (PpIX). Concentrations higher than 0.01 mm of ALA heptyl ester and higher than 1 mm of ALA were cytotoxic. The dark cytotoxicity was not related to PpIX. Intracellular localization, photocytoxicity and photobleaching rate of PpIX were the same for both drugs, although a 100 times lower concentration of ALA heptyl ester (0.01 mm) was needed in comparison with ALA (1 mm) to induce the same level of PpIX. ALA heptyl ester, topically (but not systemically) applied, is a promising candidate for fluorescence diagnosis and photodynamic therapy. Special attention must be focused on the concentrations of ALA heptyl ester; as excess may lead to cytotoxicity and inefficient PpIX generation.

— 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°C).

— 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.

Summary Background 5-Aminolevulinic acid (ALA) or its esters mediated photodynamic therapy (PDT) is the most widely practiced form of PDT in dermatology. One of its advantages is that undesirable photosensitization lasts only for 24–48 h. In order to optimize ALA–PDT it is necessary to understand the mechanisms of intracellular production and clearance of PpIX (efflux from cells into blood stream and/or its conversion into haem). The aim of this study is to investigate the factors controlling the clearance of intracellular PpIX from healthy skin of mice. Methods PpIX was induced in mouse skin by topical or systemic application of ALA or by topical application of the iron chelator ethylenediaminetetraacetic acid (EDTA). Fluorescence spectroscopy was used to study PpIX kinetics in alive and dead skin. Results Topical application of ALA or EDTA leads to porphyrin production in living skin, but not in excised skin. The clearance rates of PpIX from alive and dead skin were the same in the absence of an intracellular ALA pool. The clearance half-life of EDTA-induced PpIX was 4–7 times longer than that of PpIX after application of ALA. Conclusions Skin temperature and intracellular iron availability strongly affect PpIX clearance, while ALA application mode (topical versus systemic) and skin viability (dead versus alive) have no influence on PpIX decay. These results demonstrate that the clearance kinetics of PpIX from skin are determined mostly by the conversion of PpIX into haem, while the cellular efflux of PpIX into blood plays a minor role.

Summary Background Pain is a well-known problem associated with light exposure during topical photodynamic therapy (PDT). Different methods for dealing with the pain have been developed over the past years, ranging from cooling with air or water to nerve blocking. However, the mechanisms responsible for the pain induction have not yet been fully understood. Aim This study aims to evaluate bioimpedance in situ measurements of human skin as a method to shed light on pain-inducing real-time changes during light exposure during topical PDT. Methods Cream containing 20% aminolevulinic acid (ALA) was applied on forearms of ten healthy human volunteers. After 24 h incubation, the cream was removed and the spots were exposed to red laser light (636 nm, 300 mW/cm 2 ). During light exposure bioimpedance measurements with a 4-electrode set-up were taken at two frequencies (10 Hz and 100 kHz). Results A significant drop in skin impedance at high and low frequencies coincided with onset of pain during light exposure of spots treated with ALA. A similar drop was not observed for controls. Conclusions Bioimpedance spectroscopy can provide valuable data for real-time observation of changes in skin, and may contribute to an increased understanding of the mechanisms responsible for induction of pain during topical PDT. Future studies are needed.

The interactions of pairs of the modalities Photofrin II-sensitized photochemotherapy (PCT), ionizing radiation and an electric current were investigated by the colony formation assay in WiDr cells, a human colon adenocarcinoma cell line. When the cells were treated simultaneously with PCT and an electric current, a slightly synergistic effect was observed at low exposures (surviving fraction approximately 0.1) while a seemingly antagonist effect was found at higher exposures. The same was found to be true for the combinations of PCT plus ionizing radiation and ionizing radiation plus electric current.

Objectives The inclusion 01 chemical penetration enhancers in a novel patch-based system for the delivery of 5-aminolevulinic acid (ALA) was examined in vitro and in vivo. Poor penetration of ALA has been implicated as the primary factor for low response rates achieved with topical ALA-based photodynamic therapy of thicker neoplastic lesions. such as nodular basal cell carcinomas. Methods Several chemical permeation enhancers (dimethylsulfoxide, Labrafac CC. Labrafac PG and Labrafil M1944CS) were incorporated into bioadhesive patches tailored to deliver 19 mg ALA/cm(2). Key findings In-vitro depth penetration studies into excised porcine skin showed that high concentrations of ALA (>9 mu mol/cm(3)) could be delivered to a depth of 1.875 mm. However, inclusion of permeation enhancers did not significantly increase ALA delivery, relative to the control (P > 0.05). In-vivo studies were in strong agreement with in-vitro results, with formulations containing chemical enhancers showing no improvement in delivery compared with the control. Conclusions The patches designed in this work are suited to defineable ALA delivery without the need to immobilise patients for up to 6 h, as IS common with the cream-under-occlusion approach. Overall, permeation enhancers were not found to markedly enhance the topical delivery of ALA. 11 chemical penetration enhancers may have a greater effect on the delivery of more lipophilic ALA prodrugs, which are thought to primarily permeate the stratum corneum via the intercellular pathway.

Aminolaevulinic acid (ALA) is known to poorly penetrate into thick lesions, such as nodular basal cell carcinomas Short chain ALA esters, possessing increased lipophilicity relative to their hydrophilic parent, have previously been shown to be highly efficient at inducing protoporphyrin IX (PpIX) production in cell culture, at equimolar concentrations. In contrast, in vitro skin permeation and in vivo animal studies, which up to now have compared prodrugs on a % w/vv basis, have failed to demonstrate such benefits For the first time, equimolar concentrations of ALA, methyl-ALA (m-ALA) and hexyl-ALA (h-ALA) have been incorporated into an o/w cream preparation. In vitro penetration studies into excised porcine skin revealed that increased levels of h-ALA, compared to ALA and m-ALA were found in the upper skin layers, at all drug loadings studied. Topical application of the formulations to nude murine skin in vivo, revealed that creams containing h-ALA induced significantly higher levels of peak PpIX fluorescence (F-max = 289.0) at low concentrations compared to m-ALA (F-max = 159.2) and ALA (F-max = 191 9). Importantly, this study indicates that when compared on an equimolar basis, h-ALA has improved skin penetration, leading to enhanced PpIX production compared to the parent drug and m-ALA (C) 2010 Wiley-Liss, Inc and the American Pharmacists Association J Pharm Sci 99 3486-3498, 2010

In photodynamic therapy (PDT) a combination of visible light and a sensitising drug causes the destruction of selected cells. Aminolaevulinic acid (ALA) has been widely used in topical PDT for over 15 years. However, ALA does not possess favourable physicochemical properties for skin penetration. Consequently, the clearance rates for difficult to treat lesions, such as nodular basal cell carcinomas are relatively low. For the first time, equimolar concentrations of ALA, methyl-ALA (m-ALA) and hexyl-ALA (h-ALA) have been incorporated into a bioadhesive patch-based system. In vitro penetration studies into excised porcine skin revealed that ALA patches containing relatively high loadings (226.7 μmol cm −2 ) were associated with significantly greater tissue concentrations (70.7 μmol cm −3 ) than patches containing m-ALA (16.3 μmol cm −3 ) or h-ALA (17.4 μmol cm −3 ). ALA was also found to be the most efficient inducer of protoporphyrin (PpIX) fluorescence in mice, in vivo (maximum mean fluorescence: ALA = 236.2 a.u., m-ALA = 175.1 a.u., h-ALA = 193.5 a.u.). However, when the lipophilic hexylester was formulated in a pressure sensitive adhesive (PSA) patch, significantly higher PpIX levels were achieved compared to all bioadhesive systems tested. Of major importance, PSA patches containing relatively low h-ALA loadings induced high PpIX levels, which were localised to the application area. This study has highlighted the importance of rational selection of both the active agent and the delivery system. Bioadhesive preparations containing ALA are ideal for delivery to moist environments; whereas h-ALA-loaded PSA systems may facilitate enhanced delivery to dry areas of skin. In addition, owing to the relatively low loadings of h-ALA required in PSA patches, the costs of clinical PDT may potentially be reduced.

Summary Background Photodynamic therapy (PDT) is based on the combination of an exogenously administered precursor of photosensitizer [protoporphyrin IX (PpIX)] synthesis and exposure to light. Choosing the optimal wavelength is important. Red light penetrates deeper into tissue, while violet light is more efficient in activating PpIX but does not penetrate so deeply. Objectives We studied PpIX formation and the PDT effect after application to human skin of creams containing aminolaevulinic acid (ALA) and aminolaevulinic acid methyl ester (MAL). The aim of the study was to investigate whether the wavelength of the light used has an influence on pain sensations during topical PDT with the different prodrugs. Methods ALA cream (10%) and MAL cream (10%) were topically applied on the skin of 10 healthy volunteers. After 24 h the application site was exposed to 8 mW cm−2 violet laser or to 100 mW cm−2 red laser light. The erythema index was monitored up to 24 h after light exposure. For the first time the pain during topical ALA- and MAL-PDT was assessed by measuring the time taken for pain to occur. Also, for the first time, the intensities of the light sources were calibrated so as to have the same relative quantum efficiency. Results The pain sensation during ALA-PDT with red light came 22 s sooner than during ALA-PDT with violet light, which is statistically significant (P

It has been speculated that topical application of 5-aminolevulinic acid (ALA) or methyl 5-aminolevulinate (MAL) may be more painful during light exposure after longer application times of the compounds than after shorter times, even though the same levels of protoporphyrin IX (PpIX) is produced in both cases. The aim of our study was to investigate pain induction in the build-up and clearance phases of PpIX in the skin of healthy volunteers. 0.6 mmol/g of ALA (10% wt/wt) and MAL (11% wt/wt) creams were applied on the volunteers. The creams were maintained on the spots for 20- 24 hours and then wiped off. Subsequently, fresh creams were applied on the other arm of the volunteers for 4- 6 hours. Fluorescence emission spectra for all spots were measured every hour until the fluorescence levels were similar in both arms for ALA and MAL. Then the test areas were exposed to light until pain occurred. Time for pain to occur was recorded. The fluorescence of PpIX was measured before and after light exposure. PDT in the clearance phase seems to induce pain faster than in the build-up phase for ALA and MAL. Due to large interpersonal variations between volunteers further investigation is needed.

Folate deficiency is linked to serious birth defects, pregnancy complications, male infertility, cardiovascular diseases, and even the evolution of human skin color. Conflicting data exist on whether exposure to sun or artificial UV sources may deplete the levels of blood folate in humans. Blood contains several photosensitizers and proteins, as well as antioxidants, which when exposed to UV radiation and visible light may be involved in the degradation of folate. In this study the photodegradation of 5-methyltetrahydrofolate (5MTHF) in aqueous and deuterious solutions exposed to UVB, UVA, or visible light in the absence or presence of riboflavin, uroporphyrin, and conjugated bilirubin was investigated by absorption spectroscopy. 5MTHF is stable under exposure to visible light and UVA radiation, whereas it is slowly photooxidized under UVB exposure. However, it is rapidly oxidized by UVA or visible radiation in the presence of riboflavin or uroporphyrin, but not in the presence of conjugated bilirubin, which acts in a protective manner. Reactive oxygen species produced in type I and/or type II reactions were involved. This study suggests that 5MTHF in blood can be photodegraded in the presence of the flavins and porphyrins, but protected by bilirubins. This may have health and evolutionary implications.

Silicon microneedle (MN) arrays were used to puncture excised murine and porcine skin in vitro and transdermal and intradermal delivery of meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP) investigated using topical application of a bioadhesive patch containing 19 mg TMP cm(-2). Animal studies, using nude mice, were then conducted to investigate the in vivo performance of the bioadhesive patch following MN puncture of skin. MN puncture significantly enhanced both intradermal and transdermal delivery of TMP in vitro, though the total amounts of drug delivered (25.22% into porcine skin and 0.07% across murine skin) were still quite small in each case. Notwithstanding this, in vivo experiments showed that MN puncture was capable of permitting a prolonged increase in TMP fluorescence at the site of application. Importantly, fluorescence was negligible at distant sites, meaning systemic delivery of the drug was not sufficient to induce TMP accumulation other than at the application site. In this study we have conclusively demonstrated proof of principle; MN puncture allows true intradermal delivery of a preformed photosensitizer in animal skin models in vitro and in vivo. Importantly, transdermal delivery was much reduced in each case. Increasing MN density would allow increased amounts of photosensitizer to be delivered. However, as MNs create aqueous pores in the stratum corneum, a preformed photosensitizer must possess at least some degree of water solubility in order to permit enhanced intradermal delivery in this way. We believe that use of MN array technology in this way has the potential to significantly improve topical photodynamic therapy of skin tumors.

The main form of folate in human plasma is 5-methyltetrahydrofolate (5MTHF). The observation that folate in human serum is photosensitive supports the hypothesis that humans developed dark skin in high ultraviolet fluences areas in order to protect folate in the blood from UV radiation. However, folates alone are quite photostable. Therefore, in this study, we examined for the first time the photodegradation of 5MTHF in the presence of the endogenous photosensitizer uroporphyrin (Uro), which is sometimes present in low concentration in human serum, under UV and near-UV light exposure. We found strong indications that while 5MTHF alone is rather photostable, it is degraded quickly in the presence of Uro. Using deuterium oxide (D 2 O) as an enhancer of the lifetime of singlet oxygen and the singlet oxygen sensor green reagent (SOSG) as a scavenger of singlet oxygen, we have found that the photodegradation most likely proceeds via a type II photosensitization. Our results show that singlet oxygen is likely to be the main intermediate in the photodegradation of 5MTHF mediated by Uro. Our findings may be useful for further studies the evolution of human skin colours.

Summary The fluorescence kinetics of a new photosensitizer for photodynamic therapy, di- l -arginine protoporphyrinate (PP(Arg)2), was studied in the skin of healthy mice. Furthermore, induction of necrosis in WiDr human colon adenocarcinoma xenografts in athymic nude mice was studied after photodynamic therapy (PDT) with PP(Arg)2. After intravenous administration of PP(Arg)2 maximal fluorescence was reached after 72 h in normal mouse skin. Complete elimination of the drug from the mouse skin was not found even after 32 days. Exposure of WiDr tumours in mice to red light (λ = 632 nm, fluence 150 J/cm2, fluence rate 250 mW/cm2) 24 and 72 h after intravenous administration of 10 mg/kg of PP(Arg)2 caused extensive tumour necrosis. Epidermal damage and infiltration of inflammatory cells was seen 24 h after light exposure but not after 72 h.

Photodynamic therapy (PDT) is being evaluated in clinical trials for treatment of various oncologic and ophthalmic diseases. The main cause for cell inactivation and retardation of tumor growth after photoactivation of sensitizers is very short-lived singlet oxygen molecules that are produced and have limited diffusion distances. In this paper we show that the extent of biological damage can be modulated by using protoporphyrin, which was modified to increase its lipophilicity, and which also places the tetrapyrrole core deeper within the membrane by the carboxylate groups being anchored at the lipid:water interface. The uptake of the parent molecule (PPIX) and its diheptanoic acid analogue (PPIXC6) by WiDR and CT26 cells was investigated by fluorescence microscopy and by fluorescence intensity from the cells. The uptake of PPIXC6 increased almost linearly with incubation length for over 24 h, whereas for PPIX only 1 h was needed to reach maximal intracellular concentration. Fluorescence microscopy of both cell lines indicated that both drugs were distributed diffusely in the plasma membrane and cytoplasm, but remained outside the nucleus. The efficiency of in vitro inactivation of WiDr and CT26 cells increased with the length of the alkylcarboxylic chain. Tumors in mice that were treated with PPIX-PDT grew more slowly than control tumors. However, tumors that were given PPIXC6 followed by light exposure showed a significant delay in their growth.

The present work was carried out to investigate the role of light and darkness on the endogenous biosynthesis of porphyrins in mammalian skin (hairless BALB/c mouse) in vivo. In the skin of mice that were constantly kept in darkness (DD), increased endogenous porphyrin fluorescence was observed, which mainly originated from protoporphyrin IX (PpIX). No significant increase in the porphyrin levels was observed in mice that were kept under a normal day–night cycle (LD 12:12 h). The presence of cutaneous PpIX together with ambient light may comprise a photosensitizing mechanism by which PpIX may be a photomessenger between ambient light and internal rhythms. Copyright © 2006 John Wiley & Sons, Ltd.

Many medical applications, including photodynamic therapy for cancer (PDT), involve the use of lasers. However, the coherence of laser light is not necessary for PDT, and attempts have been made to construct non-coherent light sources for PDT, which are relatively inexpensive, stable and easy to operate, require simple maintenance but differ fundamentally from the lasers in their output characteristics. In the present work we compared two clinically used lamps, CureLight1, which is a broadband source (560–740 nm) based on a filtered halogen lamp, and CureLight2, which is a narrowband source based on light-emitting diodes (LEDs), with respect to several parameters of crucial significance for PDT efficiency in vivo: (a) depth of action in tissues, (b) heating effects, (c) pain generation, (d) photodegradation of PpIX in solution, in cells and in mouse skin and (e) photo-inactivation of cells in vitro. We conclude that CureLight2 (LED), relative to CureLight1 (halogen) has deeper PDT action in tissue, similar efficiency for bleaching PpIX in mouse skin, better efficiency for bleaching PpIX in cells and solutions and good efficiency for inactivating cells in vitro. CureLight2 gives less heating of the tissue and less pain in unsensitised human skin. All these differences are related to difference in the spectra of the lamps. Thus, PDT light sources with emissions that are visually similar have significantly different photobiological properties.

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 (

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.

The skin of nude mice was exposed to erythemogenic doses of UV radiation, which resulted in erythema with edema. An ointment containing 5-aminolevulinic acid (ALA) was topically applied on mouse and human skin. Differences in the kinetics of protoporphyrin accumulation were investigated in normal and UV-exposed skin. At 24 and 48 h after UV exposure, skin produced significantly less protoporphyrin IX (PpIX) than skin unexposed to UV. Human skin on body sites frequently exposed to solar radiation (the lower arm) also produced less PpIX than skin exposed more rarely to the sun (the upper arm). It is concluded that UV radiation introduces persisting changes in the skin, relevant to its capability of producing PpIX from ALA. The observed differences in ALA-induced PpIX fluorescence may be the result of altered penetration of ALA through the stratum corneum or altered metabolizing ability of normal and UV-exposed skin (or both).

1. 1. IsoHp reacts with 1-bromoalkanes on refluxing with a high concentration of alkali [50% (w/v) KOH] to yield porphyrin ethers in a 50–60% yield. 2. 2. Liquid secondary ion mass spectrometry (LSIMS) examination indicates that they are mono-ethers. 3. 3. These HPLC profiles show closely approximated peaks (similar for example to Hp). 4. 4. They have been separated by TLC and have the same mass, determined by LSIMS, and are, therefore, regarded as porphyrin 2, 4 position isomers. 5. 5. The reason(s) for the property of isoHp persistently to form mono-derivatives is not yet clear.