ALA and ALA hexyl ester-induced porphyrin synthesis in chemically induced skin tumours: the role of different vehicles on improving photosensitization.

Exogenous administration of 5-aminolevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of Protoporphyrin IX (PpIX) in photodynamic therapy. We analysed porphyrin formation in chemically induced squamous papillomas, after topical application of ALA and ALA hexyl ester (He-ALA) administered in different formulations, as well as the pattern of distribution in the internal organs, and the synthesis of porphyrins in distant tumoural and normal skins. A lotion formulation containing DMSO and ethanol was the best vehicle for topical ALA delivery to papillomas, whereas cream was the most efficient formulation for He-ALA application. Similar porphyrin concentration can be accumulated in the skin tumours employing either ALA or He-ALA delivered in their optimal formulations. The use of cream as a vehicle of both ALA and He-ALA, induces highest porphyrin tumour/normal skin ratios. The main advantage of using He-ALA is that porphyrins synthesized from the ester are more confined to the site of application, thus inducing low porphyrin levels in normal skin, liver, blood and spleen, as well as in papillomas distant from the point of application, independently on the vehicle employed, so reducing potential side effects of photodynamic therapy.     

Topical application of ALA and ALA hexyl ester on a subcutaneous murine mammary adenocarcinoma: tissue distribution

Although 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has proven to be clinically beneficial for the treatment of certain cancers, including a variety of skin cancers, optimal tissue localisation still remains a problem. An approach to improve the bioavailability of protoporphyrin IX (PpIX) is the use of ALA derivatives instead of ALA. In this work, we employed a subcutaneous murine mammary adenocarcinoma to study the tissue distribution pattern of the ALA hexyl ester (He-ALA) in comparison with ALA after their topical application in different vehicles. He-ALA induced porphyrin synthesis in the skin overlying the tumour (SOT), but it did not reach the tumour tissue as efficiently. Only 5 h after He-ALA lotion application, tumour porphyrin levels surpassed control values. He-ALA delivered in cream induced a substantially lower porphyrin synthesis in SOT, reinforcing the importance of the vehicle in the use of topical PDT. Porphyrin levels in internal organs remained almost within control values when He-ALA was employed. The addition of DMSO to ALA formulation slightly increased tumour and SOT porphyrin biosynthesis, but it did not when added to He-ALA lotion.     

Distribution of 5-aminolevulinic acid derivatives and induced porphyrin kinetics in mice tissues

Purpose: Porphyrins synthesised from 5-aminolevulinic acid (ALA) have been successfully used for the photodiagnosis and photodynamic treatment of cancer. To find a more efficient pro-photosensitiser, we synthesised two ALA esters: R,S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester (THP-ALA) and ALA-Undecanoyl ester (Und-ALA). Methods: In mice bearing a subcutaneous mammary adenocarcinoma, we studied the distribution of the porphyrins formed from these esters in tissues after systemic administration, to establish if these esters are retained in any specific tissue, which could potentially be targeted for photodynamic treatment with ALA derivatives. We also investigated the topical use of these esters. Results: After systemic administration, tumour and skin overlying tumour porphyrin levels were lower from the ALA esters than from ALA. Other tissues such as liver, colon, kidney, skin and spleen also accumulated less porphyrins from the esters, showing that there is no specific retention of the esters in these tissues. However, the brain was the only organ that synthesised more porphyrins from THP-ALA than from ALA. The kinetics of porphyrin synthesis from ALA esters is comparable to those from ALA in almost all tissues, showing that esterases activities are not limiting the availability of the hydrolysed ALA. Both THP-ALA and Und-ALA, applied topically on the skin over the tumour, exhibited higher selectivity than ALA for the site of application, whereas the amount of tumour porphyrin was the same from ALA and THP-ALA but lower from Und-ALA. Conclusions: THP-ALA may be useful for the treatment of brain tumours after systemic administration, whereas THP-ALA and Und-ALA may be used more suitable for the treatment of superficial tumours due to their higher selectivity.     

Topical and intratumoral photodynamic therapy with 5-aminolevulinic acid in a subcutaneous murine mammary adenocarcinoma.

One of the most promising substances used in photodynamic therapy (PDT) is 5-aminolevulinic acid (ALA), which induces endogenous synthesis and accumulation of porphyrins in malignant cells. In this paper we have shown that both topical and intratumoral administration of ALA in a subcutaneously implanted mammary carcinoma produced a significant synthesis of porphyrins and subsequent sensitization to laser light. Porphyrin accumulation was greater when ALA was administered intratumorally and tumour/normal skin porphyrin concentration ratios were higher compared with topical application. Irradiation was optimal between 2 and 3 h after topical application of 50 mg of a 20% ALA cream and 2-4 h after intratumoral administration of 30 mg ALA/cm3. The pattern of tumour response evaluated as the delay of tumour growth was similar following either route of drug administration. Applications of PDT were performed once, twice or three times in the study. The response to successive applications was constant for the same tumour, indicating that no resistance was acquired. Microscopic analysis showed both induction of foci of necrosis and haemorrhage, morphological features of apoptotic cells and total absence of cellular immune response. This paper reports on PDT with topical ALA in a subcutaneous carcinoma leading to tumour growth delay. These findings may have great relevance in the treatment of cutaneous metastasis of mammary carcinomas.     

Optimum porphyrin accumulation in epithelial skin tumours and psoriatic lesions after topical application of delta-aminolaevulinic acid

Photodynamic therapy with topically applied delta-aminolaevulinic acid is used to treat skin tumours by employing endogenously formed porphyrins as photosensitizers. This study examines the time course of porphyrin metabolite formation after topical application of delta-aminolaevulinic acid. Porphyrin biosynthesis in human skin tumours (basal cell carcinoma, squamous cell carcinoma), in psoriatic lesions, and in normal skin was investigated. Skin areas were treated with delta-aminolaevulinic acid, and levels of total porphyrins, porphyrin metabolites and proteins were measured in samples excised after 1, 2, 4, 6, 9, 12 and 24 h. There was an increase in porphyrin biosynthesis in all tissues with maximum porphyrin levels in tumours between 2 and 6 h and in psoriatic lesions 6 h after treatment. The pattern of porphyrins showed no significant difference between normal and neoplastic skin, protoporphyrin being the predominant metabolite. The results suggest that optimum irradiation time for superficial epithelial skin tumours may be as soon as 2 h after application of delta-aminolaevulinic acid, whereas for treatment of psoriatic lesions an application time of 6 h is more suitable.     

Porphyrin synthesis from ALA derivatives for photodynamic therapy. In vitro and in vivo studies

The aim of this work was to test in vitro and in vivo the efficacy of the derivatives of 5-aminolevulinic acid (ALA): hexyl-ALA (He-ALA), undecanoyl-ALA and R,S-2-(hydroximethyl)tetrahydropyranyl-ALA (THP-ALA) as pro-photosensitising agents. The compounds were assayed in a cell line derived from a murine mammary tumour, in tumour explants and after injection of the cells into mice. In vitro, undecanoyl-ALA and THP-ALA did not improve ALA efficacy in terms of porphyrin synthesis. On the other hand, half of the amount of ALA is required to obtain the same plateau amount of photosensitiser from He-ALA. However, this plateau value cannot be surpassed in spite of the four-times higher accumulation of ALA/He-ALA from the ALA derivative. This shows that He-ALA conversion to porphyrins but not He-ALA entry to the cells is limiting. Employing ionic exchange chromatography, we found that 80% of total uptake was He-ALA whereas only 20% was ALA. This suggests that the esterases, probably themselves regulated by the heme pathway, are limiting the conversion of ALA derivatives into porphyrins. A similar situation occurs with THP-ALA. Tumour explant porphyrin results correlate well with cell line data. However, i.p. injection of ALA derivatives to mice resulted in a lower porphyrin concentration in the tumour when compared to the administration of equimolar amounts of ALA, indicating that there should be retention of ALA derivatives either within the blood vessels in the initial phase of distribution and/or within the capillaries of the tumour.     

Comparative effect of ALA derivatives on protoporphyrin IX production in human and rat skin organ cultures.

Samples of human and rat skin in short-term organ culture exposed to ALA or a range of hydrophobic derivatives were examined for their effect on the accumulation of protoporphyrin IX (PpIX) measured using fluorescence spectroscopy. With the exception of carbobenzoyloxy-D-phenylalanyl-5-ALA-ethyl ester the data presented indicate that, in normal tissues, ALA derivatives generate protoporphyrin IX more slowly than ALA, suggesting that they are less rapidly taken up and/or converted to free ALA. However, the resultant depot effect may lead to the enhanced accumulation of porphyrin over long exposure periods, particularly in the case of ALA-methyl ester or ALA-hexyl ester, depending on the applied concentration and the exposed tissue. Addition of the iron chelator, CP94, greatly increased PpIX accumulation in human skin exposed to ALA, ALA-methyl ester and ALA-hexyl ester. The effect in rat skin was less marked.     

Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters

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.     

Porphyrin accumulation induced by 5-aminolaevulinic acid esters in tumour cells growing in vitro and in vivo

The ability of 5-aminolaevulinic acid and some of its esterified derivatives to induce porphyrin accumulation has been examined in CaNT murine mammary carcinoma cells growing in culture and as tumours in vivo. Topical or intravenous administration of 5-aminolaevulinic acid-esters to mice bearing subcutaneous tumours produced lower porphyrin levels in the tumour than an equimolar dose of 5-aminolaevulinic acid. Reducing the dose of intravenous hexyl- or benzyl-ALA and topical hexyl-5-aminolaevulinic acid resulted in a dose-dependent reduction in porphyrin accumulation. A number of normal tissues accumulated higher concentrations of porphyrins than tumour tissue following intravenous administration of 5-aminolaevulinic acid-esters. Esterase activity in these normal tissues was greater than that in tumour tissue. In contrast to the situation in vivo, all of the 5-aminolaevulinic acid-esters examined were at least as effective as 5-aminolaevulinic acid when applied to cloned CaNT cells in vitro, with the drug concentration required for maximum porphyrin accumulation varying with ester chain-length. Tumour cells growing in culture released esterase activity into the medium. These findings suggest that the efficacy of 5-aminolaevulinic esters may vary depending on the esterase activity of the target tissue, and suggest caution when interpreting the findings of in vitro studies using these and similar prodrugs.     

Kinetics and localisation of PpIX fluorescence after topical and systemic ALA application, observed in skin and skin tumours of UVB-treated mice.

In this study the kinetics and localisation of protoporphyrin IX (PpIX) fluorescence in skin and skin tumours were examined after topically (20% for 4h) or systemically (200 mg/kg,i.p.) administered 5-aminolaevulinic acid (ALA). As a model we used hairless mice with skin lesions (actinic keratoses and squamous cell carcinoma), which were induced by daily UVB irradiation. The epidermis of the skin surrounding the tumours (T) was altered (AS); owing to the UVB irradiation, the epidermis was thicker and less elastic. Therefore, non-UVB-irradiated mice were used to assess fluorescence of normal skin (NS). Light from a halogen lamp was used to excite at 500 +/- 20 nm and fluorescence was detected through a filter that passes light of 670 +/- 50 nm. Maximal fluorescence following i.p. ALA was observed 2 h post injection (p.i.) and was three times less than after topically applied ALA. Furthermore, after i.p. ALA a lower T selectively (T/NS) could be obtained than after topically applied ALA. Maximal fluorescence following topically applied ALA was achieved 6 h after the end of the 4 h application time. At that interval fluorescence of T was twice as high as directly after the application period. Furthermore, T selectivity (T/NS) after topical ALA at the interval of maximal fluorescence was higher than at the interval directly after application. With fluorescence cryomicroscopy localisation of fluorescence in the skin at the interval of maximal fluorescence was determined after both administration routes. For both cases fluorescence was mainly located in T, epidermis and hair follicles. Fluorescence in subcutis could only be observed at 2 h post i.p. ALA and a 6 h post topical ALA. No fluorescence could be observed in muscle. We conclude that, in this model and with these ALA doses, a higher fluorescence intensity and selectivity (T/NS) was achieved after topically applied ALA than after systemically administered ALA. These results make topically applied ALA more favourable for ALA-PDT of superficial skin tumours in this model. In general these results imply that by optimising the time after ALA application the efficacy and selectivity of topical ALA-PDT for skin tumours may be improved.     

A comparative study of tissue distribution and photodynamic therapy selectivity of chlorin e6, Photofrin II and ALA-induced protoporphyrin IX in a colon carcinoma model.

An in vivo study of tissue distribution kinetics and photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA), chlorin e6 (Chl) and Photofrin (PII) was performed to evaluate the selectivity of porphyrin accumulation and tissue damage effects in a tumour model compared with normal tissue. C26 colon carcinoma of mice transplanted to the foot was used as a model for selectivity assessment. Fluorescence measurements of porphyrin accumulation in the foot bearing the tumour and in the normal foot were performed by the laser-induced fluorescence (LIF) system. A new high-intensity pulsed light delivery system (HIPLS) was used for simultaneous irradiation of both feet by light in the range of 600-800 nm, with light doses from 120 to 300 J cm-2 (0.6 J cm-2 per pulse, 1 Hz). Photoirradiation was carried out 1 h after injection of ALA, 3 h after injection of Chl and 24 h after injection of PII. A ratio of porphyrin accumulation in tumour vs normal tissue was used as an index of accumulation selectivity for each agent. PDT selectivity was determined from the regression analysis of normal and tumour tissue responses to PDT as a function of the applied light dose. A normal tissue damage index was defined at various values (50, 80 and 100%) of antitumour effect. The results of the LIF measurements revealed different patterns of fluorescence intensity in tumour and normal tissues for ALA-induced protoporphyrin IX (ALA-PpIX), Chl and PII. The results of PDT demonstrated the differences in both anti-tumour efficiency and normal tissue damage for the agents used. The selectivity of porphyrin accumulation in the tumour at the time of photoirradiation, as obtained by the LIF measurements, was in the order ALA-PpIX > Chl > PII. PDT selectivity at an equal value of anti-tumour effect was in the order Chl > ALA-PpIX > PII. Histological examination revealed certain differences in structural changes of normal skin after PDT with the agents tested. The results of PDT selectivity assessment with respect to differences in mechanisms of action for ALA, Chl and PII are discussed.     

Distribution and photosensitizing efficiency of porphyrins induced by application of exogenous 5-aminolevulinic acid in mice bearing mammary carcinoma.

By means of a chemical extraction procedure and confocal laser scanning microscopy, we investigated the kinetic patterns of uptake and biolocalization of 5-aminolevulinic acid (ALA)-induced porphyrins in s.c. transplanted tumors, adjacent normal skin and muscle, and liver of mice bearing mammary carcinoma, after i.p. injection of 250 mg/kg ALA or topical application of ALA (20% in an oil-in-water emulsion). Furthermore, we evaluated the tumor responses after either i.p. injection or topical application of 5-ALA followed by laser irradiation (632 nm, 150 mW/cm2, 25 min) by measuring the treated tumor regression/regrowth time and by light and electron microscopy. Strong fluorescence of ALA-induced porphyrins was detected in the tumor, skin and liver tissues, while little fluorescence was seen in the adjacent muscle tissue. Moreover, the highest amounts of ALA-induced porphyrins in the tumor and skin tissues were found 1 hr after i.p. injection, whereas the amounts of the porphyrins in both tissues increased with increasing time after topical application of ALA. The fluorescence of the porphyrins was localized in several components of the skin tissue (epidermis, hair follicles and their associated sebaceous glands). Furthermore, the fluorescence was diffusely distributed in the s.c. transplanted tumor tissue. Little could be observed under a confocal laser scan microscope (CLSM) in the muscle tissue. The uptake and biolocalization data correlate well with the results of PCT efficiency of the same tumor model with ALA-induced porphyrins. Light and electron microscopy showed that the mitochondria of the tumor cells and of the endothelial cells and the basal lamina of vascular walls beneath the endothelium in the tumor tissue were initially extensively destroyed after PCT with ALA-induced porphyrins. Thereafter, diffuse degeneration followed by local and/or diffuse severe necrosis of the tumor cells was found. This may be due mainly to the initial damage to mitochondria in the cancerous and endothelial cells and also to the destruction of the vascular wall in the tumor tissue.     

On the pharmacokinetics of topically applied 5-aminolevulinic acid and two of its esters

The kinetics of protoporphyrin IX (PpIX) production in normal tissues and WiDr tumors of mice were studied after topical application of 5-aminolevulinic acid (ALA) and its methyl ester and hexyl ester. ALA and ALA esters were applied on a spot of 1.0 cm diameter on normal skin and on skin overlaying tumors. PpIX production was studied by fluorescence measurements. ALA induced PpIX not only on the spot of application but also on remote skin areas. This was not found for the ALA esters. They produced PpIX only on the spot of application. Thus, ALA, but neither its esters nor PpIX, is passing into the circulation. The time needed for ALA to enter the circulation through normal skin was about 5 hr. Even when looking normal, the skin overlaying tumors was more permeable to ALA than normal skin. Thus, when applied on the tumor, ALA induced PpIX on remote skin areas without any lag phase. Mainly, PpIX was found in all tissues although small amounts of a porphyrin with an excitation peak at about 400 nm, supposedly uroporphyrin and/or coproporphyrin, were found, notably in remote skin areas. An altered stratum corneum of the skin overlaying tumors probably contributes to the tumor-selectivity, although in the present tumor system less PpIX was found in tumors than in muscles. This is probably related to biochemical and physiological conditions in this particular tumor, since i.p. injection of ALA also leads to less PpIX formation in the tumor than in skin/muscle tissue. Nevertheless, it seems evident that ALA can diffuse more easily from the skin surface and down to the vasculature in the tumor than in the normal tissue and that this leads to a higher concentration of PpIX in the tumor than would have been found if the physiological factors relevant for drug diffusion were the same for tumors as for skin/muscles.     

Mechanistic studies on delta-aminolevulinic acid uptake and efflux in a mammary adenocarcinoma cell line

Delta-aminolevulinic acid (ALA) is the precursor in the biosynthesis of porphyrins. The knowledge of both the regulation of ALA entrance and efflux from the cells and the control of porphyrin biosynthesis is essential to improve ALA-mediated photodynamic therapy. In this work, we studied the regulation of ALA uptake and efflux by endogenously accumulated ALA and/or porphyrins in murine mammary adenocarcinoma cells. Under our set of conditions, the haem synthesis inhibitor succinyl acetone completely prevented porphobilinogen and porphyrin synthesis from ALA, and led to an increase in the intracellular ALA pool. However, neither intracellular ALA nor porphyrin pools regulate ALA uptake or efflux during the first 15 min of the process. Based on temperature dependence data, ALA but not gamma-aminobutyric acid (GABA) efflux is mediated by a diffusion mechanism. Moreover, the addition of extracellular GABA not only did not influence the rate of ALA efflux but on the contrary it affected ALA uptake, showing the contribution of a saturable mechanism for the uptake, but not for the efflux of ALA from the cells.     

Biochemical basis of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation: a study in patients with (pre)malignant lesions of the oesophagus.

Administration of 5-aminolaevulinic acid (ALA) leads to porphyrin accumulation in malignant and premalignant tissues, and ALA is used as a prodrug in photodynamic therapy (PDT). To understand the mechanism of porphyrin accumulation after the administration of ALA and to investigate whether ALA-induced protoporphyrin IX might be a suitable photosensitizer in Barrett''s oesophagus and adenocarcinoma, we determined the activities of porphobilinogen deaminase (PBG-D) and ferrochelatase (FC) in various malignant and premalignant as well as in normal tissues of the human oesophagus. A PDT power index for ALA-induced porphyrin accumulation, the ratio of PBG-D to FC normalized for the normal squamous epithelium of the oesophagus, was calculated to evaluate intertissue variation in the ability to accumulate porphyrins. In malignant and premalignant tissue a twofold increased PBG-D activity and a marginally increased FC activity was seen compared with normal squamous epithelium. A significantly increased PDT power index in Barrett''s epithelium and adenocarcinoma was found. Our results suggest that, after the administration of ALA, porphyrins will accumulate in a greater amount in Barrett''s epithelium and adenocarcinoma of the oesophagus because of an imbalance between PBG-D and FC activities. The PDT power index here defined might be a useful indicative parameter for predicting the susceptibility of these tissues to ALA-PDT.     

In vivo kinetics and spectra of 5-aminolaevulinic acid-induced fluorescence in an amelanotic melanoma of the hamster.

For successful photodynamic diagnosis (PDD) and effective photodynamic therapy (PDT) with the clinically used ''photosensitiser'' 5-aminolaevulinic acid (ALA), knowledge of the maximal fluorescence intensity and of the maximal tumour-host tissue fluorescence ratio following systemic or local application is required. Therefore, time course and type of porphyrin accumulation were investigated in neoplastic and surrounding host tissue by measuring the kinetics and spectra of ALA-induced fluorescence in vivo. Experiments were performed in the amelanotic melanoma A-Mel-3 grown in the dorsal skinfold chamber preparation of Syrian golden hamsters. The kinetics of fluorescent porphyrins was quantified up to 24 h after i.v. injection of 100 mg kg-1, 500 mg kg-1 or 1,000 mg kg-1 body weight ALA by intravital fluorescence microscopy and digital image analysis (n = 18). In separate experiments fluorescence spectra were obtained for each dose by a simultaneous optical multichannel analysing device (n = 3). A three-compartment model was developed to simulate fluorescence kinetics in tumours. Maximal fluorescence intensity (per cent of reference standard; mean +/- s.e.) in the tumour arose 150 min post injection (p.i.) (1,000 mg kg-1, 109 +/- 34%; 500 mg kg-1, 148 +/- 36%) and 120 min p.i. (100 mg kg-1, 16 +/- 8%). The fluorescence in the surrounding host tissue was far less and reached its maximum at 240 min (100 mg kg-1, 6 +/- 3%) and 360 min p.i. (500 mg kg-1, 50 +/- 8%) and (1,000 mg kg-1, 6 +/- 19%). Maximal tumour-host tissue ratio (90:1) was encountered at 90 min after injection of 500 mg kg-1. The spectra of tissue fluorescence showed maxima at 637 nm and 704 nm respectively. After 300 min (host tissue) and 360 min (tumour tissue) additional emission bands at 618 nm and 678 nm were detected. These bands indicate the presence of protoporphyrin IX (PPIX) and of another porphyrin species in the tumour not identified yet. Tumour selectivity of ALA-induced PPIX accumulation occurs only during a distinct interval depending on the administered dose. Based on the presented data the optimal time for PDD and PDT in this model following intravenous administration of 500 mg kg-1 ALA would be around 90 min and 150 min respectively. The transient selectivity is probably caused by an earlier and higher uptake of ALA in the neoplastic tissue most likely as a result of increased vascular permeability of tumours as supported by the mathematical model.     

Photosensitization and mechanism of cytotoxicity induced by the use of ALA derivatives in photodynamic therapy.

The use of more lipophilic derivatives of 5-aminolevulinic acid (ALA) is expected to have better diffusing properties, and after conversion into the parent ALA, to reach a higher protoporphyrin IX (PPIX) formation rate, thus improving the efficacy of topical photodynamic therapy (PDT). Here we have analysed the behaviour of 3 ALA derivatives (ALA methyl-ester, hexyl ester and a 2-sided derivative) regarding PPIX formation, efficiency in photosensitizing cells and mechanism of cellular death. The maximum amount of porphyrins synthesized from 0.6 mM ALA was 47 +/- 8 ng/10(5)cells. The same amount was formed by a concentration 60-fold lower of hexyl-ALA and 2-fold higher of methyl-ALA. The 2-sided derivative failed to produce PPIX accumulation. Applying a 0.6 J cm(-2)light dose, cell viability decreased to 50%. With the 1.5 J cm(-2) light dose, less than 20% of the cells survive, and higher light doses produced nearly total cell killing. Comparing the PPIX production and the induced phototoxicity, the more the amount of porphyrins, the greater the cellular killing, and PPIX formed from either ALA or ALA-esters equally sensitize the cells to photoinactivation. ALA-PDT treated cells exhibited features of apoptosis, independently on the pro-photosensitizer employed. ALA-PDT can be improved with the use of ALA derivatives, reducing the amount of ALA necessary to induce efficient photosensitization.     

ALA and ALA hexyl ester induction of porphyrins after their systemic administration to tumour bearing mice

The use of synthetic lipophilic molecules derived from 5-aminolevulinic acid (ALA) is currently under investigation to enhance cellular ALA penetration. In this work we studied the effect of systemic administration to mice of the hexyl ester of ALA (He-ALA) on porphyrin tissue synthesis as compared to ALA. In most normal tissues as well as in tumour, He-ALA induced less porphyrin synthesis than ALA after its systemic administration either intravenous or intraperitoneal, although explant organ cultures exposed to either ALA or He-ALA revealed equally active esterases. The only tissue that accumulated higher porphyrin levels from He-ALA (seven times more than ALA) was the brain, and this correlated well with a rapid increase in ALA/He-ALA content in brain after administration of He-ALA. This may be ascribed to a differential permeability to lipophilic substances controlled by the blood-brain barrier, a feature which could be further exploited to treat brain tumours.     

ALA and ALA hexyl ester in free and liposomal formulations for the photosensitisation of tumour organ cultures

In spite of the wide range of tumours successfully treated with 5-aminolevulinic acid mediated photodynamic therapy, the fact that 5-aminolevulinic acid has low lipid solubility, limits its clinical application. More lipophilic 5-aminolevulinic acid prodrugs and the use of liposomal carriers are two approaches aimed at improving 5-aminolevulinic acid transmembrane access. In this study we used both 5-aminolevulinic acid and its hexyl ester in their free and encapsulated formulations to compare their corresponding endogenous synthesis of porphyrins. Employing murine tumour cultures, we found that neither the use of hexyl ester nor the entrapment of either 5-aminolevulinic acid or hexyl ester into liposomes increase the rate of tumour porphyrin synthesis. By light and electronic microscopy it was demonstrated that exposure of tumour explants to either free or liposomal 5-aminolevulinic acid and subsequent illumination induces the same type of subcellular damage. Mitochondria, endoplasmic reticulum and plasma membrane are the structures mostly injured in the early steps of photodynamic treatment. In a later stage, cytoplasmic and nuclear disintegration are observed. By electronic microscopy the involvement of the endocytic pathway in the incorporation of liposomal 5-aminolevulinic acid into the cells was shown.     

Photobleaching during and re-appearance after photodynamic therapy of topical ALA-induced fluorescence in UVB-treated mouse skin

Photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA) seems a promising alternative for the treatment of superficial non-melanoma skin cancer and actinic keratosis. In this study, the kinetics of new PpIX fluorescence arising after a PDT treatment that had photobleached the original fluorescence were determined. Our purpose was to examine the feasibility of multiple irradiations, following a single topical ALA application, to increase PDT efficacy. In addition, photobleaching during PDT and the fluorescence spectra during and after PDT were studied. As a model we used hairless mice with and without UVB-induced skin lesions. ALA was applied to the skin for 4 hr. An illumination was delivered either immediately after application or 6 hr after the end of the application (at interval of maximum fluorescence). During PDT, the fluorescence of normal skin decreased at a faster rate than the fluorescence of the skin lesions. In the fluorescence study after PDT, the areas treated immediately post-application showed a fluorescence increase over time similar to that in non-treated areas on the same mice. A remarkable result was that the fluorescence of areas treated at maximum fluorescence increased, whereas the fluorescence of non-treated areas did not increase over time. With both treatment intervals the new fluorescence showed a characteristic PpIX spectrum. Our results demonstrate that a second illumination, when new PpIX fluorescence has been formed, may increase PDT efficacy after topical ALA application. This finding has been demonstrated previously for systemic ALA administration. Int. J. Cancer 72:110–118, 1997. © 1997 Wiley-Liss Inc.