Evaluation of the effect of 5-aminolevulinic acid hexyl ester (H-ALA) PDT on EBV LMP1 protein expression in human nasopharyngeal cells.

Nasopharyngeal carcinoma (NPC) is of high prevalence in Hong Kong and southern China. The pathogenesis of NPC is closely associated with Epstein-Barr virus (EBV) infection via regulation of viral oncoprotein latent membrane protein 1 (LMP1). The conventional treatment for NPC is chemo-radiotherapy, but the prognosis remains poor for advanced stage, recurrent and metastatic NPC. Photodynamic therapy (PDT) is a therapeutic approach to combat tumors. PDT effectiveness depends on the interaction of photosensitizers, light and molecular oxygen. 5- aminolevulinic acid hexyl derivative (H-ALA) is one of the photosensitizers derived from 5-ALA. H-ALA with improved lipophilic properties by adding a long lipophilic chain (hexyl group) to 5-ALA, resulted in better penetration into cell cytoplasm. In this study, the effect of H-ALA-PDT on NPC cells (EBV positive C666-1 and EBV negative CNE2) was investigated. The H-ALA mediated cellular uptake and cytotoxicity was revealed via flow cytometry analysis and MTT assay respectively. H-ALA PDT mediated protein modulation was analysed by western blot analysis. Our finding reported that the cellular uptake of H-ALA in C666-1 and CNE2 cells was in a time dependent manner. H-ALA PDT was effective to C666-1 and CNE2 cells. EBV LMP1 proteins was expressed in C666-1 cells only and its expression was responsive to H-ALA PDT in a dose dependent manner. This work revealed the potential of H-ALA PDT as a treatment regiment for EBV positive NPC cells. Understanding the mechanism of H-ALA mediated PDT could develop improved strategies for the treatment of NPC.

A dual-targeted hyaluronic acid-gold nanorod platform with triple-stimuli responsiveness for photodynamic/photothermal therapy of breast cancer.

Multi-stimuli-responsive theranostic nanoplatform integrating functions of both imaging and multimodal therapeutics holds great promise for improving diagnosis and therapeutic efficacy. In this study, we reported a pH, glutathione (GSH) and hyaluronidase (HAase) triple-responsive nanoplatform for HER2 and CD44 dual-targeted and fluorescence imaging-guided PDT/PTT dual-therapy against HER2-overexpressed breast cancer. The nanoplatform was fabricated by functionalizing gold nanorods (GNRs) with hyaluronic acid (HA) bearing pendant hydrazide and thiol groups via Au-S bonds, and subsequently chemically conjugating 5-aminolevulinic acid (ALA), Cy7.5 and anti-HER2 antibody onto HA moiety for PDT, fluorescence imaging and active targeting, respectively. The resulting versatile nanoplatform GNR-HA-ALA/Cy7.5-HER2 had uniform sizes, favorable dispersibility, as well as pH, GSH and HAase triple-responsive drug release manner. In vitro studies demonstrated that HER2 and CD44 receptor-mediated dual-targeting strategy could significantly enhance the cellular uptake of GNR-HA-ALA/Cy7.5-HER2. Under near-infrared (NIR) irradiation, MCF-7 cells could efficiently generate reactive oxygen species (ROS) and heat, and be more efficiently killed by a combination of PDT and PTT as compared with individual therapy. Pharmacokinetic and biodistribution studies showed that the nanoplatform possessed a circulation half-life of 1.9 h and could be specifically delivered to tumor tissues with an accumulation ratio of 12.8%. Upon the fluorescence imaging-guided PDT/PTT treatments, the tumors were completely eliminated without obvious side effects. The results suggest that the GNR-HA-ALA/Cy7.5-HER2 holds great potential for breast cancer therapy. STATEMENT OF SIGNIFICANCE: A combination of photodynamic therapy (PDT) and photothermal therapy (PTT) is emerging as a promising cancer treatment strategy. However, its therapeutic efficacy is compromised by the nonspecific delivery and unintended release of photo-responsive agents. Herein, we developed a multifunctional theranostic nanoplatform GNR-HA-ALA/Cy7.5-HER2 with pH, glutathione and hyaluronidase triple-responsive drug release for HER2 and CD44 dual-targeted and fluorescence imaging-guided PDT/PTT therapy against breast cancer. We demonstrated that HER2 and CD44 receptors-mediated dual-targeting strategy significantly enhanced the cellular uptake of GNR-HA-ALA/Cy7.5-HER2. We also demonstrated that the combined PDT/PTT treatment had significantly superior antitumor effect than PDT or PTT alone both in vitro and in vivo. Therefore, GNR-HA-ALA/Cy7.5-HER2 could serve as a promising nanoplatform for HER2-positive breast cancer therapy.

Synergetic effects of 5-aminolevulinic acid and Ascophyllum nodosum seaweed extracts on Asparagus phenolics and stress related genes under saline irrigation.

Salinity is one of the major agricultural problems that may threat food security and limit the agricultural lands expansion worldwide. Exploring novel tools controlling saline conditions and increase valuable secondary metabolites in the horticultural crops might have outstanding results that serve humanity in the current century. The current study explores the effects of weekly seaweed extracts (7 mL L-1) and/or 5-aminolevulinic acid (3, 5 and 10 ppm) sprays on Asparagus aethiopicus plants subjected to saline stress conditions (2000 and 4000 ppm) for 6 weeks in two consecutive seasons of 2016 and 2017. Under saline conditions, there were stimulatory synergetic effects of seaweed extracts (SWE) and 5-aminolevulinic acid (ALA) on branch length and number of treated plants. Similar increases were also found in fresh and the dry weight of treated plants compared to control. These morphological improvements associated with increased accumulation of specific phenols (robinin, rutin, apigein, chlorogenic acid and caffeic acid) as revealed by High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD). There were increases in the antioxidant activities of leaf extracts, chlorophyll content and sugars and proline accumulation. The transpiration and photosynthetic rates as well as the stomatal conductance were enhanced. The morphological and physiological improvements associated with increased expression of several genes responsible for water management (ANN1, ANN2 and PIP1), secondary metabolite production (P5CS1 and CHS) and antioxidants accumulation (APX1 and GPX3) in plants. Our findings indicate that SWE + ALA had stimulatory synergetic effects on the growth and secondary metabolites of A. aethiopicus subjected to saline conditions. Several mechanisms are involved in such effects including gas exchange control, sugar buildup, increasing non-enzymatic and enzymatic antioxidants control of reactive oxygen species accumulation as well as transcriptional and metabolic regulation of environmental stress.

Plum-blossom needling promoted PpIX fluorescence intensity from 5-aminolevulinic acid in porcine skin model and patients with actnic keratosis.

BACKGROUND AND OBJECTIVES: Plum-blossom needling might enhance transdermal penetration of topically applied drugs by creating vertical channels. The purpose of this study was to evaluate drug delivery assisted by plum-blossom needling comparing with CO2 laser ablative fractional resurfacing (AFR) using 5-aminolevulinic acid (5-ALA), a porphyrin precursor, as a test drug. MATERIALS AND METHODS: Ex vivo porcine skin was treated with plum-blossom needle(HWATO, Suzhou medical supplies factory Co., Ltd. China) or CO2 laser AFR before topical application of 20% 5-ALA(Sigma-Aldrich, Co., USA)cream, placebo cream and no cream. ALA-induced porphyrin fluorescence was measured by fluorescence microscopy at skin depths down to 1800µm. Needling was done by tapping the skin vertically from 5cm high above quickly. AFR was performed with a 10.6µm wavelength prototype CO2 laser, using stacked single pulses of 3 millisecond and 91.6mJ per pulse. Plum-blossom needling after ALA application was also done. Fluorescence intensity on lesion surface was examined by curalux spectrum analyzer (Laser Institute of Munich University, Germany) and VAS pain score was recorded in a randomized split-lesion clinical trial including 6 patients, 8 actinic keratosis lesions. RESULTS: AFR created regular cone-shaped channels surrounded by a 70µm thin layer of thermally coagulated dermis, respectively. The cone is approximately 200µm in diameter at the opening and 1850µm in depth. Plum-blossom needle created irregular cone-shaped channels of approximately 180µm in diameter at the opening and it always drags a tail-which was shaped from the closed deeper channels. There was no porphyrin fluorescence in placebo cream or untreated skin sites. Plum-blossom needling followed by ALA application enhanced drug delivery with significantly higher porphyrin fluorescence at the edge of hole (P<0.005) and 100µm far from the hole (P=0.000) versus AFR followed by ALA application at skin depths of 120 and 500µm. Needling after ALA application presented higher porphyrin fluorescence at the edge of hole at skin depths of 120µm (P<0.005) and lower porphyrin fluorescence at 1000µm deep hole edge, and 100µm far from the hole at 120µm, 500µm and 1000µm depths versus AFR followed by ALA application (P<0.005). Skin massage after ALA application did not affect ALA-induced porphyrin fluorescence after pretreatment of plum-blossom needling or AFR. ALA application after plum-blossom needling was better than before plum-blossom needling. The clinical trial showed that the surface fluorescence intensity was stronger in needle-pretreated-lesion than in laser-pretreated-lesion. While the VAS pain score between needle treatment and laser treatment was almost the same. CONCLUSIONS: Plum-blossom needling facilitates delivery of topical ALA into the dermis. It may help ALA to diffuse a little more broadly than AFR does in superficial dermis and obtain similar clinical effect with a much lower cost. Plum-blossom needling treatment appears to be a clinically practical and economical means for enhancing transdermal delivery of ALA, a photodynamic therapy drug, and presumably many other topical skin medications.

Light Fractionation Significantly Increases the Efficacy of Photodynamic Therapy Using BF-200 ALA in Normal Mouse Skin.

BACKGROUND: Light fractionation significantly increases the efficacy of 5-aminolevulinic acid (ALA) based photodynamic therapy (PDT) using the nano-emulsion based gel formulation BF-200. PDT using BF-200 ALA has recently been clinically approved and is under investigation in several phase III trials for the treatment of actinic keratosis. This study is the first to compare BF-200 ALA with ALA in preclinical models. RESULTS: In hairless mouse skin there is no difference in the temporal and spatial distribution of protoporphyrin IX determined by superficial imaging and fluorescence microscopy in frozen sections. In the skin-fold chamber model, BF-200 ALA leads to more PpIX fluorescence at depth in the skin compared to ALA suggesting an enhanced penetration of BF-200 ALA. Light fractionated PDT after BF-200 ALA application results in significantly more visual skin damage following PDT compared to a single illumination. Both ALA formulations show the same visual skin damage, rate of photobleaching and change in vascular volume immediately after PDT. Fluorescence immunohistochemical imaging shows loss of VE-cadherin in the vasculature at day 1 post PDT which is greater after BF-200 ALA compared to ALA and more profound after light fractionation compared to a single illumination. DISCUSSION: The present study illustrates the clinical potential of light fractionated PDT using BF-200 ALA for enhancing PDT efficacy in (pre-) malignant skin conditions such as basal cell carcinoma and vulval intraepithelial neoplasia and its application in other lesion such as cervical intraepithelial neoplasia and oral squamous cell carcinoma where current approaches have limited efficacy.

Transport of the photodynamic therapy agent 5-aminolevulinic acid by distinct H+-coupled nutrient carriers coexpressed in the small intestine.

5-Aminolevulinic acid (ALA) is a prodrug used in photodynamic therapy, fluorescent diagnosis, and fluorescent-guided resection because it leads to accumulation of the photosensitizer protoporphyrin IX (PpIX) in tumor tissues. ALA has good oral bioavailability, but high oral doses are required to obtain selective PpIX accumulation in colonic tumors because accumulation is also observed in normal gut mucosa. Structural similarities between ALA and GABA led us to test the hypothesis that the H(+)-coupled amino acid transporter PAT1 (SLC36A1) will contribute to luminal ALA uptake. Radiolabel uptake and electrophysiological measurements identified PAT1-mediated H(+)-coupled ALA symport after heterologous expression in Xenopus oocytes. The selectivity of the nontransported inhibitors 5-hydroxytryptophan and 4-aminomethylbenzoic acid for, respectively, PAT1 and the H(+)-coupled di/tripeptide transporter PepT1 (SLC15A1) were examined. 5-Hydroxytryptophan selectively inhibited PAT1-mediated amino acid uptake across the brush-border membrane of the human intestinal (Caco-2) epithelium whereas 4-aminomethylbenzoic acid selectively inhibited PepT1-mediated dipeptide uptake. The inhibitory effects of 5-hydroxytryptophan and 4-aminomethylbenzoic acid were additive, demonstrating that both PAT1 and PepT1 contribute to intestinal transport of ALA. This is the first demonstration of overlap in substrate specificity between these distinct transporters for amino acids and dipeptides. PAT1 and PepT1 expression was monitored by reverse transcriptase-polymerase chain reaction using paired samples of normal and cancer tissue from human colon. mRNA for both transporters was detected. PepT1 mRNA was increased 2.3-fold in cancer tissues. Thus, increased PepT1 expression in colonic cancer could contribute to the increased PpIX accumulation observed. Selective inhibition of PAT1 could enhance PpIX loading in tumor tissue relative to that in normal tissue.

Intravesical delivery of 5-aminolevulinic acid from water-in-oil nano/submicron-emulsion systems.

The present work reports on the development of water-in-oil (w/o) emulsions for the intravesical administration of 5-aminolevulinic acid (ALA). The physicochemical properties of droplet size, zeta potential, and viscosity of the emulsions are characterized and the ability of the emulsions to release ALA following in vitro application is tested. The delivery systems are administered intravesically for 1 and 3 h in rats to examine the drug accumulation in bladder tissue. The mean size and zeta potential of the emulsions are 50-200 nm and -3 to -14 mV, respectively. The loading of ALA into the emulsions resulted in a slower and sustained release. The release extent was found to be inversely related to the droplet size of the emulsions. The emulsions did not increase the drug permeation into tissues during short exposure duration (1 h). When the dwell time was extended to 3 h, the systems showed a 2.7-fold increase in the ALA concentration in the bladder wall. Images of confocal laser scanning microscopy demonstrated a higher and deeper fluorescence signal, with emulsion administration, as compared to the aqueous control. Intravesical emulsion delivery provides a significant advantage for drugs targeting bladder tissues.

Protoporphyrin IX enhancement by 5-aminolaevulinic acid peptide derivatives and the effect of RNA silencing on intracellular metabolism.

Intracellular generation of the photosensitiser, protoporphyrin IX, from a series of dipeptide derivatives of the haem precursor, 5-aminolaevulinic acid (ALA), was investigated in transformed PAM212 murine keratinocytes, together with studies of their intracellular metabolism. Porphyrin production was substantially increased compared with equimolar ALA using N-acetyl terminated phenylalanyl, leucinyl and methionyl ALA methyl ester derivatives in the following order: Ac-L-phenylalanyl-ALA-Me, Ac-L-methionyl-ALA-Me and Ac-L-leucinyl-ALA-Me. The enhanced porphyrin production was in good correlation with improved photocytotoxicity, with no intrinsic dark toxicity apparent. However, phenylalanyl derivatives without the acetyl/acyl group at the N terminus induced significantly less porphyrin, and the replacement of the acetyl group by a benzyloxycarbonyl group resulted in no porphyrin production. Porphyrin production was reduced in the presence of class-specific protease inhibitors, namely serine protease inhibitors. Using siRNA knockdown of acylpeptide hydrolase (ACPH) protein expression, we showed the involvement of ACPH, a member of the prolyl oligopeptidase family of serine peptidases, in the hydrolytic cleavage of ALA from the peptide derivatives. In conclusion, ALA peptide derivatives are capable of delivering ALA efficiently to cells and enhancing porphyrin synthesis and photocytotoxicity; however, the N-terminus state, whether free or substituted, plays an important role in determining the biological efficacy of ALA peptide derivatives.

Photodynamic therapy using 5-aminolaevulinic acid for the treatment of dysplasia in Barrett's oesophagus.

BACKGROUND: 5-Aminolaevulinic acid (ALA) is the naturally occurring metabolic precursor of an endogenously formed photosensitiser, protoporphyrin IX. It is used topically to treat benign and malignant skin disorders by a process called photodynamic therapy (PDT). Recently, data are emerging on its systemic use in the treatment of dysplasia in Barrett's oesophagus. OBJECTIVE: To investigate the use of ALA-PDT for the treatment of dysplasia and early cancer in Barrett's oesophagus. METHODS: A systematic literature review and synopsis is given. CONCLUSION: ALA is an attractive alternative to the currently approved photosensitiser (porfimer sodium) due to its oral route of administration, shorter generalised skin photosensitivity period and lower rate of stricture formation. The studies presented demonstrate that ALA-PDT is both safe and effective for the treatment of dysplasia and early cancer in Barrett's oesophagus.

Bioadhesive patch-based delivery of 5-aminolevulinic acid to the nail for photodynamic therapy of onychomycosis.

The in vitro penetration of 5-aminolevulinic acid (ALA) across human nail and into neonate porcine hoof when released from a novel bioadhesive patch containing 50 mg cm(-2) ALA is described. ALA is a naturally occurring precursor of the photosensitiser protoporphyrin IX (PpIX). Topical application of excess ALA bypasses negative feedback inhibition and yields photosensitising concentrations of PpIX at the application site. ALA-based photodynamic therapy (PDT) has been extensively investigated in the topical treatment of various skin neoplasias. Recently, its use has been extended to the microbiological field. If sufficient concentrations of ALA could be achieved within the nail matrix, and at the nail bed, PDT may prove to be a useful treatment for onychomycosis. Patch application for 24 h allowed an ALA concentration of 2.8 mM to be achieved on the ventral side of excised human nail. Application for 48 h induced a concentration of 6.9 mM. Application time had no significant effect on the ALA concentration at mean depths of 2.375 mm in neonate porcine, with application times of 24, 48 and 72 h all producing concentrations of 0.1 mM. Incubation of Candida albicans and Trichophyton interdigitale with ALA concentrations of 10.0 mM for 30 min and 6 h, respectively, caused reductions in viability of 87% and 42%, respectively, following irradiation with red light. Incubation with 0.1 mM ALA for 30 min and 6 h, respectively, caused reductions in viability of 32% for Candida albicans and 6% for Trichophyton interdigitale, following irradiation. Drug penetration across nail may be improved using penetration enhancers, or by filing of the impenetrable dorsal surface of the nail. Moreover, iron chelators can be used to increase PpIX production for a given ALA dose. Therefore, with suitable modifications, ALA-PDT may prove to be a viable alternative in the treatment of onychomycosis.

Influence of 6-ketocholestanol on skin permeation of 5-aminolevulinic acid and evaluation of chemical stability.

The ability of 6-ketocholestanol to increase the skin permeation of the prodrug aminolevulinic acid (5-ALA) was investigated. 6-Ketocholestanol was incorporated together with 5-ALA in four different formulations. Preparations used were a liquid solution/suspension of 5-ALA in buffer, 5-ALA in phospholipid liposomal formulations with and without gelating agent, and finally, a complex cream formulation also including phospholipids. Standard diffusion experiments of 5-ALA using Franz-type diffusion cells and porcine skin were performed. Drug stability was monitored by analyzing the 5-ALA content in the different formulations over time and viewing the preparation for microbial contamination. The analysis of 5-ALA as a nonfluorescent probe was performed after chemical reaction, leading to a fluorescent derivative. The 5-ALA permeation through porcine skin was increased threefold by 6-ketocholestanol in the cream formulation. The chemical stability of 5-ALA in the tested formulations was in the range of about 33 to 72% after an observation period of 28 days. After that time point microbial stability was no longer evident for formulations 2 and 3. Formulation 1 could be observed until day 34, and only formulation 4 showed a microbial stability over the whole observation period of 42 days.

Influence of lipophilic counter-ions in combination with phloretin and 6-ketocholestanol on the skin permeation of 5-aminolevulinic acid.

In this study, the effect of lipophilic counter-ions on the permeation of 5-aminolevulinic acid (ALA) in combination with skin impregnation by phloretin and 6-ketocholestanol was evaluated. Standard in vitro permeation experiments with porcine skin were performed analysing the ALA content by HPLC and fluorescent detection after ALA derivatisation. The shake flask method in combination with a trinitrobenzensulfonic acid test for ALA analysis was performed to calculate the apparent partition coefficient (logP(Oct)). The permeation of ALA was enhanced by cetylpyridinium chloride and benzalkonium chloride at pH 7.0 and by sodium-1-octanesulfonic acid, sodium-1-heptanesulfonic acid and sodium-1-pentanesulfonic acid monohydrate at pH 4.0. Corresponding effects of these additives were observed on the partitioning of ALA. Pre-impregnation of porcine skin with phloretin and 6-ketocholestanol increased the ALA diffusion about 1.7-fold at pH 7.0. Moreover, this transport enhancement by 6-ketocholestanol was 3.5-fold higher when a combination of ALA and cetylpyridinium chloride was used as donor.

Detection of female lower genital tract dysplasia using orally administered 5-aminolevulinic acid induced protoporphyrin IX: a preliminary study.

OBJECTIVE: Previous studies have suggested that 5-aminolevulinic acid (ALA) may be used topically on the cervix to allow optical detection of cervical dysplasia, based on the fluorescence of protoporphyrin IX (PpIX) synthesized in situ from ALA. However, the uniformity of distribution of topically applied PpIX and the sensitivity and specificity of detection are not optimal. The current study was undertaken to demonstrate the feasibility of administering ALA by mouth (po) with the hypothesis that systemic administration might provide a more reliable diagnostic tool. METHODS: Oral ALA was administered to 14 patients with abnormal Pap smears in a dose- and time-intensity design. Institutional review board approval was obtained. A starting dose of 10 mg/kg of po ALA was administered and colposcopy was performed in 3 patients at 1 h, 3 patients at 2 h, 6 patients at 3 h, and 2 patients at 4 h. The study was written with the intent to increase the dose in 10 mg/kg increments if fluorescence was not detected; however, fluorescence was detected at the first dose level. Liver function tests were checked pre and post ALA and follow-up telephone calls were made regarding possible side effects. Both white and blue light colposcopy examinations were performed by two blinded clinicians and biopsies of all abnormal areas were performed. RESULTS: All patients tolerated po ALA well, with no systemic side effects. At the 10 mg/kg dose there was no reported nausea or photosensitivity. Optimal fluorescence was achieved at the 3-h time point, with quenching noted at the 4-h time point. Excellent absorption was documented with fluorescence of the lip demonstrated with Wood's lamp. In some cases fluorescence correlated with dysplasia on biopsy. CONCLUSION: 5-ALA given via the po route and at the dose and time period studied is well tolerated and affords fluorescence of the cervix. Future study is needed to demonstrate the successful identification of dysplastic lesions, with the ultimate goal of treating dysplasia of the lower genital tract with 5-ALA and light therapy.

Clinical pharmacokinetics of 5-aminolevulinic acid in healthy volunteers and patients at high risk for recurrent bladder cancer.

5-Aminolevulinic acid (ALA) is a precursor of protoporphyrin IX (PpIX) that is being evaluated for use in photodiagnosis and phototherapy of malignant and nonmalignant disorders. Previous clinical studies using topical, oral, and intravesical administration have been conducted in attempts to determine the optimal route of administration for ALA. The purpose of these studies was to examine the systemic pharmacokinetics and elimination of ALA, the bioavailability of ALA after oral and intravesical doses, and the factors that affect ALA concentrations in the bladder during intravesical treatment. The disposition of ALA was evaluated in six healthy volunteers receiving single intravenous and oral doses (100 mg) and eight patients at high risk for recurrent bladder cancer receiving an intravesical dose (1.328 g) of ALA. The mean (+/-S.D.) plasma area under the plasma concentration-time curve from time 0 to infinity of PpIX (0.20 +/- 0.11 microg small middle dot h/ml) after intravenous administration of ALA was not significantly different from that observed after oral administration of ALA (0.15 +/- 0.11 microg*h/ml; P = 0.49). ALA terminal half-life was approximately 45 min after intravenous or oral administration. The oral bioavailability of ALA was approximately 60%. After intravesical administration, urine production was largely responsible for decreases in ALA concentration in the bladder, with less than 1% being absorbed into the systemic circulation. In summary, oral and intravenous administration of ALA at these doses results in modest plasma levels of PpIX. Regional administration (i.e., intravesical) of ALA resulted in a significant pharmacokinetic advantage, with urinary bladder being exposed to concentrations approximately 20,000-fold higher than systemic circulation.

Heterogeneity of delta-aminolevulinic acid-induced protoporphyrin IX fluorescence in human glioma cells and leukemic lymphocytes.

Delta-aminolevulinic acid (ALA)-PDT efficacy is particularly dependent on the quality of protoporphyrin IX (PpIX)-induced synthesis. The purpose of this study was to determine the ability of cells from two human cancer types to synthesise PpIX after ALA administration. Biopsies of glioma cells have been obtained from patients with glioblastomas that have or have not been given ALA IV (ex vivo incubation). Peripheral blood lymphocytes, obtained from leukemic patients, have also been ALA-incubated in vitro. In glioma cells, fluorescence heterogeneity was extensive either in ALA infused patients or in ex vivo ALA incubated cells. Mean intensities after 3 h were 110 cts (range 0-340) and 1000 cts (range 0-3600). Similar results were found in leukemic lymphocytes where cell fluorescence varied from 0 to 480 cts with a percentage of fluorescent cells varying with time and from one patient to another. Furthermore, PpIX was not detectable in two patients with CLL. These observations suggest that a marked heterogeneity of ALA uptake and/or PpIX synthesis exists in a given human cancer cell population particularly after systemic administration. Improvements for ALA transformation into PpIX are strongly recommended to ensure the efficacy of ALA/PpIX-PDT.

A vehicle for photodynamic therapy of skin cancer: influence of dimethylsulphoxide on 5-aminolevulinic acid in vitro cutaneous permeation and in vivo protoporphyrin IX accumulation determined by confocal microscopy.

Topical application of 5-aminolevulinic acid (5-ALA) followed by light irradiation is a new concept of photodynamic therapy (PDT) of skin cancers. 5-ALA is a prodrug that can be converted by the heme biosynthetic pathway into protoporphyrin IX, an effective photosensitizer. In the present work we propose the enhancement of 5-ALA-induced protoporphyrin IX accumulation by dimethylsulphoxide (DMSO) and ethylenediamine-tetraacetic acid disodium salt (EDTA). The presence of 20% DMSO (w/w) in oil-in-water emulsions increased the in vitro permeation of 5-ALA through hairless mouse skin. In vivo studies demonstrated a significant increase in the amount of protoporphyrin IX extracted from healthy hairless mouse skin after 3 h treatment with an oil-in-water emulsion containing 10% 5-ALA (w/w), 3% EDTA (w/w) and 20% DMSO (w/w). By confocal scanning laser microscopy imaging, an observed increase in red fluorescence, at 476 nm excitation and emission detected longer than 590 nm, in skin that had received this treatment, was attributed to protoporphyrin IX accumulation. Although no effect of EDTA on short-term protoporphyrin IX accumulation in skin was detected, this chelator could protect 5-ALA from decomposition during prolonged topical administration. The results obtained indicate that association of 5-ALA, EDTA and 20% DMSO may enhance the delivery of 5-ALA to the skin in the topical PDT.

Tissue distribution and kinetics of endogenous porphyrins synthesized after topical application of ALA in different vehicles.

The use of 5-aminolaevulinic acid (ALA) is gaining increasing attention for photosensitization in photodynamic therapy of superficially localized tumours. The aim of this work was to determine the kinetics of porphyrin generation in tissues after topical application of ALA delivered in different vehicles on the skin overlying the tumour and normal skin of mice. Maximal accumulation was found in tumour 3 h after ALA application in both cream and lotion preparations. Normal and overlying tumour skin tissues showed different kinetic patterns, reflecting histological changes when the latter is invaded by tumour cells. Liver, kidney, spleen and blood porphyrins also raised from basal levels, showing that ALA and/or ALA-induced porphyrins reach all tissues after topical application. During the first 24 h of ALA topical application, precursors and porphyrins are excreted by both urine and faeces. ALA lotion applied on the skin overlying the tumour induced higher accumulation of tumoural porphyrins than cream, and lotion applied on normal skin appeared to be the most efficient upon inducing total body porphyrins. This work has demonstrated the great influence of the formulation of ALA vehicle on penetration through the skin. Knowledge of the kinetics of porphyrin generation after different conditions of ALA application is needed for the optimization of diagnosis and phototherapy in human tumours.