ALA Hexyl Ester Research Articles

Enlarging the Scope of 5-Aminolevulinic Acid-Mediated Photodiagnosis towards Breast Cancers

Today, most research on treating cancers targets one single cancer, often because of the very specific operation principle of the therapy. For instance, immunotherapies require the expression of a particular antigen, which might not be expressed in all cancers or in all patients. What about metastases? Combination therapies are promising but require treatment personalization and are an expensive approach that many health systems are not willing to pay for. Resection of cancerous tissues may be conducted beforehand. However, the precise location and removal of tumors are in most cases, hurdles that require margins to prevent recurrence. Herein, we further demonstrate the wide application of aminolevulinate-based photodynamic diagnosis and therapy toward breast cancers. By selecting four breast cancer cell lines that represent the main breast tumor subtypes, we investigated their ability to accumulate the fluorescent protoporphyrin IX upon treatment with the marketed 5-aminolevulinic acid hexyl ester (ALA-Hex) or our new and more stable derivative PSI-ALA-Hex. We found that all cell lines were able to accumulate PpIX under a few hours independent of their hormonal status with both treatments. Additionally, this accumulation was less dose-dependent with PSI-ALA-Hex and induced similar or higher fluorescence intensity than ALA-Hex in three out of four cell lines. The toxicity of the two molecules was not different up to 0.33 mM. However, PSI-ALA-Hex was more toxic at 1 mM, even though lower concentrations of PSI-ALA-Hex led to the same PpIX accumulation level. Additional illumination with blue light to induce cell death by generating reactive oxygen species was also considered. The treatments led to a dramatic death of the BT-474 cells under all conditions. In SK-BR-3 and MCF-7, ALA-Hex was also very efficient at all concentrations. However, increasing doses of PSI-ALA-Hex (0.33 and 1 mM) surprisingly led to a higher viability rate. In contrast, the triple-negative breast cancer cells MDA-MB-231 showed a higher death induction with higher concentrations of ALA-Hex or PSI-ALA-Hex. Derivatives of ALA seem promising as fluorescence-guided resection tools and may enable subsequent completion of cancer cell destruction by blue light irradiation.

Superficial bladder cancer diagnosis — the deliberate choice between fluorescent diagnosis and optical biopsy

Bladder carcinoma in situ (CIS) is a potentially invasive tumor whose early detection is a key step to ensuring the preservation of the bladder, reducing mortality, and improving the quality of patients’ life. The early diagnosis of bladder cancer requires a sensitive technique that can detect the lesion to determine its stage and grade. ALA induced-PpIX makes it possible to detect tumors with 90% sensitivity. ALA hexyl ester (hALA) increases the sensitivity to 95%. Macroscopic techniques require a histological biopsy to define the tumor invasiveness. Imaging with Fibered Optic Confocal Fluorescence Microscopy allows the optical sectioning of examined tissues providing images with subcellular resolution after labeling with adequate fluorescent dye chosen based on the sensitivity of the used device. Available fluorescent agents are compatible with used devices; however, their toxicity and mutagenesis studies are unsatisfactory. During imaging, an optical fiber is introduced into the bladder via the urethra and placed in contact with the bladder wall. The distinction between the different types of epithelial cells is based on the cell size, morphology, and signal intensity. Although not fully adopted for clinical application, the FOCM represents a real asset that reduces invasiveness and complements the fluorescence-based endoscopy.

Evaluation of antimicrobial photodynamic activities of 5-aminolevulinic acid derivatives.

Antibiotic resistance is increasing day by day, thereby increase the chances of more infections by resistant bacteria. In this situation, antimicrobial photodynamic therapy (aPDT) is gaining more attraction. To evaluate the antimicrobial effect of ALA derivatives using photodynamic therapy. In this study, we evaluated the aPDT effect of different derivatives of 5-ALA. In vivo and in vitro studies were performed to measure the antimicrobial activity. Different light doses and different concentrations of drugs were used to test anti-bacterial effect of drugs as well as to detect any physiological changes in animal model after the treatment. In vivo studies revealed that ALA-methyl ester, ALA-hexyl ester, and ALA-13A are potent photosensitizers. In vitro studies involved wound healing rate, body weight, and dietary intake were evaluated, and results showed that ALA, ALA-methyl ester, ALA-hexyl ester, and ALA-13A had good anti-bacterial effects, fast healing rate, and no effect on other physical parameters. Photodynamic therapy is increasingly used to treat different types of skin infections caused by bacterial strains. Our studies revealed that ALA-methyl ester, ALA-hexyl ester, and ALA-13A are promising photosensitizers for photodynamic therapy to inhibit the growth of resistant bacterial strains.

Clinical uses of 5-aminolaevulinic acid in photodynamic treatment and photodetection of cancer: A review.

ALA-mediated Photodynamic Therapy (ALA-PDT) is one of the most promising fields in Photodynamic therapy (PDT) research for cancer treatment. 5-aminolaevulinic acid (ALA) is the prodrug of the photosensitiser Protoporphyrin IX (PpIX). After ALA administration, cells generate PpIX through the haem biosynthetic pathway. Although the exact reasons for ALA/PpIX selectivity are unknown, it is believed that due to the special regulation of haem enzymes, PpIX is accumulated in the tumours. Both ALA and its derivative ALA Methyl ester, are mainly used in dermatology. Besides, ALA-PDT has been employed for palliative and even curative treatment of endoscopically accessible tumours. Lung, oesophagus, gastric and bladder carcinomas, and also oral premalignant lesions, gynaecological intraepithelial neoplasias and Barrett's oesophagus are the conditions mostly treated with ALA-PDT. However, due to the limited penetration of ALA and light, non-dermatologic uses of ALA-PDT have not moved beyond phase I clinical trials. On the other hand, ALA-induced PpIX fluorescence is successfully employed for the Photodynamic Diagnosis (PDD) or assistance in cytoreductive surgery (Fluorescence-guided Resection, FGR). ALA has been approved for the FGR of high-grade gliomas and ALA Hexyl ester, for fluorescence cystoscopy in the diagnosis of bladder cancer. ALA-FGR is currently applied in brain, bladder, lung, colon cancers, etc. and ALA-PDD for oral premalignancies, gynaecological intraepithelial lesions and peritoneal metastases, among others. Besides, PDT can be applied concomitantly in the same diagnostic procedure. This review aimed to analyse the state of the art of clinical uses of ALA in the areas of treatment and detection in the non-dermatologic oncology fields.

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.

Improving the efficiency of bladder cancer diagnostic cystoscopy with 5-ALA hexyl ester

This article presents the results of a clinical study that examined the diagnostic efficacy of fluorescent diagnostics (FD) of non-muscularinvasive bladder cancer using a photosensitizer of FD of malignant neoplasms – 5-aminolevulinic acid hexyl ester (5-ALA HE) compared with standard cystoscopy. The study involved 110 patients. The study began with intravesical administration of 50 ml of 0.2% solution of 5-ALA HE, the exposure time was 1 hour, after which the drug was removed from the organ. During the next hour, the mucous membranes were examined in two cystoscopy modes, followed by a standard transurethral resection of all urothelium sites with suspicion for tumor lesion based on white light and visible red fluorescence, and a control blind biopsy from the visually unchanged and non-fluorescent mucous tissue in each patient. The results of the study indicate the high effectiveness of the developed FD methodology with 5-ALA HE in detecting non-muscularinvasive bladder cancer during intravesical administration of the drug, due to selective accumulation of hexasens-induced PPIX in the tumor tissue compared with healthy mucosa. Compared with the results of standard cystoscopy, fluorescence diagnostics significantly increased diagnostic sensitivity by 24.4% (from 75.1% to 99.5%), diagnostic accuracy – by 15.8% (from 82.4% to 98.2%) and a negative predictive value – by 33.2% (from 65.8% to 99%) (p≤0.05). Additionally, a total of 37 (33.6%) patients was found to have 63 foci of fluorescence with a diameter of 2.5 to 3.0 mm. 59 of these were morphologically confirmed to contain cancer cells.

Design, synthesis and in vitro evaluation of β-glucuronidase-sensitive prodrug of 5-aminolevulinic acid for photodiagnosis of breast cancer cells

Treatment of cancer cells by clinically approved hexyl ester of 5-aminolevulinic acid (ALA-Hex) induces accumulation of fluorescent porphyrins in tumors. This allows fluorescence photodiagnosis (PD) of bladder cancer by blue light illumination. However, PD of other cancers is hampered by acute toxicity of the compound limiting its use to local applications. We have designed and synthesized a new prodrug of ALA-Hex that tackles the stability-activity paradox of amino-modified 5-ALA prodrugs. The glucuronide prodrug Glu-ALA-Hex demonstrates excellent stability under physiological conditions and activation in the presence of the target enzyme. β-glucuronidase-triggered release of 5-ALA is programmed to yield fluorescence in tumor environment with elevated β-glucuronidase activity, a characteristic of many solid tumors. Glu-ALA-Hex produces similar levels of fluorescence as ALA-Hex in breast cancer MCF7 cells in vitro but with much lower non-specific cell toxicity.

FLUORESCENCE DIAGNOSIS OF BLADDER CANCER WITH AGENT HEXASENS – THE RESULTS OF MULTICENTER TRIAL

The results of multicenter trial for efficacy of fluorescence diagnosis for bladder cancer with agent hexasens (SSC Scientific Research Institute Organic Intermediates and Dyes, Russia) based on hexyl ester of 5-aminolevulinic acid compared with routine cystoscopy are represented in the article. The study included 124 patients. All patients had intravesical instillation of 50 ml of 0,2% hexasens. The exposure time accounted for 1-2 h. After removal of the drug from bladder standard cystoscopy followed by fluorescence diagnosis was performed in every patient. During the local fluorescence spectroscopy the intravesical instillation of the agent hexasens was shown to induce selective accumulation of photoactive protoporphyrin IX in tumor cells comparing with intact bladder mucosa. During 1 h after intravesical instillation of hexasens solution the level of fluorescence of hexasens-induced protoporphyrin IX in the tumor was higher than that in surrounding intact mucosa at average in 5.8 fold. As compared with results of standard cystoscopy, fluorescence diagnosis improved the sensitivity of the method by 24.4% (from 75.6% to 100%), the accuracy – by 15.2% (from 83.3% to 98.5%) and negative predictive value – by 33.5% (from 66.5% to 100%). Fluorescence diagnosis gave an opportunity to detect additional tumor foci, which were invisible in white light, in 27.4% of patients. For fluorescence diagnosis in 4.0% of patients false-positive fluorescence of bladder mucosa was detected that was probably due to inflammation. None of patients received active dose of the drug hexasens had adverse reactions, changes in general well-being and blood and urine parameters. According to the results of the study fluorescence diagnosis with hexasens for bladder cancer is recommended for use in clinical practice.

Fluorescent Diagnosis of Bladder Cancer by Hexasens as a Drug

This study was performed as a clinical trial conducted in Russia concerning the diagnostic efficacy of Hexasens drug. The photosensitizer based on hexyl ester of 5-aminolevulinic acid was used for the fluorescence diagnosis of bladder cancer. The study was conducted in 2015-2016 on the basis of the City Clinical Hospital No. 40 (Moscow, Russia). 53 patients were involved into study and were diagnosed as bladder cancer in T1N0M0 stage. Fluorescence diagnosis was carried out for 1 hour after the intravesical induction of 0.2% solution (50 mL or 100 mg) of Hexasens drug (FSUE SSC NIOPIK, Russia). The results of the fluorescence diagnosis were compared with the results of the standard routine cystoscopy. The found results showed that the performance of fluorescent diagnostics (FD) led to improve diagnostic sensitivity as 13.2% compared to standard cystoscopy (from 86.8% to 100%); the diagnosis accuracy increased as 8.8% (from 91.2% to 100%) and a negative predictive value increased as 20.9% (from 79.1% to 100%). During the carrying out of fluorescence diagnosis, 8 patients (15.0%) out of 53 ones also revealed 14 fluorescing foci that not identified in white light, in which the tumorous process in them was confirmed during morphological study. The induction of Hexasens solution into a bladder and the carrying out of a fluorescent diagnosis was not accompanied by the development of any adverse reactions. Patients showed a subjective discomfort only during a prolonged exposure of the drug solution in a bladder (up to 2 hours). The developed diagnosis methods is recommended for the applying in advanced clinical diagnosis.

Organization of Clinical Trials of Photosensitizer based on 5-Aminolevulinic Acid Hexyl Ester

Background: This paper deals with the statistical and methodological study conducted in order to assess the possibility of combining in one study, Phase 2 and 3 clinical trials of the Russian drug Hexasense based on 5-aminolevulinic acid hexyl ester. Methods: Hexasense drug is intended for fluorescence diagnosis of bladder cancer (the route of administration - intravesical instillation of solution). The clinical trial involved a comparative study of the effectiveness of diagnostic fluorescence depending on the exposure time of the drug solution in the bladder (1 and 2 hours). This is due to the fact that the time interval of 2 hours checked on the research Phase 1 used to cause inconvenience to patients. Results: After selecting the optimal diagnostic mode (a task of Phase 2) it is necessary to evaluate the effectiveness of the selected mode as compared to standard white-light cystoscopy (a task of Phase 3). Comparison of the effectiveness of two diagnostic fluorescence diagnostic modes with different exposure time of solution of 1 and 2 hours has been planned. At the second stage, the selected fluorescence diagnosis mode will be compared with the results of a standard white-light cystoscopy obtained in the same group of patients. A difference of 10% will be taken for significant differences in values of sensitivity and specificity. The statistical analysis showed that the required number of patients at a significance level of 2.5%, power of 80%, and for minimum difference in proportions of 10% subject to the correction of 10% for the dropout and unassessed patients would be 134 people (two groups of 67 patients). In this case, the same sample of patients allows comparing reliably the effectiveness of two diagnoses with the correction for multiplicity of comparison. Application: This will help to minimize the number of patients involved in the study and not reduce the accuracy of the results at the same time. The Ministry of Health of the Russian Federation authorized the clinical trial of the drug Hexasense with Phases 2 and 3 combined.

Intermediate Filament Proteins of Lamin A/C and Cytokeratin 18 are involved in Apoptotic Induction by Photodynamic Therapy with Hexaminolevulinate in Human Colon Carcinoma Cells

Hexaminolevulinate (HAL), a hexylester of 5-aminolevulinic acid, is a precursor of the photosensitizer protoporphyrin IX (PpIX) and clinically used for photodynamic therapy (PDT) of cancer, an established modality with a light-activated drug. However, the mechanism involved in the killing cancer cells is still not fully understood. Our previous report (Cancer Lett., 2013; 339: 25-32) has shown a crucial role of caspase-6-mediated cleavage of lamin A/C, a member of type-V intermediate filaments (IFs), in the HAL-PDT-induced apoptosis in human B-cell lymphoma cells. Lamin A/C is present in all cell types; while cytokeratin 18, a major component of type-I IFs, is expressed only in the epithelial cells and carcinoma. This study has focused upon the roles of the two IF proteins, lamin A/C and cytokeratin 18 in the HAL-PDT-mediated apoptotic induction in the human colon carcinoma COLO 205 and HCC2998 cell lines. HAL-PDT-induce apoptosis was confirmed by fluorescence microscopy, electron microscopy and M30 CytoDeath™ ELISA in both cell lines. Fluorescence microscopy, immunoblots and immunocytochemistry showed that both lamin A/C and cytokeratin 18 were involved in the apoptotic induction and the specific caspase-6 inhibitor stopped not only the cleavages of the two IF proteins, but also the apoptotic induction. Knockdown of both lamin A/C and cytokeratin 18 by siRNAs induced the cells to be apoptotic, further supporting the hypothesis that the disruption of both lamin A/C and cytokeratin 18 is required in the apoptotic induction by HAL-PDT in the human carcinoma cells.

Real-time analysis of endogenous protoporphyrin IX fluorescence from δ-aminolevulinic acid and its derivatives reveals distinct time- and dose-dependent characteristicsin vitro

Photodynamic therapy (PDT) and photodiagnosis based on the intracellular production of the photosensitizer protoporphyrin IX (PPIX) by administration of its metabolic precursor -aminolevulinic acid (ALA) achieved their breakthrough upon the clinical approval of MAL (ALA methyl ester) and HAL (ALA hexyl ester). For newly developed ALA derivatives or application in new tumor types, in vitro determination of PPIX formation involves multiparametric experiments covering variable pro-drug concentrations, medium composition, time points of analysis, and cell type(s). This study uses a fluorescence microplate reader with a built-in temperature and atmosphere control to investigate the high-resolution long-term kinetics (72 h) of cellular PPIX fueled by administration of either ALA, MAL, or HAL for each 10 different concentrations. For simultaneous proliferation correction, A431 cells were stably transfected with green fluorescent protein. The results indicate that the peak PPIX level is a function of both, incubation concentration and period: maximal PPIX is generated with 1 to 2-mM ALA/MAL or 0.125-mM HAL; also, the PPIX peak shifts to longer incubation periods with increasing pro-drug concentrations. The results underline the need for detailed temporal analysis of PPIX formation to optimize ALA (derivative)-based PDT or photodiagnosis and highlight the value of environment-controlled microplate readers for automated in vitro analysis.

Lamin A/C cleavage by caspase-6 activation is crucial for apoptotic induction by photodynamic therapy with hexaminolevulinate in human B-cell lymphoma cells

Photodynamic therapy (PDT) with a light-activated drug is an approved modality for cancer treatment. Hexaminolevulinate (HAL), a hexylester of 5-aminolevulinic acid as the photosensitising protoporphyrin IX (PpIX) precursor, is clinically used for both PDT and photodetection. Our previous studies have shown that HAL–PDT can effectively induce apoptosis in several human blood malignant cell lines. However, the mechanisms involved in the apoptotic induction are still not fully elucidated. In this study we have focused on the role of cellular lamin A/C in the apoptotic induction. HAL–PDT-mediated apoptosis was confirmed by various techniques including fluorescence microscopy and electron microscopy in both human B-cell lymphoma Ramos and Daudi cell lines. The lamin A/C, together with caspases-6 and -3, was cleaved during the apoptosis. Western blots, immunocytochemistry, fluorescence microscopy and electron microscopy demonstrated that the specific caspase-6inhibitor abrogated the HAL-PDT-mediated cleavages of both caspase-6 and lamin A/C and subsequent apoptosis in these two cell lines, suggesting that the cleavage of lamin A/C by the caspase-6 activation is crucial for such apoptotic induction.

Matrix mediated fluorescence enhancement — why nanoparticles and PPIX make a friendly couple

The hexyl ester of 5-aminolevulinic acid (HAL) is an efficient inducer of the endogenous photosensitizer protoporphyrin IX (PPIX) in neoplastic tissue and approved for the fluorescence photodetection of bladder cancer. It is, however, limited to topical application due to unsuitable pharmacokinetic and pharmacodynamic properties following systemic administration. In this study we sought to prepare HAL laden polymeric nanoparticles made from polylactic acid (PLA) as possible means to improve the systemic bioavailability of HAL. Nanoparticles were prepared by nanoprecipitation. Particle size and morphology of freshly prepared and dehydrated nanoparticles were assessed and the ability to generate PPIX fluorescence was tested on a T24 human bladder carcinoma cell line. Fluorescence intensities of cells incubated with nanoencapsulated HAL were significantly higher than in presence of free HAL. Interestingly, the effect was — at least partly — provoked by the direct interaction of exogenously induced PPIX with the nanoparticles.

Hyperthermia enhances the antitumor effect of photodynamic therapy with ALA hexyl ester in a squamous cell carcinoma tumor model

Photodynamic therapy (PDT) using 5-aminolevulinic acid is considered to be ineffective in the treatment of tumors with progression to the deep layer. Therefore, for such tumors, a method is required which can enhance the effectiveness of this therapy. We examined the anti tumor effect of the combination of PDT with 5-aminolevulinic acid hexyl ester (hALA) and hyperthermia (HT) in a squamous cell carcinoma (SCC) tumor model. A tumor model was prepared by subcutaneously implanting SCC into nude mice, and treated with HT, PDT with hALA (hALA-PDT), or hALA-PDT combined with HT (PDT+HT). The treatment was performed by remodeled near infra-red irradiator which allows the generation of two types of rays for PDT and HT. With HT, the tumor was irradiated for raising the temperature with a light dose of 437.5 J/cm(2). With hALA-PDT, the tumor treated with 250 mg/kg hALA was irradiated with a light dose of 50 J/cm(2). With PDT+HT, the tumor was treated as for hALA-PDT except that the temperature was raised during irradiation with a light dose of 437.5 J/cm(2) (including light dose of 50 J/cm(2) for PDT). The tumor growth rates on Day 12 were 97.10% in HT, 67.55% in hALA-PDT and 33.90% in PDT+HT, and PDT+HT showed significant inhibitory effects on tumor growth, although the anti-tumoral effects of HT and hALA-PDT were not seen. hALA-PDT combined with HT demonstrated a significant inhibitory effect on the tumor growth of squamous cell carcinoma showing a progression in the deep layer. This suggests that this therapy is useful for tumors showing progression to the deep layer, which hALA-PDT alone is generally ineffective in treating.

Development of Novel Oral Formulations Prepared via Hot Melt Extrusion for Targeted Delivery of Photosensitizer to the Colon

Colon-residing bacteria, such as vancomycin-resistant Enterococcus faecalis and Bacteroides fragilis, can cause a range of serious clinical infections. Photodynamic antimicrobial chemotherapy (PACT) may be a novel treatment option for these multidrug resistant organisms. The aim of this study was to formulate a Eudragit®-based drug delivery system, via hot melt extrusion (HME), for targeting colonic release of photosensitizer. The susceptibility of E. faecalis and B. fragilis to PACT mediated by methylene blue (MB), meso-tetra(N-methyl-4-pyridyl)porphine tetra-tosylate (TMP), or 5-aminolevulinic acid hexyl-ester (h-ALA) was determined, with tetrachlorodecaoxide (TCDO), an oxygen-releasing compound, added in some studies. Results show that, for MB, an average of 30% of the total drug load was released over a 6-h period. For TMP and h-ALA, these values were 50% and 16% respectively. No drug was released in the acidic media. Levels of E. faecalis and B. fragilis were reduced by up to 4.67 and 7.73 logs, respectively, on PACT exposure under anaerobic conditions, with increased kill associated with TCDO. With these formulations, photosensitizer release could potentially be targeted to the colon, and colon-residing pathogens killed by PACT. TCDO could be used in vivo to generate oxygen, which could significantly impact on the success of PACT in the clinic.

Synergistic Increase in Osteosarcoma Cell Sensitivity to Photodynamic Therapy with Aminolevulinic Acid Hexyl Ester in the Presence of Hyperthermia

We observed that two osteosarcoma cell lines from the same tumor displayed marked differences in their sensitivities to photodynamic therapy (PDT) with aminolevulinic acid hexyl ester (hALA-PDT). We investigated why these two closely related lines had different hALA-PDT sensitivities and whether the PDT phototoxicity of the less sensitive cell line could be increased by a simultaneous application of hyperthermia (HT). Flow cytometry was used to evaluate the intracellular accumulation of protoporphyrin IX (PpIX), a metabolic product of aminolevulinic acid, in two human mandibular osteosarcoma cell lines (HOSM-1 and HOSM-2) treated with HT, hALA-PDT, or hALA-PDT combined with HT (PDT + HT). With hALA-PDT, cells treated with 0.2 mM hALA were irradiated with a light dose of 10-80 J/cm(2) from a near-infrared irradiator. With PDT + HT, the cells were treated as for hALA-PDT except that the temperature was raised to 43.5 degrees C during irradiation. At 6 h after hALA treatment, HOSM-2 cells carried about 1.53-fold more PpIX than HOSM-1 cells. With hALA-PDT, the survival rate for HOSM-1 cells treated with 80 J/cm(2) irradiation was 35.7%, while that for HOSM-2 cells treated with 40-80 J/cm(2) was below 12%. With PDT + HT, the survival rate for HOSM-1 and HOSM-2 cells treated with 80 J/cm(2) irradiation was 14.1% and 10.7%, respectively. A combination therapy comprising hALA-PDT + HT treatment may be very useful for the treatment of tumors containing cells that are insensitive to hALA-PDT, such as the HOSM-1 cell line described in this study.

Porphyrin synthesis from aminolevulinic acid esters in endothelial cells and its role in photodynamic therapy

Photodynamic therapy (PDT) may cause tumour cell destruction by direct toxicity or by inducing microcirculatory shutdown. Protoporphyrin IX generated from 5-aminolevulinic acid (ALA) has been widely used as an endogenous photosensitiser in PDT. However, the hydrophilic nature of the ALA molecule limits its penetration through the stratum corneum of the skin and cell membranes and thus, ALA alkyl-esters have been developed to improve ALA permeation. The aim of this work was to study Protoporphyrin IX synthesis from ALA and its derivatives ALA methyl ester (Me-ALA) and ALA hexyl ester (He-ALA) in the microvascular endothelial cell line HMEC-1 derived from normal skin, and to evaluate their response to PDT. We found that lower light doses are required to photosensitise HMEC-1 endothelial cells than to photosensitise PAM212 transformed keratinocytes, showing some possible selectivity of ALA-PDT for vascularisation in skin. Employed at concentrations leading to equal Protoporphyrin IX synthesis, ALA, He-ALA and Me-ALA presented the same efficacy of HMEC-1 photosensitisation. However, He-ALA was a promising compound for the use in the enhancement of Protoporphyrin IX in HMEC-1 cells employed at low concentrations at both short and long time exposures whereas Me-ALA should be employed at high concentrations and longer time periods in order to surpass the Protoporphyrin IX levels obtained with ALA. The advantage of Me-ALA over ALA was based on its lower dark toxicity. This is the first work to report vascular cell photosensitisation employing alkyl-esters of ALA, and we demonstrated that these derivatives could exert the same effect as ALA and under certain conditions enhance photosensitisation of vasculature.

Analysis of differential PDT effect in rat bladder tumor models according to concentrations of intravesical hexyl-aminolevulinate

The hexylester of 5-aminolevulinic acid (HAL) is a very efficient precursor of the photosensitizer protoporphyrin IX (PpIX) for photodynamic therapy (PDT). Our previous study, performed in rat orthotopic bladder tumors, indicated an opposite effect of HAL/PpIX-PDT according to HAL concentration. The present study investigated possible reasons for this differential effect considering the impact of extracted amounts of PpIX in normal and tumor bearing bladders along with PpIX distribution in distinctive histopathological layers. High performance liquid chromatography (HPLC) analysis of tumor and normal bladder tissues after 8 mM and 16 mM HAL instillation showed that PpIX was the main porphyrin species. The PpIX production in tumor bladders instilled with 8 mM HAL was significantly higher than after 16 mM HAL. Fluorescence confocal microscopy demonstrated a punctuate bright fluorescence pattern in tumor zones of bladders instilled with 8 mM HAL, whereas a more diffuse cytoplasmatic fluorescence distribution was observed after 16 mM HAL instillation. Immunofluorescence staining together with transmission electron microscopy showed severe mitochondrial damage in tumor zones of bladders treated with 8 mM HAL/PpIX PDT, with intact mitochondria in tumor zones of bladders treated with 16 mM HAL/PpIX PDT. We conclude that the differential response to HAL/PpIX PDT in function of HAL concentrations could be attributed to diminished PpIX synthesis and differential intracellular localisation of PpIX. Mitochondria were shown to be the critical photodamaged sites of HAL/PpIX PDT and as such tissue sensitivity to treatment can be estimated through investigation of intracellular PpIX distribution.

Effects of photoactivated 5-aminolevulinic acid hexyl ester on MDR1 over-expressing human uterine sarcoma cells

The role of multi-drug resistance ( MDR1) and its product, P-glycoprotein (P-gp) on 5-aminolevulinic acid hexyl ester (Hexyl-ALA) mediated phototoxicity was determined with human uterine sarcoma cells, MES-SA control and MDR1 expressing MES-SA-Dx5. MDR1 expression reduced intracellular levels of the Hexyl-ALA metabolite, protoporphyrin IX (PpIX) to a limited degree and could be reversed with a P-gp inhibitor, verapamil. P-gp expression also reduced Hexyl-ALA photosensitivity. More importantly, photoactivated Hexyl-ALA reduced at the mRNA and protein levels without altering housekeeping GAPDH mRNA. These findings suggest that Hexyl-ALA could be used to selectively reduce P-gp expression in overcoming resistance to chemotherapy agents such as doxorubicin and paclitaxel.