Aminolevulinic acid derivatives and liposome delivery as strategies for improving 5-aminolevulinic acid-mediated photodynamic therapy

Photodynamic Therapy employing 5-aminolevulinic acid (ALA) as a precursor of the photosensitizer Protoporphyrin IX has become a promising approach to treat superficial cancers. However, the hydrophilic nature of the ALA molecule somewhat limits the penetration through the skin as well as all cell membranes. Different attempts are currently under investigation to enhance ALA penetration, such as the development of new synthetic and more lipophilic molecules derived from ALA and the incorporation of ALA into lipophilic vehicles such as liposomes. Among the new synthesized molecules, we can find ALA esters, ALA aminoacid derivatives and ALA dendrimers. In general, there is consensus that the promising results obtained in vitro with ALA esters cannot be reproduced in vivo. However, ALA methyl ester (1) has been widely used for treatment of skin malignancies and ALA hexyl ester (15) proved to be more powerful than ALA in bladder imaging. ALA aminoacid derivatives have been designed to use specific cellular aminopeptidases to targeting tumors, and it was shown that they can be metabolized to ALA with some specificity.     

Topical delivery of 5-aminolevulinic acid-encapsulated ethosomes in a hyperproliferative skin animal model using the CLSM technique to evaluate the penetration behavior

Psoriasis, an inflammatory skin disease, exhibits recurring itching, soreness, and cracked and bleeding skin. Currently, the topical delivery of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT) is an optional treatment for psoriasis which provides long-term therapeutic effects, is non-toxic and enjoys better compliance with patients. However, the precursor of ALA is hydrophilic, and thus its ability to penetrate the skin is limited. Also, little research has provided a platform to investigate the penetration behavior in disordered skin. We employed a highly potent ethosomal carrier (phosphatidylethanolamine; PE) to investigate the penetration behavior of ALA and the recovery of skin in a hyperproliferative murine model. We found that the application of ethosomes produced a significant increase in cumulative amounts of 5-26-fold in normal and hyperproliferative murine skin samples when compared to an ALA aqueous solution; and the ALA aqueous solution appeared less precise in terms of the penetration mode in hyperproliferative murine skin. After the ethosomes had been applied, the protoporphyrin IX (PpIX) intensity increased about 3.64-fold compared with that of the ALA aqueous solution, and the penetration depth reached 30-80 μm. The results demonstrated that the ethosomal carrier significantly improved the delivery of ALA and the formation of PpIX in both normal and hyperproliferative murine skin samples, and the expression level of tumor necrosis factor (TNF)-α was reduced after the ALA-ethosomes were applied to treat hyperproliferative murine skin. Furthermore, the results of present study encourage more investigations on the mechanism of the interaction with ethosomes and hyperproliferative murine skin.     

Topical delivery of 5-aminolevulinic acid-encapsulated ethosomes in a hyperproliferative skin animal model using the CLSM technique to evaluate the penetration behavior

Psoriasis, an inflammatory skin disease, exhibits recurring itching, soreness, and cracked and bleeding skin. Currently, the topical delivery of 5-aminolevulinic acid-photodynamic therapy (ALA–PDT) is an optional treatment for psoriasis which provides long-term therapeutic effects, is non-toxic and enjoys better compliance with patients. However, the precursor of ALA is hydrophilic, and thus its ability to penetrate the skin is limited. Also, little research has provided a platform to investigate the penetration behavior in disordered skin. We employed a highly potent ethosomal carrier (phosphatidylethanolamine; PE) to investigate the penetration behavior of ALA and the recovery of skin in a hyperproliferative murine model. We found that the application of ethosomes produced a significant increase in cumulative amounts of 5–26-fold in normal and hyperproliferative murine skin samples when compared to an ALA aqueous solution; and the ALA aqueous solution appeared less precise in terms of the penetration mode in hyperproliferative murine skin. After the ethosomes had been applied, the protoporphyrin IX (PpIX) intensity increased about 3.64-fold compared with that of the ALA aqueous solution, and the penetration depth reached 30–80 μm. The results demonstrated that the ethosomal carrier significantly improved the delivery of ALA and the formation of PpIX in both normal and hyperproliferative murine skin samples, and the expression level of tumor necrosis factor (TNF)-α was reduced after the ALA–ethosomes were applied to treat hyperproliferative murine skin. Furthermore, the results of present study encourage more investigations on the mechanism of the interaction with ethosomes and hyperproliferative murine skin.     

Rational design of 5-aminolevulinic acid derivatives aimed at improving photodynamic therapy

5-aminolevulinic acid (ALA) is the first intermediate in heme biosynthesis and is therefore a precursor of protoporphyrin IX (PpIX). PpIX is used as an endogenous photosensitizer in photodynamic therapy (PDT). Several chemical modifications have been made, both on the amino and carboxyl groups of ALA to induce higher PpIX production and photosensitisation. Esterification of ALA with aliphatic lineal and cyclic alcohols was found to reduce the amount of ALA required for photosensitization. Esterification by aliphatic alcohols with carbohydrate chains equal or lower than C4 leads to porphyrin accumulation lower than ALA, whereas equal or longer than C6 chains leads to greater synthesis of porphyrin. A branch point in the alcohol located next to the site of ester cleavage limits access of the esters to the esterase active site, resulting in lower PpIX production. ALA esters of the polyethylenglycol family can induce high levels of PpIX, with some selectivity for endothelial cells toward tumor cells. On the basis of the differential expression of some aminopeptidases in tumor vasculature when compared to normal vasculature, some ALA-pseudopeptides were synthesized. In a rational design of ALA derivatives, the transport mechanism of these aminoacids into the cell is central. Due to the similar characteristics between ALA and GABA transport, a novel approach for designing new ALA derivatives which could penetrate more easily into tumoral cells, would be to take into account the structures of the inhibitors of GABA transport.     

Development of a keratoacanthoma after topical photodynamic therapy with 5-aminolevulinic acid

Since its FDA approval in 1999, photodynamic therapy (PDT) with topical 5-aminolevulinic acid has become an increasingly popular modality for the treatment of actinic keratosis (AKs). It is hoped that in addition to improving clinical signs and symptoms of AKs, PDT might prevent the development of skin cancer. We present a case of a patient developing a keratoacanthoma immediately following PDT for AKs.     

5-氨基-3-乙酰丙酸光动力疗法在肿瘤诊断和治疗中的应用进展

5-氨基-3-乙酰丙酸（5-aminolevulinic acid,5-ALA）是一种内源性的光敏剂,肿瘤细胞和其他恶性细胞可以选择性地吸收外源性5-ALA,使细胞内积聚过量的原卟啉。原卟啉在一定波长的光照射下,发生化学反应,使肿瘤细胞或其他增生活跃的细胞坏死、凋亡,这一过程被称为氨基-3-乙酰丙酸-光动力疗法（aminolevulinic acid-photodynamic therapy,ALA-PDT）。本文对ALA-PDT近年来在肿瘤的诊断和治疗中的应用进展作一综述。     

乙醇脂质体透皮作用的研究与发展

目的：介绍乙醇脂质体透皮作用的研究与发展。方法：综合分析乙醇脂质体的组成及一般特性、制备及乙醇脂质体载药系统透皮给药的药剂学促透机制、最新的基础实验和研究进展。结果：作为各种药物的载体，乙醇脂质体具有其良好的药物包裹率和显著的促进药物透皮吸收特性。结论：乙醇脂质体新剂型透皮给药具有广阔的应用前景。     

5-氨基酮戊酸光动力疗法治疗寻常痤疮临床应用进展

寻常痤疮(acne vulgaris)是一种毛囊皮脂腺单元的慢性炎症性皮肤病,好发于青年人群,病因及发病机制复杂.临床上多根据皮损性质将痤疮分为轻、中、重度分级治疗.近年来,5-氨基酮戊酸光动力疗法(5-aminolevulinic acid photodynamic therapy,ALA-PDT)被广泛应用于中、重...     

Treatment of actinic cheilitis by photodynamic therapy with 5-aminolevulinic acid and blue light activation

Actinic cheilitis (AC), a common disorder of the lower lip, should be treated early to prevent progression to invasive squamous cell carcinoma. This study evaluated the safety and efficacy of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) activated by blue light for the treatment of AC. Fifteen patients with clinically evident or biopsy-proven AC received two treatments with ALA PDT with blue light activation. Treatments were spaced three to five weeks apart. Most patients achieved 65% to 75% clearance three to five weeks after the first treatment and all achieved more than 75% clearance one month after the second treatment. Three patients achieved complete clearance. Pain and burning during irradiation were absent or mild. All patients said they would repeat the procedure. ALA PDT with 417 nm blue light is a promising option for the treatment of AC of the lower lip.     

5-氨基酮戊酸光动力疗法联合手术治疗巨大尖锐湿疣的疗效观察

目的观察5-氨基酮戊酸光动力疗法(ALA-PDT)联合手术治疗巨大尖锐湿疣的临床疗效。方法对22例外生殖器、肛周、肛管、会阴部、腹股沟巨大尖锐湿疣患者进行ALA-PDT联合手术治疗,其中男性15例,女性7例。通过手术切削的方法将疣体突出皮面的部分去除,之后病变部位及其周围至少1 cm的范围内使用ALA-PDT治疗,7~10 d治疗1次,共治疗3~5次,对每次治疗后的不良反应、疗效等作出评价,并进行6~12个月的随访,对其预后及复发情况作出评判。结果 22例巨大型尖锐湿疣患者经手术联合ALA-PDT治疗3~5次后,20例患者疣体完全消失,治愈率91%;术后局部均会出现红肿、疼痛、渗出、少量渗血,病变靠近尿道部位治疗后1~4 d内排尿时可出现轻微的烧灼感,无需特殊处理,可自行消退;其中4例患者出现局部明显红肿、糜烂、少量脓性渗出。但是所有患者均未发生严重感染、溃疡、瘢痕和尿道、肛管狭窄等不良反应;随访6~12个月,原位复发2例,复发率为9%。结论 ALA-PDT疗法联合手术是治疗巨大型尖锐湿疣的新疗法,它具有安全、有效、痛苦小、不良反应小、术中出血少、术后无需特殊护理、对局部结构及功能影响小等特点,同时与传统治疗方法相比,可明显提高治愈率,降低复发率。     

NF-kappaB inhibition improves the sensitivity of human glioblastoma cells to 5-aminolevulinic acid-based photodynamic therapy

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most cases, this type of tumor is characterized by a constitutive activation of the nuclear factor-kappaB (NF-κB). This factor is known to be a key regulator of various physiological processes such as inflammation, immune response, cell growth or apoptosis. In the present study, we explored the role of NF-κB activation in the sensitivity of human glioblastoma cells to a treatment by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). 5-ALA is a physiological compound widely used in PDT as well as in tumor photodetection (PDD). Our results show that inhibition of NF-κB improves glioblastoma cell death in response to 5-ALA-PDT. We then studied the molecular mechanisms underlying the cell death induced by PDT combined or not with NF-κB inhibition. We found that apoptosis was induced by PDT but in an incomplete manner and that, unexpectedly, NF-κB inhibition reduced its level. Oppositely PDT mainly induces necrosis in glioblastoma cells and NF-κB is found to have anti-necrotic functions in this context. The autophagic flux was also enhanced as a result of 5-ALA-PDT and we demonstrate that stimulation of autophagy acts as a pro-survival mechanism confering protection against PDT-mediated necrosis. These data point out that 5-ALA-PDT has an interesting potential as a mean to treat glioblastoma and that inhibition of NF-κB renders glioblastoma cells more sensitive to the treatment.     

5-氨基酮戊酸对人舌麟癌Tea8113细胞株体外光动力杀伤作用的基础研究

目的初步研究体外5-氨基酮戊酸介导的光动力疗法对人舌鳞癌Tea8113细胞的杀伤效应。方法体外培养Tca8113细胞,以5-氨基酮戊酸为光敏剂,半导体激光治疗仪给予光动力疗法,采用MTr法检测光敏剂不同孵育时间、不同光敏剂浓度对Tea8113细胞抑制率的影响。结果5-AIA—PDT作用后,Tea8113细胞生长受到抑制,孵育时间和浓度效应关系显著（P〈0．05）。药物最佳作用浓度为1mmoL／l,最佳孵育时间为6h。结论5-氨基酮戊酸一光动力疗法能有效杀伤Tea8113细胞,光敏剂孵育时间、光敏剂浓度是影响疗效的重要因素。     

5-氨基酮戊酸光动力疗法联合电灼术治疗肛周尖锐湿疣疗效观察

目的 评价5-氨基酮戊酸光动力疗法（ALA-PDT）联合电灼术治疗肛周尖锐湿疣的疗效和复发率.方法 将67例肛周尖锐湿疣患者按门诊就诊顺序随机分为三组,联合治疗组22例用电灼术去除显性疣体后立即进行ALA-PDT治疗,每周1次,连续治疗3～4次;光动力组22例,用ALA-PDT治疗,每周1次,1个月为1个疗程;电灼术组23例,用传统方法电灼术对所有皮损逐个进行电灼气化治疗,每周1次的分批治疗,1个月为1个疗程.末次治疗后随访6个月判定疗效及观察复发率.结果 联合治疗组痊愈率为90.9％（20/22）,复发率为9.1％ （2/22）;光动力组痊愈率为54.5％ （12/22）,复发率为22.7％ （5/22）;电灼术组痊愈率为39.1％ （9/23）,复发率为43.5％ （10/23）.联合治疗组的痊愈率和复发率与电灼术治疗组差异有统计学意义（P =0.000;P =0.017）.结论 ALA-PDT治疗肛周尖锐湿疣治愈率高,复发率低,副作用小.     

Self-adhesive thin films for topical delivery of 5-aminolevulinic acid.

Self-adhesive thin-films have been developed as a topical delivery system for 5-aminolevulinic acid (ALA). The thin films are suitable for use during the photodynamic therapy of epithelial skin tumors. They are composed of a combination of the lipophilic polymer Eudragit NE and the lipophilic plasticiser acetyl tributyl citrate (ATBC). Because of its hydrophilicity, ALA forms suspension systems within these thin films, as evidenced by light microscopy. ALA release measured using Franz cells is very rapid from a Eudragit NE thin film loaded with 10% w/w ALA (200 microg ALA after 2.5 h), and even higher when ATBC is included. A Eudragit NE/ATBC (1: 2) thin film loaded with 20% w/w ALA releases 2000 microg ALA after 3.5 h. Combined release/permeation of ALA through excised membranes of human stratum corneum plus epidermis yielded fluxes of 50-100 microg ALA within 5 h for the Eudragit NE/ATBC (1: 2) thin film. The ATBC acts as a permeation enhancer for ALA. Scanning electron microscopy of the thin film surface shows protruding ALA particles which rapidly dissolve on contact with an aqueous medium. This surface dissolution mechanism is the cause of the rapid ALA release and hence also the high skin permeation in vitro. The mechanical properties of the thin films were also briefly examined. Adhesive strength increases with higher ATBC loading and decreases with higher ALA loading. Internal cohesion decreases with greater ATBC loading and increases with higher ALA loading. As part of this project, an improved derivatisation assay for gradient HPLC of ALA with 9Fluorenylmethyloxycarbonylchloride is also presented.     

Photosensitizer delivery for photodynamic therapy of choroidal neovascularization.

The present review examines the importance of improving photosensitizer delivery for choroidal neovascularization (CNV) in light of the clinical impact of photodynamic therapy (PDT) for CNV. An overview of the classes of available photosensitizers is provided and the properties governing photosensitizer uptake and circulation in serum are discussed. Current delivery systems, for example liposomal formulations as well as the use of the promising strategy of antibody targeted delivery as a strategy to improve PDT selectivity and efficiency for CNV treatment are described. A summary of the work using Verteporfin, tin ethyl purpurin and Lu-Tex--photosensitizers currently in clinical trials for CNV--is given.     

The synthesis and applications of 5-aminolevulinic acid (ALA) derivatives in photodynamic therapy and photodiagnosis

A route has been developed to high-purity precursors, viz., ALA esters, to be used in photodiagnosis and photodynamic therapy. Hexyl, butyl and methyl 5-aminolevulinates are similar to the ALA acid in chemical stability and efficacy in producing the appropriate photosensitizer PpIX. Tests carried out on animal models showed the method based on the esters to be the more selective.     

5-氨基酮戊酸光动力疗法治疗中重度痤疮的临床效果

目的研究中重度痤疮患者应用5-氨基酮戊酸光动力疗法的治疗效果。方法104例中重度痤疮患者,经随机数字编号后,52例奇数患者编入对照组,52例偶数患者纳入观察组。对照组采用红蓝光照射治疗,观察组采用5-氨基酮戊酸光动力疗法治疗。比较两组患者的治疗效果及不良反应发生情况。结果观察组患者治疗2、4、6周后的总有效率分别为28.85%、75.00%、86.54%,均高于对照组的9.62%、51.92%、69.23%,差异均具有统计学意义(χ²=6.1905、5.9713、4.5217,P=0.0128、0.0145、0.0335<0.05)。观察组患者的不良反应发生率为5.77%,低于对照组的32.69%,差异具有统计学意义(χ²=12.1333,P=0.0005<0.05)。两组患者的不良反应经对症处理后均缓解消退。结论中重度痤疮患者采用5-氨基酮戊酸光动力疗法治疗,效果理想,值得推行。     

Rheological characterization and permeation behavior of poloxamer 407-based systems containing 5-aminolevulinic acid for potential application in photodynamic therapy

Although anti-retroviral therapy is the most efficient disease management strategy for HIV-AIDS, its applications are limited by several factors including the low bioavailability and first pass metabolism of the drugs. Nanocarriers such as liposomes have been developed to circumvent some of these problems. We report here preparation of novel liposome formulations for efficient delivery of anti-retroviral drugs to mammalian cells in culture. The liposomes were prepared and surface was modified using poly (ethylene glycol). Encapsulation efficiency of the anti-retroviral drug saquinavir was found to be approximately 33% and also exhibited sustained release of the drug. Although PEGylated liposomes were more stable in protein-supplemented media, had better colloidal stability and exhibited lesser sonochemical stability due to lower cavitation threshold. The cell viability studies using Jurkat T-cells revealed that the PEGylated liposomes loaded with saquinavir were less cytotoxic as compared to the non-PEGylated liposomes or free drug confirming the potential of the liposomes as a sustained drug-release system. The drug delivery potential of the liposomes loaded with Alexa flour 647 was evaluated using Jurkat T-cells and flow cytometry showing uptake upto 74%. Collectively, our data demonstrate efficient targeting of mammalian cells using novel liposome formulations with insignificant levels of cytotoxicity.     

Cellulitis as a postprocedural complication of topical 5-aminolevulinic acid photodynamic therapy in the treatment of actinic keratosis

BACKGROUND: Actinic keratoses (AKs) are a common premalignant tumor of the skin. Several treatment modalities exist for broad-area therapy. Photodynamic therapy (PDT) is one such treatment modality. Disruption of squamous epithelium locally compromises the normal physical barrier of the skin, potentially allowing bacteria penetration into the dermis. This may occur subsequent to PDT, resulting in cellulitis and its sequelae. Undiagnosed and untreated this can prolong recovery times and increase patient discomfort. OBJECTIVE: We report 4 cases of cellulitis that developed after treatment of AKs with PDT. These cases developed 1 to 4 days after PDT. METHODS: Standard short-contact 2-hour incubation is performed on patients receiving treatment on the face or scalp. Patients are contacted by telephone on day 1 and day 3 postprocedure. Patients are asked to call immediately if they experience no resolution of discomfort or an abrupt increase in pain in the days following treatment. Those patients reporting an increase in pain and discomfort on either of these days are asked to come to the office for examination. A culture and sensitivity is performed on those presenting with cellulitis clinically and empiric antibiotic therapy is initiated. Antibiotic therapy is adjusted, if necessary, based on the culture and sensitivity report. RESULTS: All cases of cellulitis presented with an increase in pain and burning as the primary symptom between day 1 and 4 following PDT. The appearance of impetiginized areas or pustules was clearly evident in 2 out of the 4 patients. A culture and sensitivity confirmed the growth of staphylococcus aureus in all 4 patients. CONCLUSION: Cellulitis should be considered as a possible complication in patients reporting an abrupt increase in pain or those who do not experience a gradual resolution of pain and discomfort following PDT.     

Comparison of the uptake of 5-aminolevulinic acid and its methyl ester in keratinocytes and skin

Photodynamic therapy is widely used in the treatment of superficial skin cancers. 5-Aminolevulinic acid (ALA) and its methylated form, methyl-ALA (MAL), are frequently used as precursors to photosensitizing substances. Nevertheless, the mechanism of the uptake of ALA and MAL in keratinocytes and of their skin penetration is still controversial. Since both compounds are not sufficiently lipophilic to penetrate through lipid membranes, they must employ specific uptake systems which may vary between different cell types. Here, we studied ALA and MAL uptake in keratinocyte cell lines originating from healthy cells (CCD 1106 KERTr cells) or keratinocyte tumors (A431 cells). ALA uptake resulted in faster protoporphyrin IX (PpIX) production than MAL uptake. A pharmacological characterization of the uptake systems revealed that PpIX formation was most efficiently reduced with GABA transporter (GAT) substrates. GABA, β-alanine, and (S)-SNAP-5114 reduced ALA uptake and, to a lesser extent, MAL uptake in the cell lines. The pharmacology of these compounds indicates that ALA and MAL are taken up by normal and pathological keratinocytes via GAT-3. Furthermore, the amino acids arginine, cysteine, and histidine also inhibited the uptake of ALA, and even more so MAL, suggestive of an additional involvement of amino acid transporters. To show that PpIX formation in vivo is restricted to the application site, which has been questioned for ALA in one other report, we applied clinically used ALA and MAL formulations to the skin of nude mice. Contrary to the results of these previous authors, the resulting PpIX fluorescence increased over time and was restricted to the application site for both preparations.