Administration Of 5-aminolaevulinic Acid Research Articles

The use of dipeptide derivatives of 5-aminolaevulinic acid promotes their entry to tumor cells and improves tumor selectivity of photodynamic therapy.

The use of endogenous protoporphyrin IX generated after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). However, the bioavailability of ALA is limited by its hydrophilic properties and limited cell uptake. A promising approach to optimize the efficacy of ALA-PDT is to deliver ALA in the form of prodrugs to mask its hydrophilic nature. The aim of this work was to evaluate the potential of two ALA dipeptide derivatives, N-acetyl terminated leucinyl-ALA methyl ester (Ac-Leu-ALA-Me) and phenylalanyl-ALA methyl ester (Ac-Phe-ALA-Me), for their use in PDT of cancer, by investigating the generation of protoporphyrin IX in an oncogenic cell line (PAM212-Ras), and in a subcutaneous tumor model. In our in vitro studies, both derivatives were more effective than ALA in PDT treatment, at inducing the same protoporphyrin IX levels but at 50- to 100-fold lower concentrations, with the phenylalanyl derivative being the most effective. The efficient release of ALA from Ac-Phe-ALA-Me appears to be consistent with the reported substrate and inhibitor preferences of acylpeptide hydrolase. In vivo studies revealed that topical application of the peptide prodrug Ac-Phe-ALA-Me gave greater selectivity than with ALA itself, and induced tumor photodamage, whereas systemic administration improved ALA-induced porphyrin generation in terms of equivalent doses administered, without induction of toxic effects. Our data support the possibility of using particularly Ac-Phe-ALA-Me both for topical treatment of basal cell carcinomas and for systemic administration. Further chemical fine-tuning of this prodrug template should yield additional compounds for enhanced ALA-PDT with potential for translation to the clinic.

Sustained and efficient porphyrin generation in vivo using dendrimer conjugates of 5-ALA for photodynamic therapy

The use of endogenous protoporphyrin IX (PpIX) after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). However the efficacy of ALA-PDT is sub-optimal for thicker tumours and improved ALA delivery and therapeutic response are required. We have investigated the conjugation of ALA to a second-generation dxcendrimer for enhancing porphyrin synthesis in vitro and in vivo in a murine tumour model using systemic i.p. administration. In vitro, the dendrimer was more efficient than ALA for porphyrin synthesis at low concentrations in good correlation with higher cellular ALA dendrimer accumulation. In vivo, the porphyrin kinetics from ALA exhibited an early peak between 3 and 4 h in most tissues, whereas the dendrimer induced sustained porphyrin production for over 24 h and basal values were not reached until 48 h after administration. Integrated porphyrin accumulation from the dendrimer and ALA, at equivalent molar ratios, was comparable showing that the majority of ALA residues were liberated from the dendrimer. The porphyrin kinetics appear to be governed by the rate of enzymatic cleavage of ALA from the dendrimer, which is consistent with in vitro results. ALA dendrimers may be useful for metronomic PDT, and multiple low-dose ALA-PDT treatments.

5-Aminolaevulinic acid peptide prodrugs enhance photosensitization for photodynamic therapy

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (ALA) has been widely used in photodynamic therapy for a range of malignant and nonmalignant lesions. However, ALA is relatively hydrophilic and lacks stability at physiologic pH, limiting its bioavailability. We have investigated more lipophilic, uncharged ALA-peptide prodrugs based on phenylalanyl-ALA conjugates, which are water soluble and chemically stable for improving ALA delivery. Pharmacokinetics of the induced protoporphyrin IX (PpIX) were studied in transformed PAM212 keratinocyte cells and pig skin explants. The intracellular porphyrin production was substantially increased with Ac-L-Phe-ALA-Me (compound 1) and Ac-L-Phe-ALA (compound 3) compared with equimolar ALA: after 6-h incubation, the PpIX fluorescence measured using 0.01 mmol/L of compound 1 was enhanced by a factor of 5 compared with ALA. Phototoxicity results showed good correlation with PpIX levels, giving a LD(50) (2.5 J/cm(2)) of 25 micromol/L for ALA, 6 micromol/L for 5-aminolaevulinic hexyl ester, and 2.6 micromol/L for compound 1, which exhibited the highest phototoxicity. However, these results were stereospecific because the corresponding D-enantiomer, Ac-D-Phe-ALA-Me (compound 2), induced neither porphyrin synthesis nor phototoxicity. PpIX levels were considerably reduced when cells were incubated with compound 1 at low temperatures, consistent with active transport. In pig skin explants, compound 1 induced higher porphyrin fluorescence than ALA by a factor of 3. These results show that water-soluble peptide prodrugs of ALA can greatly increase its cellular uptake, generating more intracellular PpIX and improved tumor cell photosensitization. The derivatives are comparable in efficacy with 5-aminolaevulinic hexyl ester but less toxic and more stable at physiologic pH.

Macromolecular delivery of 5-aminolaevulinic acid for photodynamic therapy using dendrimer conjugates

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (5-ALA) has been widely used in photodynamic therapy. However, cellular uptake of 5-ALA is limited by its hydrophilicity, and improved means of delivery are therefore being sought. Highly branched polymeric drug carriers known as dendrimers present a promising new approach to drug delivery because they have a well-defined structure capable of incorporating a high drug payload. In this work, a dendrimer conjugate was investigated, which incorporated 18 aminolaevulinic acid residues attached via ester linkages to a multipodent aromatic core. The ability of the dendrimer to deliver and release 5-ALA intracellularly for metabolism to the photosensitizer, protoporphyrin IX, was studied in the transformed PAM 212 murine keratinocyte and A431 human epidermoid carcinoma cell lines. Up to an optimum concentration of 0.1 mmol/L, the dendrimer was significantly more efficient compared with 5-ALA for porphyrin synthesis. The intracellular porphyrin fluorescence levels showed good correlation with cellular phototoxicity following light exposure, together with minimal dark toxicity. Cellular uptake of the dendrimer occurs through endocytic routes predominantly via a macropinocytosis pathway. In conclusion, macromolecular dendritic derivatives are capable of delivering 5-ALA efficiently to cells for sustained porphyrin synthesis.

Enhanced porphyrin accumulation using dendritic derivatives of 5-aminolaevulinic acid for photodynamic therapy: An in vitro study

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid has been widely used in photodynamic therapy for a range of malignant and certain non-malignant lesions. However, cellular uptake of 5-aminolaevulinic acid is limited by its hydrophilic nature and improved means of delivery are therefore being sought. Highly branched polymeric drug carriers known as dendrimers are a promising new approach to drug delivery. The aim of this study was to investigate the efficacy of dendrimers conjugated with 5-aminolaevulinic acid for porphyrin production in the transformed PAM 212 keratinocyte cell line and skin explants. Each dendritic derivative incorporated three 5-aminolaevulinic acid residues which were conjugated as esters via methyl or propyl linkers to a central tertiary carbon whose remaining terminal bore an amino, aminobenzyloxycarbonyl or nitro group. In the cell line, all compounds were more efficient at low concentrations compared to equimolar 5-aminolaevulinic acid for porphyrin production, with the most efficient incorporating the longer propyl linker. This compound was also the most lipophilic according to partition coefficient measurements. The intracellular porphyrin fluorescence levels showed good correlation with cellular phototoxicity following light exposure for all the compounds, together with minimal dark toxicity. Our findings indicate that the key factors influencing the efficacy of the dendritic derivatives are lipophilicity and steric hindrance within the dendritic structure which could restrict access to intracellular esterases for liberation of 5-aminolaevulinic acid. These findings should be taken into account in the design of larger dendrimers of 5-aminolaevulinic acid.

Acrylate yellow filters in operating lights protect against photosensitization tissue damage.

Photosensitized patients are exposed to bright lights when undergoing intraoperative photodynamic therapy or fluorescence measurements. Acrylate yellow filters might reduce unwanted tissue damage. To investigate the protective value of these filters, the spectral power distribution of the operating lights and light energy densities with and without an acrylate yellow filter were measured. Subsequently the effects of light exposure on the survival of a human hepatocellular carcinoma cell line and the photodamage induced in pig tissues after the administration of 5-aminolaevulinic acid were also studied. The light energy density in the ultraviolet and blue region of the light spectrum emitted by the operating light was reduced up to 50 per cent by the acrylate yellow filter. The survival of photosensitized cells was longer and photodamage induced in pig tissues was less when exposed to filtered light. Photodamage induced by operating lights can be reduced by filtering out ultraviolet and blue light by means of acrylate yellow filters.

Review article: the potential role for photodynamic therapy in the management of upper gastrointestinal disease.

Photodynamic therapy involves the activation of an exogenously administered, or an endogenously generated, photosensitizer with light to produce localized tissue destruction. It is an attractive, predominantly endoscopic technique for the palliation of advanced upper gastrointestinal cancer and the eradication of early neoplastic and pre-neoplastic lesions. The nature of the biological response allowing safe healing and the exploitation of tissue threshold effects mean that adjacent tissue damage can be minimized. This review used a database of 368 papers. The nature of the photosensitizer is critical to the depth of tissue damage and the risk of adjacent tissue damage and stricture formation. The generation of protoporphyrin IX following administration of 5-aminolaevulinic acid has proved useful for the treatment of high-grade dysplasia in Barrett's oesophagus. A double-blind randomized placebo controlled trial has confirmed that it is a safe and effective method for the ablation of low-grade dysplasia. The treatment of more advanced lesions requires exogenously administered photo-sensitizers. However, recent data indicate that the neoplastic potential remains in some patients and continued follow-up is necessary. Photodynamic therapy can be used to eradicate early neoplasia and palliate advanced cancer, but caution is required before a definitive cure can be claimed.

7130 The value of a new fluorescence endoscopy equipment in upper gastrointestinal endoscopy.

Background: With time-gated detection of protoporphyrin IX (PpIX) fluorescence by spectroscopy (LIFS) we were able to detect dysplasia in Barrett`s esophagus with a sensitivity of 71% and a specificity of 64%. Aimof this study was to evaluate the capability of simultaneouslaserinduced fluorescence imaging (LIFE) and white-light endoscopy for detection of dysplasia and early tumors. Methods:In fifteen patients, with a routine endosocpy one to two weeks earlier, fluorescence endoscopy was performed after administration of 5-aminolaevulinic acid. The experimental set-up consists of an optical parametric oscillator (GWU, Germany), pumped by the third harmonic of a Q-switched Nd:YAG laser (GCR-11, Spectra Physics, USA). The laser beam (λ = 505 nm) was coupled into the illumination fiber bundle of the endoscope. For detection of the fluorescence images the double channel gastroscope (GIF 2 T 20, Olympus Co, Japan) was equipped with an auxiliary image guide (Medisecur, Germany) adapted to an intensified CCD camera (Princeton Instruments, USA). Optical filters were used to select the fluorescence bands of PpIX. The biopsies were taken at locations with high and for control at locations with low PpIX fluorescence. Subsequently these samples were analyzed by routine pathology. Results:Short-Barrett`s esophagus was newly detected in 5 patients and excluded in 4 patients. In one Short-Barrett`s esophagus a early carcinoma was found. Furthermore, malignancy in a duodenal polyp and one early cancer in the stomach were newly diagnosed. In one patient with a known localized squamous carcinoma of the esophagus tumor spread in the esophagus was seen. Interestingly not only malignancies and specialized intestinal metaplasia in Barrett`s showed significantly higher fluorescence, but also intestinal metaplasia in the stomach (median fluorescence intensity: intestinal metaplasia: 1.28 (68% CI:.27 - 3.07); inflamed stomach mucosa: 0.10 (68 % CI: 0.01 - 1.08); Sen: 83% Spez: 77%). Therefore we were able to visualize intestinal metaplasia in 3 patients, not detected by prior endoscopic biopsies. Furthermore, due to visibility of the increased PpIX fluorescence, localization of lesions was possible. Therefore, LIFE was time saving and we needed much less biopsy samples for diagnosis than usually. Conclusion:Fluorescence endoscopy improves sensitivity and decreases endoscopy time. However, up until now spectroscopy is more sensitive, due to a higher spatial resolution.

Kidney-preserving tumour resection in renal cell carcinoma with photodynamic detection by 5-aminolaevulinic acid: preclinical and preliminary clinical results.

To identify the outer border of a renal cell carcinoma (RCC) during a kidney-preserving tumour resection by photodynamically detecting RCC after the exogenous administration of 5-aminolaevulinic acid (ALA) in an animal model and in humans. Human RCC was xenotransplanted subcutaneously and orthotopically into the kidneys of nude mice. ALA was then administered orally or intravenously (100-400 mg/kg body weight). The tumours were removed and examined macroscopically and microscopically for fluorescence, and the concentrations of protoporphyrin IX (PPIX) evaluated in the subcutaneous tumours. In a pilot study, nine patients with RCC of <4 cm diameter underwent partial nephrectomy; 20 mg ALA/kg body weight was given orally 4 h before surgery. During the operation, the macroscopic fluorescence of the tumours was evaluated and any side-effects recorded. In the mouse model, the RCC fluoresced in all tumours, both macro- and microscopically, reaching a maximum 1.5 h after intravenous and 4 h after oral administration. The tissue concentrations of PPIX in the subcutaneous tumours were also maximal 4 h after oral administration. In the patients undergoing surgery, the RCC also fluoresced clearly and was sufficiently intense to identify the outer margins of the tumours for kidney-preserving tumour resection. There were no side-effects of the ALA with the dosages and methods of administration used. The photodynamic detection of RCC with ALA facilitates the identification of tumour margins in man and in the mouse model, producing no undesirable side-effects. It may be helpful in determining the boundaries of the resected tissue when carrying out conservative kidney-preserving surgery.

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.

Photodynamic therapy of normal rat arteries after photosensitisation using disulphonated aluminium phthalocyanine and 5-aminolaevulinic acid.

Photodynamic therapy of cancer exposes adjacent arteries to the risk of injury and the possibility of haemorrhage and thrombosis. The nature of photodynamic injury to normal arteries has not been satisfactorily defined, and the ability of arteries to recover with time is unclear. To clarify these issues, we have investigated the effects of PDT on rat femoral arteries, using a second-generation photosensitiser, disulphonated aluminium phthalocyanine, and a new method of photosensitisation, using endogenous synthesis of protoporphyrin IX following systemic administration of 5-aminolaevulinic acid (ALA). Pharmacokinetic studies of sensitiser fluorescence were carried out to determine peak levels of sensitiser. Subsequently photodynamic therapy at times corresponding to maximal fluorescence was performed using two light doses, 100 and 250 J cm-2. The nature of injury sustained and recovery over a 6 month period was investigated. Three days following PDT, all vessels treated showed complete loss of endothelium, with death of all medial smooth muscle cells, leaving an acellular flaccid artery wall. No vascular occlusion, haemorrhage or thrombosis was found. A striking feature was the lack of inflammatory response in the vessel wall at any time studied. Re-endothelialisation occurred in all vessels by 2 weeks. The phthalocyanine group showed repopulation of the media with smooth muscle cells to be almost complete by 3 months. However, the ALA group failed to redevelop a muscular wall and remained dilated at 6 months. Luminal cross-sectional area of the ALA-treated group was significantly greater than both control and phthalocyanine groups at 6 months. All vessels remained patent. This study indicates that arteries exposed to PDT are not at risk of catastrophic haemorrhage or occlusion, a finding that is of significance for both the local treatment of tumours and the use of PDT as an intraoperative adjunct to surgery for the ablation of microscopic residual malignant disease.

Endogenous porphyrin distribution induced by 5-aminolaevulinic acid in the tissue layers of the gastrointestinal tract

The accumulation of endogenous porphyrins in rats following systemic administration of 5-aminolaevulinic acid (ALA) has been examined to assess the photosensitization characteristics of this technique for photodynamic therapy (PDT) and chemical extraction assays with fluorescence and absorbance detection of the porphyrin content have been carried out. We compared the results obtained using quantitative microfluorimetry on normal gastric and colonic tissues in rats at 0.5 1, 2, 4 and 6 h and chemically induced duodenal tumours 2 and 4.5 h after intravenous administration of ALA at a dose of 200 mg kg −1. With chemical extraction followed by high performance liquid chromatography analysis, protoporphyrin IX (PpIX) was found to be the predominant porphyrin present, reaching peak levels of several microgrammes per gramme at 2–4 h in each type of tissue; a small amount of coproporphyrin was detected at 0.5 and 2 h in normal gastric mucosa and duodenal tumour respectively. Both the extraction assay and quantitative microfluorimetry showed that the porphyrin fluorescence builds up rapidly in the mucosal layers of the colon and stomach, reaching a maximum at 2 h, whereas lower fluorescence levels were found with a slower rate of accumulation in the corresponding muscularis layers. A significant PpIX content was found in the duodenal tumour, with a maximum of 7.1 μg g −1 4.5 h after ALA administration. We conclude that systemic administration of ALA can induce effective tissue sensitization with protoporphyrin IX and appears to be a promising technique for PDT.

Genetic variation of iron-induced uroporphyria in mice

Iron overload causes inhibition of hepatic uroporphyrinogen decarboxylase (UROD) and uroporphyria in C57BL/10ScSn but not DBA/2 mice [Smith, Cabral, Carthew, Francis and Manson (1989) Int. J. Cancer 43, 492-496]. We have investigated the induction of uroporphyria in 12 inbred strains of mice 25 weeks after iron treatment (600 mg/kg) to determine if there was any correlation with the Ah locus. Under these conditions, inhibition of UROD occurred to varying degrees in Ahd mice (SWR and AKR) as well as nominally Ahb-1 (C57BL/6J, C57BL/10ScSn and C57BL/10-cc) and Ahb-2 strains (BALB/c and C3H/HeJ). Five other Ahb or Ahd strains (C57BL/Ks, A/J, CBA/J, LP and DBA/2) were unaffected. Thus there appeared to be no correlation with the Ah phenotype and this illustrated that some other variable inherited factors are involved. Comparisons between another susceptible strain, A2G, and the congenic A2G-hr/+strain (carrying the recessive hr gene) showed a modulating influence associated with the hr locus. In contrast with individual mice of inbred strains, which showed consistent responses to iron, those of the outbred MF1 strain showed a spectrum of sensitivities as might be expected for a heterogeneic stock. The rate of porphyria development was accelerated by administration of 5-aminolaevulinic acid (5-ALA) in the drinking water, but this did not overcome strain differences. Among four strains the order of susceptibility was SWR > C57BL/10ScSn > C57B1/6J > DBA/2 (the last strain was completely resistant). With degrees of iron loading greater than 600 mg of Fe/kg (1200-1800 mg of Fe/kg) C57BL/10ScSn mice (after 20 weeks) and SWR mice (after 5 weeks which included 4 weeks of 5-ALA treatment) had less inhibition of UROD and a lower uroporphyric response, showing that there was an optimum level of liver iron concentration. Studies on selected microsomal enzyme activities associated with cytochrome P-450 showed no correlation with the propensities of strains to develop porphyria. These activities included the NADPH-dependent oxidation of uroporphyrinogen I to uroporphyrin I.