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A comparative experimental study of the specific activity of drugs based on 5-aminolevulinic acid (5-ALA) and its hexyl ester (5-ALA HE) was carried out. Their ability to induce the synthesis of photoactive protoporphyrin IX in the healthy tissues of the rabbit bladder when instilling the drug solutions at various concentrations has been estimated. It was shown that 5-ALA HE results in the induction and accumulation of PPIX in the rabbit bladder epithelium at much lower concentrations than 5-ALA. Thus, a significant increase in the fluorescence intensity in comparison with the control was achieved by instillation of 5-ALA HE solution in the rabbit’ bladder at a concentration of only 0.0001% (fluorescence intensity 2.20±0.60 a.u.), and for 5-ALA – only when using a solution at a concentration of 0.3% (fluorescence intensity 2.60±1.02 a.u.).

Results of autofluorescence dermatoscopy using videodermatoscope «EcoSkin» in patients with different dermatologic diseases are represented. The study included 64 patients, 27 of them had different types of psoriasis, 11 – acne, 3 – rosacea, 5 – eczema, 3 – mite-induced dermatitis, 4 – superficial mycosis of the glabrous skin, 7 – atopic dermatitis, 4 – seborrheic dermatitis. The major changes of autofluorescence were detected in pathological foci in patients with psoriasis. Authors suppose that increased autofluorescence of porphyrins in psoriatic plaques in the study is associated with enhanced prolyferation in pathological tissues and with liver dysfunction. The analysis of autofluorescence images in patients with rosacea, eczema, atopic and mite-induced dermatitis did not show significant difference in autofluorescence images of affected skin comparing with intact skin in same patients. In patients with acne a pattern of spot red-orange autofluorescence of skin in seborrheic areas corresponding with location of openings of pilosebaceous apparatus was detected that was associated by authors with Propionibacterium acnes infection.

The therapeutic properties of 5-aminolevulinic acid (5-ALA) have been extensively studied for cancer detection and treatment using photodynamic therapy (PDT). When administered externally, 5-ALA is converted to protoporphyrin IX (PpIX) in cancer cells, which generates reactive oxygen species (ROS) upon exposure to light. This process enables targeted cell death induction and cancer detection. Given the highly conserved nature of heme biosynthesis over billions of years, we hypothesized that natural mechanisms might exist to prevent excessive accumulation of PpIX or heme resulting from 5-ALA overload. Therefore, we anticipated alterations in protein expression profiles upon exogenous administration of 5-ALA. To understand cellular responses to 5-ALA, we investigated protein expression changes and identified OR1B1 as a promising target in bladder, prostate, lung, and cervical cancer cells. OR1B1 expression was observed only with 5-ALA and ferrous chloride, highlighting the central role of heme in this discovery. Immunofluorescence and electron microscopy confirmed OR1B1's sub-cellular localization. These findings suggest that 5-ALA transformation in cancer cells and OR1B1 expression have potential for enhancing cancer detection and developing alternative treatments, including immunotherapy. This approach overcomes the limitations of PDT and opens new avenues for effective and targeted cancer interventions. • This paper represents first complete proteomic analysis of the effects on cancer cells following exposure to 5-aminolevulinic acid (5-ALA). • 5-ALA triggers the down- or upregulation of >100 proteins. • Upon the analysis of significantly upregulated proteins, OR1B1 stands out, showing significant and specific upregulation following 5-ALA treatment without light with respect to its expression at the cellular membrane. • Heme and not protoporphyrin IX triggers the expression of OR1B1. • OR1B1 is a potential target for other imaging or treatment modalities in combination with 5-ALA. [ABSTRACT FROM AUTHOR]

The technique of intraoperative fluorescence diagnosis with alasens for brain metastases includes visual assessment of alasens-induced protoporphyrin IX fluorescence and local spectroscopy. The technique allows to reduce the rate of misdiagnosis, to assess accurate local extent of brain metastases and to improve surgical radicality. When applying this technique the sensitivity of fluorescence diagnosis is 96.7%, the specificity is 100%. The technique is designed for neurosurgeons specialized on neurooncology.

Background The establishment of a precise and rapid method to detect metastatic lymph nodes (LNs) is essential to perform less invasive surgery with reduced gastrectomy along with reduced lymph node dissection. We herein describe a novel imaging strategy to detect 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence in excised LNs specifically with reduced effects of tissue autofluorescence based on photo-oxidation of PpIX. We applied the method in a clinical setting, and evaluated its feasibility. Methods To reduce the unfavorable effect of autofluorescence, we focused on photo-oxidation of PpIX: Following light irradiation, PpIX changes into another substance, photo-protoporphyrin, via an oxidative process, which has a different spectral peak, at 675 nm, whereas PpIX has its spectral peak at 635 nm. Based on the unique spectral alteration, fluorescence spectral imaging before and after light irradiation and subsequent originally-developed image processing was performed. Following in vitro study, we applied this method to a total of 662 excised LNs obtained from 30 gastric cancer patients administered 5-ALA preoperatively. Results Specific visualization of PpIX was achieved in in vitro study. The method allowed highly sensitive detection of metastatic LNs, with sensitivity of 91.9% and specificity of 90.8% in the in vivo clinical trial. Receiver operating characteristic analysis indicated high diagnostic accuracy, with the area under the curve of 0.926. Conclusions We established a highly sensitive and specific 5-ALA-induced fluorescence imaging method applicable in clinical settings. The novel method has a potential to become a useful tool for intraoperative rapid diagnosis of LN metastasis. [ABSTRACT FROM AUTHOR]

Summary: Background: As carcinogenesis is often accompanied by inflammation, fluorescence diagnosis (FD) of tumours reportedly leads to false positive results. However, an influence of inflammation on the efficiency of photodynamic therapy (PDT) is not known. Objective: Therefore we investigated whether LPS-induced expression of the pro-inflammatory cytokine interleukin-6 (IL-6) influences hypericin and protoporphyrin IX (PpIX) accumulation, and their photocytotoxicity. As in vitro models served normal and malignant human skin cell lines. Additionally we analyzed whether low-dose photodynamic treatment as found during FD and sublethal PDT could modulate the expression of IL-6 and other cytokines as low-dose PDT has the potential for treatment of inflammatory skin diseases. Methods: Cells were pre-incubated with LPS to induce a pro-inflammatory milieu. Photosensitizer accumulation and toxicity were measured by a microplate reader, IL-6 via Sandwich ELISA and the other cytokines via a Multiplex Immunoassay. Results and conclusions: Neither hypericin uptake nor PpIX accumulation was influenced by the proinflammatory state. The false positive classification in FD of patients could rather be due to sensitizer-loaded immune cells. On the other hand, low dose PDT is able to significantly reduce the level of IL-6 in all cell lines. Under differing PDT-conditions, PDT can enhance IL-6 in malignant cells, which could elicit a specific anti-tumour immune response. Furthermore, hypericin alone is able to up-regulate the anti-inflammatory cytokines IL-4, -5 and -10, which could be useful to reduce excessive inflammation. LPS-induction has no additional influence in most cases. Some of these findings could contribute to abate inflammatory diseases on skin or promote wound healing. [Copyright &y& Elsevier]

Background Basal cell carcinoma (BCC) is the most common malignant skin carcinoma. Fluorescence diagnosis (FD) has been suggested as a promising method for noninvasive detection of subclinical tumour cell dissemination in BCC. Objectives In this prospective study, we evaluated the clinical performance of a preoperative definition of the lateral borders of BCC by FD in comparison with its definition by purely clinical diagnosis (CD). The fluorescence intensity on the skin was recorded using a digital light-emitting diode-based fluorescence imaging system. Methods Twenty-six patients with BCC (22 with nodular subtype) of the H-zone were included. The tumour area was determined 3 h after application of methyl aminolaevulinate by inspection and photographic documentation (CD) and FD. Subsequently, BCCs were excised according to the complete area defined by CD and FD with a security margin of 3 mm; surgical specimens were sectioned horizontally and subjected to meticulous histological mapping. The tumour areas as determined by FD, CD and histology were superimposed to map the entire lateral tumour margin. Results The tumour area could be visualized by FD in 24 of 26 patients. The mean tumour area as determined by FD was significantly smaller than the tumour area as determined by CD [80 mm2, 95% confidence interval (CI) 50–110 mm2 vs. 101 mm2, 95% CI 76–125 mm2; P < 0·012]. The superimposition of FD and histology showed in 10 of 26 patients a complete detection of the tumour margin by FD; thus sensitivity of FD was calculated as 38·5%. In only three of 26 patients FD revealed a tumour extent greater than determined by CD. Specificity of FD was calculated as 88·4%. Conclusions On aggregate, this study suggests that preoperative FD of nodular BCC localized in the H-zone offers no additional benefit to define subclinical tumour infiltration compared with CD alone. [ABSTRACT FROM AUTHOR]

Summary: Generation of singlet oxygen and estimation of its lifetime, the oxygen effect, the subcellular localization of photosensitizers and their photodegradation during photodynamic therapy (PDT), the effects of PDT on DNA and chromosomes were described in the first part of our review. In this review we discuss PDT in combination with other treatments (hyperthermia, ionizing radiation, electrotherapy, chemotherapeutic drugs) or with other agents; cell interactions and bystander effects in PDT; the influence of PDT on the cytoskeleton; a novel technology, named photochemical internalisation (PCI), for light-induced delivery of macromolecules and 5-aminolevulinic acid (ALA) PDT. Lipophilic derivatives of ALA, instead of ALA itself, were proposed for induction of protoporphyrin IX (PpIX) in the treatment of superficial lesions. Based on this research a Norwegian company PhotoCure ASA was founded in 1993 with the aim to commercialise photodynamic technologies developed at the Norwegian Radium Hospital, the largest comprehensive cancer centre in Northern Europe. The company has two products on the market: Metvix® for treatment of basal cell carcinomas and actinic keratoses, and Hexvix® for fluorescence diagnosis of bladder cancer. New ALA derivatives for fluorescence diagnosis and treatment of early-stage cancers in internal organs, e.g. colon cancer, are currently being investigated. [Copyright &y& Elsevier]

Background Topical 5-aminolaevulinic acid (ALA)-photodynamic therapy (PDT) for the treatment of psoriasis has been evaluated in a few studies. In these studies different treatment parameters were used, there was a variable clinical response, and a nonhomogeneous fluorescence was seen after irradiation with Wood's light. Objectives To study the clinical effectiveness, immunohistochemical changes and protoporphyrin IX accumulation in ALA-PDT in patients with psoriasis. Methods Eight patients with stable plaque psoriasis with symmetrical involvement were included in the study. Two symmetrical plaques were randomly allocated to PDT either with 10% ALA or with placebo. Irradiation consisted of 2 and 8 J cm−2 with a dark interval of 2 h (Waldmann PDT 1200 L, 600–750 nm, 40 mW cm−2) once weekly for 4 weeks. Before, during and after irradiation, fluorescence diagnosis was performed. Biopsies were taken at baseline, week 1 and week 6 for immunohistochemical assessment. Psoriatic plaques were clinically assessed using the plaque severity (sum) score. Fluorescence diagnosis was performed and expression of immunohistochemical markers for proliferation, differentiation and T-cell infiltration [Ki67, keratin 10 (K10), CD4, CD8 and CD45RO] was assessed. Results From week 1 up to week 6, ALA-PDT gave a significant reduction in the number of Ki67+ nuclei, while the K10 expression increased. After 6 weeks significant improvement was observed for CD8 and CD45RO. These changes were absent in the placebo-treated lesions. The sum scores were also significantly lower in the ALA-treated plaques. Heterogeneity of macroscopic fluorescence was observed during treatment despite keratolytic treatment. Conclusions The present study shows that clinical improvement during fractionated ALA-PDT in psoriasis parallels histological improvement as seen in normalization of epidermal proliferation, differentiation and infiltration of relevant T-cell subsets. Optimizing the current treatment protocol may increase clinical efficacy further. [ABSTRACT FROM AUTHOR]

Abstract: Background: Early detection of premalignant/malignant lesions in the oral cavity can certainly improve the patient’s prognosis. This study presents fluorescence imaging with the topical application of 5-aminolevulinic as a way to improve detection of various oral tissue pathologies. This procedure depends mainly on comparing the intensity of red and green fluorescence emitted from tissues during examination. Materials and methods: Seventy-one patients who presented with clinically suspicious oral leukoplakia were recruited for this study. Each of the patients was required to have 5-aminolevulinic acid in the form of mouth rinse prior to fluorescence imaging. Following this a surgical biopsy was acquired from the exact examination site. The results of the fluorescence spectroscopy have been compared with histopathology. Results: A Student’s t-test was applied to test the viability of the ratio between red and green fluorescence. The red-to-green ratio was found to increase significantly when the lesion was identified as dysplastic or carcinoma in situ. By applying a threshold line to discriminate between normal and dysplastic lesions; a sensitivity of 83–90% and specificity of 79–89% were obtained. Conclusion: Fluorescence spectroscopy combined with 5-aminolevulinic acid-induced protoporphyrin IX was found as a valuable tool in the diagnosis of oral premalignancy. This technique offers the potential to be advantageous over other non-optical techniques in terms of providing real-time diagnosis, in situ monitoring, cost effectiveness and more tolerated by patient compared to surgical biopsy. [Copyright &y& Elsevier]

Background Fluorescence imaging is an attractive diagnostic technique for skin tumour demarcation with potential to move to clinical use. Bispectral fluorescence imaging combines skin autofluorescence with δ-aminolaevulinic acid-induced fluorescence. To evaluate the technique, fluorescence data must be compared with the histopathological extent of the tumour, which is the purpose of the current study. Objectives To investigate the agreement between bispectral fluorescence images and the histopathological tumour boundary of ill-defined basal cell carcinomas (BCCs). After fluorescence imaging the tumours were removed using Mohs micrographic surgery (MMS) to obtain histopathological maps of the tumour boundaries. Methods Twelve patients with aggressive BCC of mean diameter 16 mm (range 5–32) in the face were included in the study. The patients were subjected to bispectral fluorescence imaging within the 2 months prior to MMS. The fluorescence images and histopathological maps were aligned using image warping. Results Five patients (42%) showed good agreement with the histopathological mapping and the remaining seven patients (58%) showed partial agreement. Bispectral investigation combining autofluorescence with protoporphyrin IX (PpIX) fluorescence generally yielded better agreement with the histopathological boundaries of the tumours compared with using only the PpIX fluorescence. Conclusions In this preliminary study the fluorescence has been compared with the histopathological tumour boundaries. The result implies that the technique can be applied as a useful tool for indicating tumour boundary of aggressive BCCs. Further refinement is needed to be able to indicate the exact tumour border. [ABSTRACT FROM AUTHOR]

In fluorescence diagnosis with 5-aminolevulinic acid (ALA)-induced porphyrins (FDAP), protoporphyrin IX (PpIX) accumulation can be macroscopically visualized. Interpretation of these data is still problematic because of the low reproducibility of the procedure and poor understanding of the mechanisms involved in PpIX tumor selectivity. In this study, PpIX accumulation is investigated in patients with psoriasis and actinic keratosis (AK) following FDAP. For this purpose, desquamated lesional and non-lesional skin were incubated with 20% ALA ointment for 3 h, FDAP was performed, and highly fluorescing lesional skin and non-lesional skin were biopsied. In extracts from these biopsies, PpIX, protein, and dsDNA were quantified by spectrofluorometry. Digital images acquired with FDAP were analyzed using image analysis software. PpIX per biopsy in lesional skin in both psoriasis and AK was significantly higher than in non-lesional skin (p<0.05). When corrected for epidermal involvement, only lesional psoriatic skin showed significantly higher PpIX levels than non-lesional skin. The PpIX-ratio lesional:non-lesional skin (mean(pmol per mL)±SEM) was 4.12±0.91 in psoriasis and 1.96±0.24 in AK. In FDAP, the ratio of lesional:non-lesional skin was 1.77±0.06 in psoriasis and 1.37±0.07 in AK. Macroscopic fluorescence and PpIX content appeared to be well correlated (r=0.73), thus making FDAP a good predictor of PpIX content. [ABSTRACT FROM AUTHOR]

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[Display omitted] • Efficient synthesis of peptide-targeted prodrug with multiple ALA units. • Prodrug shows sustained and enhanced PpIX production vs ALA. • Enhanced PpIX production vs non-targeted species in MDA-MB-231 cells. Photodynamic therapy (PDT) is a promising approach for the targeted treatment of cancer and various other human disorders. An effective, clinically approved approach in PDT involves the administration of 5-aminolevulinic acid (ALA) to generate elevated levels of the natural photosensitiser protoporphyrin IX (PpIX). The development of prodrugs of ALA is of considerable interest as a means to enhance the efficiency and cell selectivity of PpIX accumulation for PDT applications. In this work a novel peptide-targeted dendrimeric prodrug of 5-aminolevulinic acid (ALA) 13 was synthesised which displays nine copies of ALA on a core structure that is linked to a homing peptide for targeted delivery to a specific cancer cell type. The synthesis was accomplished effectively via a flexible, modular solid phase and solution phase route, using a combination of solid phase peptide synthesis and copper-catalysed azide-alkyne cycloaddition chemistry. The prodrug system shows a sustained and enhanced production of protoporphyrin IX (PpIX) in the MDA-MB-231 cell line that over-expresses the epidernal growth factor receptor (EGFR+) in comparison to equimolar ALA and the corresponding non-targeted ALA dendrimer (nine copies of ALA). This study provides a proof of concept for the development of a new generation of prodrugs for ALA-based photodynamic therapy that can deliver an enhanced ALA payload to specific tissue types. [ABSTRACT FROM AUTHOR]

We performed a pathological study to identify the locus of production of protoporphyrin IX (PPIX) in human metastatic brain tumors. Patients with metastatic brain tumors ( n = 11) received 1 g of 5-aminolevulinic acid (5-ALA) perorally 2 h before undergoing surgery. The target region was exposed to laser light with a peak wavelength of 405 ± 1 nm and an output of 40 mW. Tissue samples from the tumor bulk and surrounding areas were examined by histological and fluorescence methods. Of the 11 tumors, 9 manifested PPIX fluorescence in the tumor bulk and peritumoral brain tissue. Our findings indicate that PPIX fluorescence can be observed in peritumoral edematous areas that are free of neoplastic cells, because PPIX produced by neoplastic cells leaks into the surrounding edematous area. [ABSTRACT FROM AUTHOR]