A study comparing endogenous protoporphyrin IX induced by 5-ALA and ALA-methyl ester with exogenous PpIX and PpIX dimethyl ester in photodynamic diagnosis of human nasopharyngeal carcinoma xenografts.

5-Aminolevulinic acid (5-ALA) and its esters have been under intense investigation to enhance the endogenous production of protoporphyrin IX (PpIX) in tumour cells for the purpose of photodynamic diagnosis. In this study we have investigated the use of exogenous PpIX and its dimethyl ester (PME) and compared the results with endogenous PpIX produced via 5-ALA and ALA methyl ester (AME) in poorly differentiated NPC/CNE-2 nasopharyngeal carcinoma cells in both in vivo and in vitro systems. All prodrugs and photosensitizers were administered to tumour bearing balb/c nude mice either intravenously or topically. in vitro results show that 5-ALA induced more PpIX fluorescence when compared with AME in NPC/CNE-2 cells and PME showed better uptake than PpIX. in vivo results show that exogenous PpIX and PME show promise as good candidates as photosensitizers for photodynamic diagnosis as they exhibit significant selectivity between tumour tissue and normal tissue at 3 h. Modification of delivery vehicle used for application of exogenous PpIX and PME could allow for rapid uptake; better selectivity and localization of the photosensitizer.

Photodynamic diagnosis of a human nasopharyngeal carcinoma xenograft model using the novel Chlorin e6 photosensitizer Fotolon.

The current study investigates the potential of the photosensitizer Fotolon as a photodynamic diagnostic agent in an in vivo system of human nasopharyngeal carcinoma (NPC) cells. Fotolon (formerly known as Photolon) represents a complex of trisodium salt Chlorin e6 and polyvinylpyrrolidone (PVP). It is a photosensitizing agent that selectively accumulates in tumour tissues. A poorly differentiated human nasopharyngeal carcinoma cell line CNE-2 was xenografted on 6-8 weeks old male balb/c nude mice for our photodynamic diagnostic (PDD) studies. A fluorescence endoscope system was used to perform the in vivo macroscopic fluorescence digital imaging of tumours on the mice. The macroscopic images were further analyzed for distribution of fluorescence intensity. Laser fluorescence confocal microscopy was used to capture microscopic fluorescence images of the tumour tissue. In our PDD studies, we observed intense fluorescence in the tumour and tumour vasculature of human NPC xenografts on nude mice as early as 15-min post administration of Fotolon. High fluorescence selectivity in the tumour tissue was observed between 3-h and 6-h time points. The tumour to normal tissue selectivity ratio was highest at 6 h. The microfluorescence tumour imaging shows similar trends confirming the macroscopic fluorescence data. Fotolon shows much promise as a good photodiagnostic agent.

Novel photodynamic diagnosis of bladder cancer: ex vivo fluorescence cytology using hypericin.

In this study we have evaluated the use of hypericin ex vivo urine fluorescence cytology as a non-invasive method for detecting early bladder cancers. To date this is the first study reported using this technique with hypericin. Urine samples from patients with early bladder cancers were processed for fluorescence cytology by incubation with hypericin, a novel photosensitizer. Normal urine samples incubated with hypericin served as normal controls. Laser confocal microscopy and spectroscopy was used to detect the fluorescence in the exfoliated low-grade urothelial tumor cells. Fluorescence cytology was considered positive if hypericin fluorescence of the low-grade urothelial tumor cells was detected to be stronger (>8.5 times) compared to the baseline fluorescence established for normal urine samples. Automated analysis for an objective reproducible outcome appears possible. The possibility of detection of malignant urothelial cells in early cancer makes ex vivo fluorescence cytology promising for routine diagnostic screening.

Macro-microscopic fluorescence of human bladder cancer using hypericin fluorescence cystoscopy and laser confocal microscopy.

The early detection of carcinoma is very essential for the diagnosis and prognosis of a bladder cancer patient. In this study we have investigated the use of hypericin as a fluorescent tumour marker and laser confocal microscopy as a diagnostic tool to aid the diagnosis of such cancers. Both cellular and clinical studies have been conducted. In the cellular studies, we have compared two bladder cell lines for the uptake and sub-cellular localization of hypericin. It was found that there was a rapid uptake and clearance of hypericin and significant localization in mitochondria and lysosomes. The study also revealed that there was a time-dependent increase in fluorescence intensity in bladder cells. The optimum localization was found to be 2-4 h post drug incubation. In the clinical study, consisting of 30 patients, both white light and fluorescence cystoscopy were performed after hypericin instillation. Biopsies taken from fluorescing regions were imaged using the confocal microscope. The order of fluorescence was detected to be as follows: normal < inflammation < grade 1 < grade 2 < CIS < grade 3. It was also found that the fluorescence intensity increased with the stage of the disease thereby enabling the determination of the degree of invasiveness of cancer. This enables the use of hypericin as a prognostic marker and laser confocal microscopy as a tool to aid in diagnosis of bladder cancer.

Fluorescence confocal microscopy and image analysis of bladder cancer using 5-aminolevulinic acid.

5-aminolevulinic acid mediated changes in tissue specific fluorescence were studied in bladder cancer. Bladders of normal patients and also patients diagnosed with cancer were instilled with 5-aminolevulinic acid and the resultant protoporphyrin IX mediated fluorescence intensity was imaged and quantified with confocal laser microscopy and fluorescence image analysis. Urothelial tumour cells were observed to fluoresce more intensely than normal urothelial cells. Submucosa and muscle tissues exhibited minimal fluorescence compared to urothelial cells of malignant origin and also normal urothelial cells. Degree of fluorescence intensity was in the order of malignant urothelium > normal urothelium > normal submucosa > normal muscles. Fluorescence intensity was also found to increase with duration of ALA instillation. Grade 3 malignant cells produced more fluorescence compared to grade 2 and grade 1. Similarly, T1 transitional cell carcinoma (TCC) showed increased fluorescence intensity than that of Ta TCC. Also, tumour blood vessels fluoresced more intensely compared to blood vessels found in normal bladder tissue. Tissue specific ALA mediated PpIX micro fluorescence can be used as a diagnostic technique for early detection of neoplasms and confocal laser microscopy and fluorescence image analysis are advantageous diagnostic tools for the photodynamic diagnosis of bladder neoplasms in vivo.

A study of 5-aminolevulinic acid and its methyl ester used in in vitro and in vivo systems of human bladder cancer.

The use of 5-aminolevulinic acid and its esters to induce endogenous porphyrins for the purpose of detection of epithelial cancers is being studied extensively in many centres around the world. The challenge is to prepare an efficacious formulation for the purpose of cancer detection. Photodynamic diagnosis of cancer using 5-aminolevulinic acid (ALA) and its ester derivatives is being actively investigated. In this study, we compared ALA with ALA methyl ester (AME) derivative in terms of PpIX fluorescence intensity in in vitro and in vivo systems of bladder carcinoma. For the in vivo system consisting of RT112 xenografts, the modes of drug administration compared were intravenous administration and topical application. The Karl Storz fluorescence endoscopy system was used to obtain macroscopic fluorescence images. The macroscopic images were further analysed for fluorescence intensity distribution. For the intravenous administration, over all time points studied (1, 3, 6 h), AME-PpIX fluorescence was lower than ALA-PpIX fluorescence and was cleared at a faster rate than the ALA-PpIX when administered intravenously. Topical application with two different polymers, Gantrez and Polyvinyl pyrrolidone (PVP) which are fast releasing polymers was found to be comparable in inducing PpIX fluorescence. Topical AME-PpIX fluorescence was found to be comparable with ALA-PpIX fluorescence. The results of this study suggest that the AME can also be used as a good diagnostic agent.

Macro-microscopic fluorescence imaging of human NPC xenografts in a murine model using topical vs intravenous administration of 5-aminolevulinic acid.

The use of 5-aminolevulinic acid to induce endogenous porphyrins for the purpose of detection of epithelial cancers is being studied extensively in many centres around the world. The challenge is to prepare an efficacious formulation of 5-ALA for the purpose of cancer detection. In this study, we compared two formulations of topical 5-ALA applications with intravenous administration in NPC/CNE-2 xenografts on balb/c nude mice. One of the formulations was a gantrez muco-adhesive patch and the other was a polyvinyl-pyrolidone muco-adhesive patch. The Karl Storz fluorescence endoscopy system was used to obtain macroscopic fluorescence images. Microscopic fluorescence imaging was done by laser confocal microscopy. The macroscopic images were further analysed for fluorescence intensity distribution. It was found that between the two formulations of topical application of 5-ALA; there was very little difference in the fluorescence biodistribution. When the topical applications were compared with the intravenous administration, the tumour to normal differential in biodistribution was significantly higher with the topical application compared to the intravenous application.