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Purpose: We studied how to avoid irritative bladder symptoms after bladder photodynamic therapy, such as urgency, frequency and pain, which are associated with the inflammation and destruction of normal urothelium., Materials and Methods: Rats bearing orthotopic bladder tumors were instilled with hexyl-aminolevulinate and illuminated with red light at a high vs low (100 vs 15 mW/cm(2)) fluence rate. Cystectomy specimens 48 hours after treatment were subjected to anatomopathological examination. Inflammatory reaction and apoptosis were evaluated. In vivo photobleaching was assessed during illumination at each fluence rate., Results: All superficial tumors were eradicated irrespective of light dose and fluence rate. High fluence rates induced necrosis with inflammatory reaction and absent normal urothelium. Low fluence rates did not provoke inflammation and resulted in apoptotic cell death with preserved urothelial integrity. This could be attributable to faster photobleaching of the photosensitizer in normal urothelium at low fluence rates., Conclusions: Bladder photodynamic therapy at a low fluence rate minimizes side effects without hampering therapeutic efficacy., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Unlabelled: What's known on the subject? and What does the study add? Fluorescence cystoscopy with hexylaminolevulinate (h-ALA, Hexvix®) is known to improve tumour detection in non-muscle-invasive bladder cancer. However, specificity is relatively low and the intensity of the observed fluorescence signal decreases over time due to protoporphyrin IX (PpIX) efflux. This study evaluates in an in vivo model the use of a dendritic 5-aminolevulinic acid compound for fluorescence diagnosis. Fluorescence ratios between tumour and urothelium as well as muscle were significantly better as compared with h-ALA. Sustained synthesis of PpIX accounts for preservation of fluorescence for >24 h., Objective: • To overcome the relative lack of tumour selectivity of fluorescence-guided cystoscopy using 5-aminolevulinic acid (ALA) or its ester derivative (e.g. hexylaminolevulinate, h-ALA; Hexvix®), we evaluated the use of dendrimers bearing different ALA loads in rats bearing orthotopic bladder tumours., Materials and Methods: • Rat bladders were instilled with h-ALA or ALA dendrimers and fluorescence ratio between tumour and normal urothelium, as well as tumour and muscle and depth of fluorescence were determined with Image J software. • Quantification of ALA and/or esters systemic reabsorption was evaluated by high-performance liquid chromatography., Results: • Slow hydrolysis of ALA from dendrimers as observed in vitro implies a higher initial ALA load and longer resting times in vivo. Sustained synthesis of protoporphyrin IX (PpIX) explains persistence of fluorescence for >24 h. • There were significantly better fluorescence ratios with dendrimers, as well as higher penetration depths and absence of systemic reabsorption., Conclusion: • The prolonged and sustained PpIX synthesis, the improved tumour selectivity with a deeper penetration and the absence of systemic reabsorption are primary indicators that ALA dendrimers could be an alternative to h-ALA in fluorescence-guided cystoscopy., (© 2012 THE AUTHORS. BJU INTERNATIONAL © 2012 BJU INTERNATIONAL.)

mTHPC is a non polar photosensitizer used in photodynamic therapy. To improve its solubility and pharmacokinetic properties, liposomes were proposed as drug carriers. Binding of liposomal mTHPC to serum proteins and stability of drug carriers in serum are of major importance for PDT efficacy; however, neither was reported before. We studied drug binding to human serum proteins using size-exclusion chromatography. Liposomes destruction in human serum was measured by nanoparticle tracking analysis (NTA). Inclusion of mTHPC into conventional (Foslip(®)) and PEGylated (Fospeg(®)) liposomes does not affect equilibrium serum protein binding compared with solvent-based mTHPC. At short incubation times the redistribution of mTHPC from Foslip(®) and Fospeg(®) proceeds by both drug release and liposomes destruction. At longer incubation times, the drug redistributes only by release. The release of mTHPC from PEGylated vesicles is delayed compared with conventional liposomes, alongside with greatly decreased liposomes destruction. Thus, for long-circulation times the pharmacokinetic behavior of Fospeg(®) could be influenced by a combination of protein- and liposome-bound drug. The study highlights the modes of interaction of photosensitizer-loaded nanovesicles in serum to predict optimal drug delivery and behavior in vivo in preclinical models, as well as the novel application of NTA to assess the destruction of liposomes., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

Background: Collagen matrices as substitution for connective tissue are known to promote wound healing. Photodynamic therapy has been anecdotally associated with improved wound healing and reduced scarring. The present study investigates the impact of collagen based scaffolding material, embedded with a liposomal formulation of meta-tetra (hydroxyphenyl) chlorin (mTHPC, Foslip(®)) and photodynamic therapy on wound healing in mice., Methods: After incision in the neck region, two different types of collagen material, previously incubated with Foslip(®) at different concentrations, were implanted followed by illumination at 652nm (10J/cm(2), 100mW/cm(2)). Mice were imaged daily up to two weeks, whereafter excision was performed and pathological analysis., Results: Scab detachment was observed at day seven for controls whereas it occurred as early as three days for PDT at the lowest concentrations. In the latter conditions, final matrix remodelling could be observed as evidenced by elastin neosynthesis., Conclusions: Topical application of low dose Foslip(®) in a collagen matrix followed by illumination considerably accelerates wound healing., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We used the phenomenon of previously described photoinduced fluorescence quenching and fluorescence polarization to evaluate the transfer of meta-tetra(hydroxyphenyl)chlorin (m-THPC) from commercial high-drug load liposomes to plasma proteins and model membranes. Fluorescence quenching of m-THPC in liposomes by iodide indicates that part of m-THPC in PEGylated liposomes is localized in the PEG shell, while the rest is bound to the lipid bilayer. It was shown that the two molecule pools in the commercial PEGylated liposomal formulation Fospeg® condition the characteristics of the m-THPC release kinetics. A substantial percentage of m-THPC from Fospeg® is released much faster than from the conventional liposomal formulation Foslip®. Using the technique of resonance light scattering, it was shown that partial m-THPC aggregation is present in liposomes with very high drug loads, higher in PEGylated liposomes compared to conventional ones.

Liposomal formulations of meso-tetra(hydroxyphenyl)chlorin (mTHPC) have already been proposed with the aim to optimize photodynamic therapy. Spectral modifications of these compounds upon irradiation have not yet been investigated. The objective of this study was to evaluate photobleaching properties of mTHPC encapsulated into dipalmitoylphosphatidylcholine (DPPC) liposomes, Foslip. Fluorescence measurements in DPPC liposomes with different DPPC:mTHPC ratios demonstrated a dramatic decrease in fluorescence anisotropy with increasing local mTHPC concentration, thus suggesting strong interactions between mTHPC molecules in lipid bulk medium. Exposure of Foslip suspensions to small light doses (<50 mJ/cm(2)) resulted in a substantial drop in fluorescence, which, however, was restored after addition to the sample of a non-ionic surfactant Triton X-100. We attributed this behavior to photoinduced fluorescence quenching. This effect depended strongly on the molar DPPC:mTHPC ratio and was revealed only for high local mTHPC concentrations. The results were interpreted supposing energy migration between closely located mTHPC molecules with its subsequent dissipation by the molecules of photoproduct acting as excitation energy traps. We further assessed the effect of photoinduced quenching in plasma protein solution. Relatively slow kinetics of photoinduced Foslip response during incubation in the presence of proteins was attributed to mTHPC redistribution from liposomal formulations to proteins. Therefore, changes in mTHPC distribution pattern in biological systems would be consistent with changes in photoinduced quenching and would provide valuable information on mTHPC interactions with a biological environment.

Foslip is a recently designed third generation photosensitiser based on unilamellar dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) liposomal formulations of meta-tetra(hydroxyphenyl)chlorine (mTHPC). The present study investigates Foslip behaviour and its photodynamic efficiency in EMT6 xenografted nude mice at different times following i.v. administration of 0.3 mg kg(-1) mTHPC in a Foslip formulation. Plasma pharmacokinetics and biodistribution were studied by high performance liquid chromatography and were described by a three compartments analysis with half-lifes of 0.13, 4.31 and 35.7 h. The highest tumour to muscle ratios were observed at 6 and 15 h post-administration. Intratumoral distribution was carried out using two photon excitation confocal microscopy. Progressive efflux from the vascular compartment was noted in favour of tumour parenchyma, which was almost completed at 15 h. The best tumour response was obtained for a drug-light interval of 6 h, interval for which mTHPC was present in both endothelial and parenchyma cells. Tumour and plasma concentrations however were far below their maximal values. Based on these observations, we assume that the presence of mTHPC in both vasculature and tumour cells is required for optimal PDT efficacy.

Purpose: Fluorescence guided transurethral resection has gained acknowledgment from the urological community and it is progressively becoming more applied. It has been shown to decrease the recurrence rate of nonmuscle invasive bladder cancer due to incomplete resection due to lack of visualization. The implantation of viable tumor cells seeded during transurethral resection is another reason for recurrence. We investigated whether applying photodynamic therapy on sensitized tumor cells would decrease the amount of viable intraluminal cells and tumor cell implantation., Material and Methods: Two models were designed to mimic the situation after fluorescence guided transurethral resection, including partly or fully de-epithelialized bladders and circulating tumor cells loaded with protoporphyrin IX. Photodynamic therapy was performed. Controls consisted of no drug with no light, light only and drug only. Immediately after photodynamic therapy the intravesical contents were retrieved and clonogenic assays were performed on cells. Bladders were harvested 10 days after cell administration and subjected to pathological analysis., Results: In the photodynamic therapy and control groups tumor volume was proportional to the instilled cell load. Clonogenic assays showed that viable cells were decreased a tenth of the initial administered amount. Tumor implantation decreased to less than a fifth of control values., Conclusions: Photodynamic therapy can effectively decrease the amount of viable tumor cells in the bladder lumen. This results in a significant decrease in tumor implantation. This technique could possibly be used to further decrease the recurrence rate of nonmuscle invasive bladder cancer.

Background and Objectives: Generalized skin sensitization is a main drawback of photodynamic therapy with systemic administration of photosensitizers. We have evaluated the potential use of an intratumoral injection of a liposomal formulation of mTHPC (Foslip) in a mouse model of local recurrence of breast cancer., Materials and Methods: Mice were directly injected into the tumor (IT) with 25 microl of a Foslip suspension (0.15 mg/ml) and illumination (652 nm, 20 J/cm(2)) was performed at different time points with pathological assessment after 48 hours. In a parallel mice series plasma samples were obtained at different endpoints after IT Foslip injection for HPLC analysis and the tumors were subjected in toto to macrofluorescence imaging. Fluorescence polarization measurements were conducted in vitro to estimate the rate of sensitizer redistribution from liposomes., Results: Optimal, albeit partial, cure rates were obtained at 24 hours post-sensitizer and uninistration. Inhomogeneous and weak fluorescence was observed at early time points and became maximal at 24 hours. Plasma levels of mTHPC increased until 15 hours. Fluorescence polarization measurements showed a slow sensitizer transfer from liposomes to model membranes., Discussion and Conclusion: The weak intratumoral fluorescence at early time points could be explained by concentration quenching within the liposomes as evidenced from fluorescence polarization studies. Progressive mTHPC redistribution from liposomes and its further incorporation into tumor tissue resulted in fluorescence build-up over time with a maximum at 24 hours post-injection. This correlates perfectly with the best therapeutic effect at this time point. The absence of total cure can be attributed to inhomogeneous photosensitizer distribution. mTHPC is reabsorbed into the blood stream but the total administered amount is much reduced as opposed to systemic administration so that repeated PDT sessions might be favorable in terms of side effects and tumor response., ((c) 2008 Wiley-Liss, Inc.)

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.

Photodynamic therapy is a complex treatment modality where a large array of factors can influence therapeutic outcome. Vascularization, vessel permeability, oxygenation and light distribution in the tissue as well as immune response play a key role in the photodynamic process. Each of these factors can be influenced by the choice of the animal model. It is therefore of the utmost importance to choose an appropriate model for pre-clinical oncologic PDT studies. Orthotopic tumor models present the closest resemblance to the clinical situation with regard to the elements involved in PDT. We present here a brief organ specific overview of the different orthotopic animal models that can be used for in vivo photodynamic therapy studies.

The present study addresses the impact of different aggregation states of meta-tetra(hydroxyphenyl)chlorin (mTHPC) on the photoinactivation of cells. Measurements of the photophysical properties of mTHPC in MCF-7 cells showed progressive sensitizer aggregation with increasing incubation time. Reconstructed absorption spectra of intracellular mTHPC showed a significant decrease in the molar extinction coefficient and broadening of the Soret band at 24 h incubation compared to 3 h. Intracellular photobleaching of mTHPC slowed down, and the profile changed from mono- to bi-exponential upon incubation. Fluorescence lifetime imaging (FLIM) measurements revealed a substantial decrease in the lifetime of mTHPC fluorescence at 24 h compared to 3 h. In addition, the intracellular localization of mTHPC as observed by fluorescence microscopy changed from a diffuse homogeneous fluorescence pattern at short incubation times to a punctiform pattern at 24 h. The efficiency of photodynamic therapy (PDT) assessed by a clonogenic assay was three times greater at 24 h. However, when the survival curves were replotted as a function of the number of absorbed photons, the efficiency was 1.8 times greater at 3 h than at 24 h. The loss of photosensitizing efficiency at higher mTHPC concentrations was attributed to self-quenching of the triplet states of the sensitizers.

An ideal bladder tumor model consists in an orthotopic solitary tumor with a well-defined localization and stage, as well as unaltered normal mucosa. None of the existing models covers all these requirements. We have created a new model, suitable for diagnostic and therapeutic purposes. Female Fisher rats were divided into different groups according to bladder preconditioning and tumor cell (AY27) administration. Generalized desepithelialization was obtained by an intravesical instillation of HCl, neutralized by NaOH. Localized desepithelialization of the bladder fundus was the result of application of a micro-swab, imbibed with the same chemicals. Tumor cells administration was either generalized (intravesical instillation) or focal (micro swab). No bladder perforations were observed. Generalized desquamation always produced multifocal tumors, whereas focal application of HCl/NaOH resulted in solitary tumors of the bladder fundus, irrespective of the method of tumor cell administration. Only hyperplasia could be detected at day 3. AY27 cells were covered by umbrella cells at day 5 and subepithelial AY 27 tumor nests, covered by full thickness epithelium were observed day 7.

The present study investigates the interaction of the second generation photosensitizer Foscan with plasma albumin and lipoproteins. Spectroscopic studies indicated the presence of monomeric and aggregated Foscan species upon addition to plasma protein solutions. Kinetics of Foscan disaggregation in albumin-enriched solutions were very sensitive to the protein concentration and incubation temperature. Kinetic analysis demonstrated that two types of Foscan aggregated species could be involved in disaggregation: dimers with a rate constant of k1 = (2.30+/-0.15) x 10(-3) s(-1) and higher aggregates with rate constants varying from (0.55+/-0.04) x 10(-3) s(-1) for the lowest to the (0.17+/-0.02) x 10(-3) s(-1) for the highest albumin concentration. Disaggregation considerably increased with the temperature rise from 15 degrees C to 37 degrees C. Compared to albumin, Foscan disaggregation kinetics in the presence of lipoproteins displayed poorer dependency on lipoprotein concentrations and smaller variations in disaggregation rate constants. Gel-filtration chromatography analysis of Foscan in albumin solutions demonstrated the presence of aggregated fraction of free, non-bound to protein Foscan and monomeric Foscan, bound to protein.

Purpose: We evaluated the possibility of performing endoscopic fiber-optic confocal microscopy in a rat bladder model and we distinguished different cell types., Material and Methods: Rhodamine 123 (Molecular Probes, Eugene, Oregon) (100 microM) was instilled for 30 minutes in 5 tumor bearing rat bladders (AY27). Five normal rats served as controls. A Cell-viziotrade mark confocal microscopy fiber was placed transurethrally in contact with normal or transformed bladder wall. Frozen sections were obtained from the same spots and subjected to conventional fluorescence microscopy and anatomical-pathological analysis., Results: The different cells types present in rat epithelium (umbrella, intermediate and basal cells) could easily be identified with the Cell-viziotrade mark device due to their differences in morphology and fluorescence intensity. Individual AY-27 cells could not be demarcated due to the strong fluorescence signal but the entire tumor appeared as a brightly homogenous fluorescent blot surrounded by small inflammatory cells., Conclusions: We report the feasibility of endoscopic, in vivo, fiber-optic confocal microscopy in the rat bladder. We distinguished tumors from normal epithelium and visualized the different epithelial cell types in nontransformed rat bladder epithelium.

Photodynamic therapy (PDT) is an approved anticancer treatment modality that eliminates unwanted cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which is selectively taken up by tumor cells. Present study reports the modalities of cell death after photosensitization of human adenocarcinoma HT29 monolayer and spheroid cells with a second generation photosensitizer Foscan. Kinetics of apoptosis and necrosis after Foscan-PDT in monolayer cells determined by flow cytometry using labeling of cleaved poly(ADP-ribose) polymerase (PARP) and staining with propidium iodide (PI) demonstrated that Foscan was not a strong inducer of apoptosis and necrosis was a prevailing mode of cell death. Cytochrome c release (cyt c) and mitochondrial membrane potential (Deltapsim) addressed by flow cytometry technique at different time points post-Foscan-PDT demonstrated that cell photoinactivation was governed by these mitochondrial events. Foscan-loaded HT29 multicell spheroids, subjected to irradiation with different fluence rates and equivalent light doses, displayed much better tumoricidal activity at the lowest fluence rate used. Apoptosis, measured by caspase-3 activation was evidenced only in spheroids irradiated with the lowest fluence rate and moderate fluence inducing 65% of cell death. Application of higher fluence rates for the same level of photocytotoxicity did not result in caspase-3 activation. The observation of the fluence rate-dependent modulation of caspase-3 activity in spheroids offers the possibility of regulating the mechanism of direct cell photodamage and could be of great potential in the clinical context.

In the present study, the systemic absorption of hypericin was investigated after intravesical instillation of the compound in nine patients with superficial transitional cell carcinoma (TCC) bladder tumors. Hypericin (8 microM) was instilled in the bladder for 2-3 h before photodynamic diagnosis of bladder tumors. Blood was then collected from a peripheral vein 1 h after termination of the instillation. Solid phase extraction with ammonium acetate buffer and methanol was used to extract hypericin from the plasma. A reversed-phase high performance liquid chromatographic method with fluorescence detection was used to identify and quantify hypericin in the extracts from plasma samples. Analysis of standard plasma samples, which were spiked with known amounts of hypericin, indicated that the pH of the buffer was a determining factor in the extraction yield. The results obtained using ammonium buffer (pH 3.5) and methanol showed the mean extraction recovery of hypericin to be 64% (RSD=12%, n=6). The limits of detection and quantification were 6 and 20 nM, respectively. Extraction and analysis of the plasma of patients after intravesical administration showed hypericin concentrations below the detection limit (<6 nM). In addition, photodynamic treatment of in vitro cultured HeLa cells incubated with 1-100 nM hypericin concentrations showed that lower concentrations (1-20 nM) of hypericin do not induce significant photocytotoxic effects. Taken together, these results imply that photosensitization or other systemic side effects in patients are not to be expected after photodynamic diagnosis of TCC bladder tumors with hypericin.

Whole-bladder wall photodynamic therapy (PDT) is a promising treatment for carcinoma in situ (CIS) and diffuse premalignant changes of the bladder. After the results of our clinical studies showing that intravesical hypericin selectively accumulates in superficial bladder tumors, we investigated the hypericin-PDT efficacy in an AY-27 orthotopic transitional cell carcinoma rat bladder tumor model. After the instillation of hypericin (30 microM, 2 hr) in the bladder, tumors were irradiated (25-50 mW/cm 6-48 J/cm(2)) using 595 nm laser light. Data demonstrate that light doses of 12-48 J/cm(2) resulted in selective PDT-induced urothelial tumor damage without damaging detrusor musculature. Histological assessment of bladder sections 2 days after PDT showed tumor destruction, with tumor cells shrinking and detaching from the bladder wall. There were tumor regrowth 1-3 weeks after treatment. The in vivo/in vitro clonogenic assay results revealed up to 98% of tumor cell kill by hypericin PDT. In conclusion, hypericin PDT can be used to safely induce a selective urothelial tumor damage without damaging detrusor musculature, when optimum hypericin concentration and light fluences are used. A small percentage (2-5%) of tumor cells that survive the photodynamic treatment resulting in tumor regrowth after a prolonged period of time is likely due to oxygen depletion during light irradiation., (Copyright 2003 Wiley-Liss, Inc.)

To determine the initial photodamage sites of Foscan-mediated photodynamic treatment, we evaluated the enzymatic activities in selected organelles immediately after light exposure of MCF-7 cells. The measurements indicated that the enzymes located in the Golgi apparatus (uridine 5'-diphosphate galactosyl transferase) and in the endoplasmic reticulum (ER) (nicotinamide adenine dinucleotide [reduced] [NADH] cytochrome c [cyt c] reductase) are inactivated by the treatment, whereas mitochondrial marker enzymes (cyt c oxidase and dehydrogenases) were unaffected. This indicates that the ER and the Golgi apparatus are the primary intracellular sites damaged by Foscan-mediated PDT in MCF-7 cells. We further investigated whether the specific mitochondria events could be associated with Foscan photoinduced cell death. The dose response profiles of mitochondrial depolarization and cytochrome c release immediately after Foscan-based PDT were very different from that of overall cell death. By 24 h post-PDT the fluence dependency was strikingly similar for both mitochondrial alterations and cell death. Therefore, although mitochondria are not directly affected by the treatment, they can be strongly implicated in Foscan-mediated MCF-7 cell death by late and indirect mechanism.

An effective therapeutic outcome in the treatment of bladder cancer is largely defined by its early detection. In this context, big expectations have been placed on the fluorescence-guided diagnosis of bladder cancer. This paper reviews the applications of endo- and exogenous fluorescence for early diagnosis of in situ carcinoma of the bladder. Despite certain advantages of autofluorescence, exogenous fluorescence, based on the intravesical instillation of fluorophores with the following visible light excitation, has been shown to be more effective in terms of sensitivity and specificity for detecting carcinoma in situ. The equipment consists of a slightly modified light source in order to choose between white (conventional endoscopy) or blue light (fluorescence endoscopy) excitation, and specific lenses, in order to enhance maximally the contrast between normal (blue) autofluorescence and red fluorescence from malignancies. Among exogenous fluorophores, a particular emphasis will be put on the 5-aminolevulinic acid (ALA), its ester derivative (h-ALA) and hypericin. These dyes demonstrated an excellent sensitivity above 90% and specificity ranging from 70% to 90%.

Objective: To determine the use of hypericin instillation for the fluorescent detection of papillary bladder cancer and carcinoma in situ., Patients and Methods: Eighty-seven patients with papillary bladder cancer and/or carcinoma in situ received instillations with 40 mL of an 8 micromol/L hypericin solution for at least 2 h. Fluorescent excitation with blue light was effective for up to 16 h, and biopsies were examined by fluorescence microscopy., Results: There were no side-effects reported, no photobleaching and all papillary lesions fluoresced red. The sensitivity and specificity for detecting carcinoma in situ was 94% and 95%, respectively. An interval of 4 months is recommended after BCG instillations before using this test. Fluorescence microscopy showed that hypericin was selectively localized in the epithelium., Conclusions: Hypericin-induced fluorescence has a high sensitivity and specificity for detecting bladder cancer. After 4 months there are few false-positive results in patients treated with BCG.

In a recent clinical study, we reported a selective uptake of hypericin in superficial bladder tumors. The results suggested that hypericin, a potent photosensitizer, could be used not only for diagnosis but also for photodynamic therapy (PDT) of superficial bladder tumors. In the present study, we investigated the biodistribution of hypericin in an orthotopic rat bladder tumor model by assessing the extent of hypericin penetration and the kinetics of accumulation into rat bladder tumors and normal bladder wall. Hypericin (8 or 30 microM) was instilled into the bladder via the catheter for 1, 2 or 4 hr. The fluorescence of hypericin in the bladder tumors and normal bladder was documented using fluorescence microscopy. In situ quantification of hypericin fluorescence in the tumor or normal bladder was performed using the laser-induced fluorescence technique. There was much more hypericin fluorescence in the tumor than in the normal bladder, with the tumor-to-normal-bladder ratio mounting to 12:1 after 4 hr of hypericin (30 microM) instillation. Moreover, hypericin was retained in the tumor for at least 1 hr before it was gradually lost from the tissue. Microscopically, the fluorescence of hypericin was restricted to the urothelial tumor and normal urothelium without fluorescence in the submucosa and the muscle layers. Subsequently no hypericin was detected in plasma, indicating that under these conditions systemic side effects should not be expected. Because the conditions used in this study were similar to those used in our previous clinical study, it is therefore likely that whole bladder wall PDT in the clinic under these conditions will produce selective urothelial tumor destruction without causing damage to the underlying muscle layers., (Copyright 2002 Wiley-Liss, Inc.)

In a recent clinical study we showed that hypericin accumulates selectively in urothelial lesions following intravesical administration of the compound to patients. In the present study the efficacy of hypericin as a photochemotherapeutic tool against urinary bladder carcinoma was investigated using the AY-27 cells (chemically induced rat bladder carcinoma cells). The uptake of hypericin by the cells increased by prolonging the incubation time and increasing the extracellular hypericin concentration. Photodynamic treatment of the cells incubated with 0.8 and 1.6 microM hypericin concentrations resulted in remarkable cytotoxic effects the extent of which depended on the fluence rates. Photoactivation of 1.6 microM hypericin by 0.5, 1.0 or 2.0 mW/cm2 for 15 min resulted in 3, 30 and 95% of the antiproliferative effect, respectively. Increasing the photoactivating light dose from 0.45 to 3.6 J/cm2 resulted in a five-fold increase in hypericin photodynamic activity. Irrespective of the fluence rates and irradiation times incubation of the cells with 10 microM hypericin induced rapid and extensive cell death in all conditions. The type of cell death (apoptosis or necrosis) induced by photoactivated hypericin depended largely on the hypericin concentration and the postirradiation time. At lower hypericin concentrations and shorter postirradiation times apoptosis was the prominent mode of cell death; increasing the hypericin concentration and/or prolonging the postirradiation time resulted in increased necrotic cell death. Cell pretreatment with the singlet oxygen quencher histidine, but not with the free-radical quenchers, significantly protected the cells from photoactivated hypericin-induced apoptosis, at least when a relatively low concentration (1.25 microM) was used. This result suggests the involvement of a Type-II photosensitization process. However, cells treated with higher hypericin concentrations (2.5-5 microM) were inadequately protected by histidine. Since hypericin is thus shown to be a potent and efficient photosensitizer, and since the conditions used were the same as when hypericin is used clinically to locate early-stage urothelial carcinoma lesions, hypericin may well become very important for the photodynamic treatment of superficial bladder carcinoma.

In a recent clinical study, we showed that hypericin accumulates selectively in urothelial lesions of the bladder following intravesical administration of the compound in patients. This observation infers that hypericin, a potent photosensitizer, could be used as a selective photodynamic therapy (PDT) tool against superficial bladder cancer. In the present study we investigated the in vivo PDT activity of hypericin in transition cell carcinoma (TCC) tumors of the bladder. Both the distribution and tumor PDT response were carried out using subcutaneous heterotopic AY-27 TCC tumors in syngeneic rats. For both PDT and distribution studies, hypericin (1 or 5 mg/kg) was injected intravenously 0.5, 6 or 24 h before PDT or distribution evaluation. The data show that hypericin is a potent photosensitizer in the treatment of TCC tumors in vivo and that the interval between drug administration and photo-irradiation has a dramatic effect on the PDT outcome. Using a 0.5 h interval between drug administration and photo-irradiation the tumor regrowth study indicated that no tumor mass could me measured 9-10 days after PDT. On the contrary, lengthening the time interval between drug administration and photo-irradiation resulted in a gradual loss of PDT efficiency in these tumors. For instance, while the 6 h drug interval protocol produced a moderate PDT activity in which the tumor sizes decreased to about 50% of their original sizes 11-16 days after photo-irradiation, the 24 h interval protocol was even less effective. The distribution data indicate that the PDT efficiency of hypericin in TCC tumors corresponded to the plasma concentrations rather than to the over all concentrations in the tumor. It is therefore conceivable that the mechanism of PDT efficacy of hypericin in TCC tumors is through indirect (vascular effects) rather than through direct effects (cellular destruction) of hypericin in these tumors. In conclusion, our data indicate that hypericin is a potent photosensitizer against AY-27 TCC tumors and that the PDT efficacy of hypericin is largely determined by photosensitizer distribution in the tumor at the time of photo-irradiation.

Purpose: We determined the sensitivity and specificity of detecting flat bladder carcinoma in situ through fluorescent detection after intravesical hypericin instillations., Materials and Methods: The study included 40 patients, of whom 26 presented with macroscopic visible tumor, 9 had a positive cytology without visible tumor and 5 underwent cystoscopy after bacillus Calmette-Guerin instillations (4) or radiotherapy (1). We instilled 40 ml. of a 8 microM. solution of hypericin intravesically for at least 2 hours. Fluorescence excitation with blue light was effective up to 16 hours after termination of the instillation., Results: All visible papillary tumors showed red fluorescence. In addition, 134 flat fluorescent areas were detected. Analysis of 281 biopsies from flat bladder wall indicated 93% sensitivity and 98.5% specificity for detecting carcinoma in situ. Visible lesions resulting from radiotherapy, chemotherapy or immunotherapy did not show any fluorescent signs and, therefore, did not induce false-positive readings. There were no signs of photobleaching during inspection and resection., Conclusions: We report a simple yet comprehensive endoscopic method for early detection of bladder cancer, including carcinoma in situ. Hypericin induced fluorescence has a high sensitivity and specificity for detection of bladder transitional cell carcinoma, papillary and flat carcinoma in situ. When carcinoma in situ is suspected, this technique is highly recommended.

Recently, we reported the selective accumulation of hypericin in transitional cell carcinoma cells following intravesical instillation of hypericin in humans. This observation infers that hypericin, a potent photosensitizer, could be used as a selective PDT (photodynamic therapy) tool against superficial bladder cancer. The aim of the present study was to investigate in vitro whether hypericin exhibits specific affinity for TCC transitional cell carcinoma) bladder cells and to assess its photocytotoxic effect. Three human TCC cell lines (J-82, T-24 and RT-4), a chemically induced rat TCC cell line (NBT-II), but also non-bladder carcinoma cells (HeLa, A431, MCF-7 and MCF-***ADR) and normal cells (HEL229, RPE and PHK), were used in this comparative study. Flow cytometric analysis of cells treated with different hypericin-containing vehicles for various incubation times (2 hours or 24 hours) indicated that short exposure of the cells (2 hours) to hypericin in the absence of serum results in the highest intracellular accumulation of the compound. As expected, prolonging the incubation time increased both the cellular accumulation and photocytoxicity of hypericia. With the exception of the RT-4 and MCF-7 cells (which were less sensitive to hypericin), all the other carcinoma cell lines examined showed equal sensitivity to the photoactivated hypericia, independently of their histological origin (bladder or non-bladder). Moreover, normal cells exhibited the same pattern of hypericin photosensitivity as shown by the cancer cells, indicating that, in cultured cells, hypericin cellular uptake and subsequent photokilling is not selective. This suggests that in vivo factors other than the cancer cells themselves are responsible for the specific accumulation of hypericin in urothelial carcinoma lesions.

Carcinoma in situ (CIS) of the bladder is a confounding disease that is difficult to recognize endoscopically because it is a flat cancer. Many studies have suggested its relationship with subsequent invasive disease. Early recognition of CIS therefore is essential in offering the patient the most appropriate treatment and the highest cure rate. Because white light cystoscopic examination is not sufficient to reveal areas of dysplasia or CIS, random biopsies are recommended. The authors evaluate whether amino levulinic acid (ALA) fluorescence detection could be helpful in diagnosing CIS and if the specificity could be enhanced by reducing the ALA dose. Sixteen patients with papillary bladder cancer, and CIS and dysplasia were given low-dose ALA. Fluorescence detection of the metabolized ALA was performed 3 hours later, with the naked eye, after blue light illumination. Carcinoma in situ or dysplasia was found in 50 biopsies. The sensitivity for detecting CIS was 94% with a specificity of 54%. Carcinoma in situ can be diagnosed with a very high accuracy through fluorescence detection after ALA instillation. Fluorescence detection can be achieved with the naked eye and does not necessitate either complex equipment or specially trained personnel.

The early diagnosis of bladder cancer is central to the effective treatment of the disease. Presently, the detection of bladder tumors relies on cystoscopy and there are no methods available to easily and specifically identify the presence of bladder cancer cells. A variety of new technologies and potential tumor markers are being studied in bladder cancer and some are being translated into clinical use. It is important to realise that all available results on the diagnostic value of tumor markers do not allow firm clinical recommendations, but tests based on biomarkers will undoubtedly influence the management of bladder cancer in the near future.

Purpose: We analyzed the value of the Bard bladder tumor antigen (BTA*) test for the diagnosis of stage Ta superficial bladder cancer and carcinoma in situ, and compared it to the highly sensitive bladder washing cytology., Materials and Methods: The BTA test is a latex agglutination test that qualitatively detects the presence of basement membrane complexes in the urine. A total of 60 patients with superficial bladder cancer underwent voided urine BTA analysis and bladder washing cytologies., Results: Of the patients 65% were correctly diagnosed with the BTA test compared to 32% with bladder washings, which is statistically significant (p < 0.001)., Conclusions: The BTA test is a noninvasive diagnostic tool that is superior to bladder washing cytology for diagnosing superficial bladder cancer.

Objectives: This article evaluates the results of whole bladder wall photodynamic therapy (PDT) for multifocal carcinoma in situ after a mean follow-up time of 3 years., Methods: Photofrin II was used as a photosensitizer (2 mg/kg) and in situ dosimetry to obtain the best possible central positioning of the light diffuser as well as to know exact dosimetry data (scattered plus nonscattered light)., Results: Major classical drawbacks of PDT, such as severe bladder irritative symptoms and bladder shrinking, can be minimized with the help of in situ dosimetry. The success rate after 3-year follow-up is 60%. Fifty percent of the recurrences occurred in the prostatic urethra without evidence of disease of the bladder., Conclusions: A success rate of 60% is comparable to the results obtained after bacille Calmette-Guérin (BCG). Side effects such as loss of bladder capacity can be minimized with adequate light dosimetry but they are still higher than with BCG (9% versus 1% cystectomy).

Fluorescence tagging of tumors by sensitizing agents such as hematoporphyrin derivatives is an efficient method for diagnosing tumors but presents, even at low doses, a number of serious drawbacks for the patients. We demonstrate a fiberoptic instrument for the diagnosis of bladder cancer, based on a tripled frequency Nd YAG laser, without need for sensitizing agents, based on the autofluorescence of tissues. The contrast demarcation obtained for CIS and TCC respectively is 2.2 and 2.7, which is about 30% higher than what can be obtained after photosensitizing. The integration of the diagnostic method with a reliable therapeutic technique for tumor cell destruction, opens the way for cost-effective preventive care of high risk patients.

The diagnosis of transitional cell bladder carcinoma and especially carcinoma in situ of the bladder in spinal cord disordered persons is often made difficult by catheters, infections or stone-induced chronic inflammation. Fluorescence tagging of tumors by sensitizing agents such as hematoporphyrin derivatives enhances visualization but presents a number of drawbacks for the patients, even at low doses. We have developed a cystoscopic fiber optic instrument, based on a mercury arc lamp, for in vivo detection of human bladder carcinoma without sensitizing agents. The tissue autofluorescence upon UV excitation (365 nm) is detected and a demarcation contrast ratio for carcinoma in situ and transitional cell carcinoma of 2.6 and 3.2 respectively is obtained. This demarcation ratio is 60 percent higher than the contrast ratio obtained after photosensitizer injection. The integration of a reliable diagnostic method with a known efficient therapeutic technique (Nd YAG laser irridiation) opens the way for cost-effective preventive care of high-risk patients.

Photodynamic therapy (PDT) using Photofrin IIR (PII) as a photosensitizer is currently being evaluated as a new treatment modality for superficial bladder cancer. An optimum therapeutic ratio requires uniform illumination of the whole bladder wall and accurate light dosimetry. The first clinical light dosimetry results (16 patients) are reported, obtained using a system which allows in situ measurement and control of the light fluence at the bladder wall. The true light fluence at the bladder wall (i.e. non-scattered incident light plus scattered light) appeared to be on the average a factor beta = 4.8 +/- 1.2 (mean +/- SD) larger than the non-scattered incident light fluence. The latter is often, but incorrectly, used in reporting light fluence. The factor beta varied between patients with extremes of 2.5 and 7.1. Because such large variations were unexpected, but may have significant clinical consequences, experiments in plastic bladder models were performed to study separately the various factors (e.g. bladder shape, air bubble) affecting the dosimetry in clinical treatments. The results imply that the clinical variations are most likely to be the result of variations in optical properties of the bladder wall mucosa, probably due to the disease and prior treatments. If light dosage is based on non-scattered light only (without in situ light dosimetry, according to a current clinical protocol) the present results indicate variations in the true (total) light fluence between patients by a factor of at least 2. At the least this may cause unnecessary discomfort to a number of patients.

A point-monitoring fluorescence diagnostic system based on a low-energy pulsed laser, fiber transmission optics, and an optical multichannel analyzer was used for diagnosis of patients with bladder malignancies. Twenty-four patients with bladder carcinoma, carcinoma in situ, and/or dysplasia were injected with hematoporphyrin derivative, Photofrin, 0.35 or 0.5 mg/kg body weight, forty-eight hours prior to the investigation. The ratio between the red sensitizer emission and the bluish tissue autofluorescence provided excellent demarcation between papillary tumors and normal bladder wall. Certain cases of dysplasia also could be differentiated from normal mucosa. Benign exophytic lesions such as malakoplakia appeared different from malignant tumors in fluorescence. Flat suspicious bladder mucosa such as seen in infectious diseases or after radiation therapy appeared normal on fluorescence.

We report on the preliminary results of 12 patients with multifocal carcinoma in situ of the bladder treated with whole bladder wall photodynamic therapy. The total light dose (scattered plus nonscattered light) measured in situ was 100 joules per cm.2 in the first 6 patients (group 1) and 75 joules per cm.2 in the remaining 6 (group 2). These light doses correspond on the average to 27 joules per cm.2 and 15.5 joules per cm.2 nonscattered light as reported by other investigators. Followup ranged from 6 to 22 months (average 11.5). In group 1, 2 tumors recurred after 6 and 9 months, respectively, and 2 other patients had a permanently shrunken bladder without evidence of disease. In group 2, 1 tumor recurred 5 months after photodynamic therapy. In this group the bladder capacity increased on the average to 135% of the pretreatment value 3 months after photodynamic therapy. All recurrences were in patients with a history of invasive bladder cancer (stages T1 and T2). These preliminary results demonstrate the importance of in situ scattered light dosimetry for minimizing local side effects of whole bladder photodynamic therapy.

The authors present a series of 175 femoropopliteal (below-knee) and 65 femorotibial reconstructions with the human umbilical vein (HUV) graft performed over an eight year period. With a mean follow-up of 36.7 months (range one month to 84 months) the early patency rate of 89% decreased to 54% after five years. Long-term patency was found to be primarily related to the location of the distal anastomosis and the quality of the outflow, showing a statistically significant decrease after repeat revascularization. Early thrombosis, even in the absence of a technical failure and late aneurysmal degeneration remain the major problems associated with the use of the HUV graft. It is therefore recommended that these reconstructions be preserved for patients with advanced ischemia or a limited life-expectancy.

A case report of large-bowel perforation after intravesical neodymium-yttrium aluminum garnet laser irradiation is presented. Recommendations to prevent bowel injury are discussed.