Evans blue as a selective dye marker for white-light diagnosis of non-muscle-invasive bladder cancer: an in vitro study.

OBJECTIVE: To develop a diagnostic method relying on the preferential accumulation of a dye in non-muscle-invasive bladder cancer (NMIBC) that is visible in conjunction with white-light cystoscopy (WLC). MATERIALS AND METHODS: We investigated in detail the permeation of Evans blue in urothelial cell carcinoma (UCC) spheroids prepared from T24, J82 and RT-112 human cell lines and spheroids composed of normal human urothelial (NHU) cells. To gain more insight into the differential accumulation, all spheroids were investigated ultrastructurally using transmission electron microscopy (TEM). RESULTS: We found that, after exposure to Evans blue for 2 h, UCC spheroids accumulated dramatically more dye than spheroids composed of NHU cells. Using TEM it was found that the malignant spheroids contain similar ultrastructural characteristics, i.e. a wide intercellular space and a decreased number of desmosome-like cell attachments, to those from clinical samples of non-papillary carcinoma in situ of the bladder. CONCLUSION: We believe the present findings could be important for future developments in clinical diagnostics for early bladder cancer detection, staging and grading involving WLC.

Biodistribution and photodynamic effects of polyvinylpyrrolidone-hypericin using multicellular spheroids composed of normal human urothelial and T24 transitional cell carcinoma cells.

Polyvinylpyrrolidone (PVP)-hypericin is a potent photosensitizer that is used in the urological clinic to photodiagnose with high-sensitivity nonmuscle invasive bladder cancer (NMIBC). We examined the differential accumulation and therapeutic effects of PVP-hypericin using spheroids composed of a human urothelial cell carcinoma cell line (T24) and normal human urothelial (NHU) cells. The in vitro biodistribution was assessed using fluorescence image analysis of 5-µm cryostat sections of spheroids that were incubated with PVP-hypericin. The results show that PVP-hypericin accumulated to a much higher extent in T24 spheroids as compared to NHU spheroids, thereby reproducing the clinical situation. Subsequently, spheroids were exposed to different PDT regimes with a light dose ranging from 0.3 to 18 J∕cm2. When using low fluence rates, only minor differences in cell survival were seen between normal and malignant spheroids. High light fluence rates induced a substantial difference in cell survival between the two spheroid types, killing ∼80% of the cells present in the T24 spheroids. It was concluded that further in vivo experiments are required to fully evaluate the potential of PVP-hypericin as a phototherapeutic for NMIBC, focusing on the combination of the compound with methods that enhance the oxygenation of the urothelium.

Use of fluorescein isothiocyanate-human serum albumin for the intravesical photodiagnosis of non-muscle-invasive bladder cancer: an in vitro study using multicellular spheroids composed of normal human urothelial and urothelial cell carcinoma cell lines.

OBJECTIVE: • To evaluate human serum albumin (HSA), fluorescently labelled with fluorescein isothiocyanate (FITC), as a potential intravesical photodiagnostic method for the early detection of non-muscle-invasive bladder cancer. PATIENTS AND METHODS: • By using multicellular spheroids prepared from normal human urothelial (NHU) cells and from different urothelial cell carcinoma (UCC) cell lines (T24, J82), we simulated three-dimensionally the normal urothelium and non-muscle-invasive UCCs present in the bladder of patients. • The distribution of FITC-HSA in these spheroids was investigated. RESULTS: • Our data showed that fluorescently labelled albumin is quite evenly dispersed throughout the spheroids. However, in the case of the 10 mg/mL incubations, the fluorescence intensity seems to increase slightly towards the spheroid core. • Using 1 mg/mL, the penetration of FITC-HSA in T24 differed significantly from the penetration in NHU spheroids, but this was not the case for J82 spheroids. • When the concentration of FITC-HSA was increased 10-fold, all UCC spheroids exhibited a significantly different accumulation of FITC-HSA. CONCLUSIONS: • As spheroids represent a suitable in vitro model for predicting the in vivo behaviour of compounds, our data suggest that FITC-HSA could be used for the early detection of non-muscle-invasive bladder cancer. • Human serum albumin conjugates of new or already available intravesical drugs could be generated to create alternative bladder cancer therapies with increased selectivity.

Human serum albumin as key mediator of the differential accumulation of hypericin in normal urothelial cell spheroids versus urothelial cell carcinoma spheroids.

Hypericin is a bright red fluorescent compound that can be used in urological medicine as a photodiagnostic to detect non-muscle-invasive bladder cancer lesions. To this end a bladder instillation fluid is prepared in which the water-insoluble hypericin is solubilized by the presence of human serum albumin (HSA) to which the compound binds. In the present study, we explored the possibility that besides acting as a passive hypericin carrier, HSA also actively contributes to the selective localization of the compound. By using multicellular spheroids prepared from normal human urothelial (NHU) cells and from different urothelial carcinoma cell (UCC) lines (T24, RT-112 and RT-4), we simulated three-dimensionally the normal urothelium and urothelial cell carcinomas present in the bladder of patients. The distribution of hypericin in these spheroids was investigated in the presence or absence of HSA. Our data show that when hypericin is solubilized by HSA, an excellent differentiation in distribution of hypericin in normal urothelial spheroids and malignant spheroids is observed, clearly suggesting a key role for albumin in the specific localization of hypericin in non-muscle-invasive bladder tumours. Furthermore, PDT results show that both the hypericin-PDT effect on tumour spheroids and the selective character of the treatment can significantly be increased by the presence of HSA. Interestingly, we also observed that the presence of HSA did not convey tumouritropic characteristics to other photosensitizers like pheophorbide a and mTHPP, implying that both the particular characteristics of the photosensitizer and HSA contribute to the final selective accumulation of the compound in tumoural tissue.

In vitro study of the photocytotoxicity of bathochromically-shifted hypericin derivatives.

Hypericin has excellent photosensitizing properties and displays favorable tumouritropic characteristics, but at the same time exhibits minimal dark toxicity. As such, the compound is a promising photosensitizer in the context of clinical photodynamic therapy (PDT). The present study was undertaken to investigate whether a newly-synthesized series of hypericin derivatives with a bathochromic shift shows promise for future PDT applications. Potentially these structures offer an advantage over the parent compound by being photo-activated by red light, which penetrates deeper into tumour tissue. Our results show that 3 compounds (a dibenzoxazole, a pyridazinone, and especially a dibenzthiazole derivative of hypericin), designed to exhibit a bathochromic shift in their absorption spectrum, demonstrated an efficient singlet oxygen yield and intracellular uptake, and concomitantly a potent photocytotoxic effect under white-light conditions. These results indicate that it is possible to synthesize bathochromically-shifted compounds based on hypericin chemistry which maintain their PDT potential. However, the data also show that the present derivatives are only poor photosensitizers when used under red-light conditions.

Orlistat treatment is safe in overweight and obese liver transplant recipients: a prospective, open label trial.

Obesity is a frequent complication following liver transplantation and is insufficiently responsive to dietary and life style advice. We studied the safety of orlistat treatment in obese and overweight liver transplant recipients (n = 15) on a stable tacrolimus-based immunosuppressive regimen. For safety reasons, the treatment period was restricted (6 months 120 mg t.i.d., 3 months 120 mg daily). Three patients dropped out, tacrolimus dose was adjusted in six of 12 remaining patients (dose reduction in 4, increase in 2, P = N.S.). All dose adjustments occurred during the 6 months of orlistat 120 mg t.i.d. therapy. No drug intolerance, adverse events or episodes of rejection occurred during the study. Efficacy of orlistat treatment in this population could not be shown, because a formal control population was not included in this safety trial. Moreover, only a significant decrease of waist circumference (P < 0.01 versus start of the study), but not of weight or body mass index, was achieved in the treated group. Orlistat treatment is well tolerated in liver transplant recipients and can be started safely, provided immunosuppressive drug levels and dietary adherence are closely monitored.