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.

Biodistribution of PVP-hypericin and hexaminolevulinate-induced PpIX in normal and orthotopic tumor-bearing rat urinary bladder.

PURPOSE: In this preclinical study, we examined the biodistribution of hypericin formulated as its water-soluble PVP-hypericin complex in the different layers (urothelium, submucosa, muscle) of a normal rat bladder and a rat bladder bearing a malignant urothelium composed of syngeneic AY-27 tumor cells. The results were compared with the biodistribution of hexaminolevulinate (HAL)-induced protoporphyrin IX (PpIX). METHODS: Freshly prepared PVP-hypericin and HAL solutions were instilled in both normal as well as tumor-bearing rat bladders. Following instillation, bladders were removed and snap frozen in liquid nitrogen. Fluorescence of PVP-hypericin or PpIX-induced HAL was measured in the bladder layers and quantified using image analysis software. RESULTS: The results of these experiments show that PVP-hypericin (30 µM) accumulated about 3.5-fold more in malignant urothelial tissue when compared to normal urothelium, whereas PpIX accumulated to the same extent in malignant and normal urothelium, both after intrabladder instillation of 8 or 16 mM HAL. Besides, PVP-hypericin and PpIX accumulated selectively in the urothelium with a tumor-to-muscle ratio of 30.6 for PVP-hypericin and 3.7-8.3 for 16 and 8 mM HAL, respectively. CONCLUSIONS: This study shows that PVP-hypericin appears to have great potential as a photodynamic agent against non-muscle-invasive bladder cancers after intravesical administration, with a limited risk of affecting the deeper layers of the bladder.

An improved orthotopic rat bladder tumor model using Dil-loaded fluorescent AY-27 cells.

Here we evaluate an improved orthotopic rat bladder tumor model, to be used for the evaluation of the therapeutic potential of novel cancer therapeutics. Before instilling AY-27 tumor cells into chemically denudated rat bladders, AY-27 cells were labeled with the fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). We found that the presence of Dil did not alter the in vitro AY-27 cell proliferation and that the Dil label was strongly associated with the cells. We further provide evidence that the use of fluorescently labeled AY-27 tumor cells allows the visualization and hence the validation of the orthotopic tumor inoculation process. Using this technique it was possible to track down the tumor cells after inoculation into the bladder, which makes it straightforward to distinguish tumor cells from remaining or regenerated normal urothelium over a period of 5 d. The results also demonstrated that malignant AY-27 tissue exists as an intact non-muscle invasive bladder tumor only for 1-3 d after cell implantation. Accordingly the AY-27 bladder tumor model was used to evaluate the antitumoral effect of a single intravesical MM-C instillation. All rats instilled with 1 mM MM-C survived the final endpoint of 30 d after intravesical MM-C. Moreover, 10 and 30 d after treatment the urothelium of the MM-C-treated animals was completely restored. Remarkably, after MM-C treatment distinct patchy fluorescent dots were found into the submucosa and the regenerated urothelium, suggesting that dye retention is secondary to the digestion of Dil-loaded AY-27 cells and cellular debris by macrophages and related immune cells.

Prevention of tumor cell reimplantation during transurethral resection: the in-vitro antiadhesive and cytotoxic properties of an irrigant containing polyethylene glycol 400.

A major challenge to urologists with respect to bladder cancer is the high rate of tumor recurrence after transurethral resection (TUR). Implantation of resected tumor cells on traumatized bladder urothelium is believed to be the main cause of tumor recurrence. The aim of this study was to find a safe irrigant fluid and modality that prevents reimplantation of malignant cells during TUR. Therefore, the cytotoxicity and antiadherence effects of polyethylene glycol 400 (PEG400) and PEG4000 were investigated and compared with currently used irrigant fluids, water and 1.5% glycine (G-IF), on the RT112 urothelial cell carcinoma cell line. PEG400 (20%), G-IF, water and to a lesser extent 10% PEG400 and PEG4000 showed dramatic cytotoxic effects, besides exhibiting interesting antiadherence characteristics. The presence of serum proteins did not interfere with the activity of PEG400. In a clonogenic assay, both water and 20% PEG400 showed a better cytotoxic profile than G-IF, and it was found that these two fluids were able to induce a 5-log kill. This study shows that a solution of 20% PEG400 in water is a promising irrigant with antiadhesive and cytotoxic properties, which could be used to prevent tumor cell reimplantation during TUR. The irrigant remains active in the presence of serum proteins, is transparent, inexpensive and possesses an excellent safety profile.

TRPV1 is involved in stretch-evoked contractile changes in the rat autonomous bladder model: a study with piperine, a new TRPV1 agonist.

AIM: Vanilloids like capsaicin and resiniferatoxin (RTX) have been used for more than a decade in the treatment of neurogenic detrusor overactivity. Recently, the vanilloid molecule piperine (PIP) has been shown to have similar pharmacological properties as these drugs. In this study, we looked at PIP-effects on autonomous bladder contractile activity, with particular interest for its selectivity for the transient receptor potential channel 1 (TRPV1) receptor. Additionally, we studied the role of TRPV1 in volume-induced contractile changes using selective and non-selective TRPV1 antagonists. METHODS: The acute and prolonged effects of PIP were studied on rat bladders. Each bladder was excised and placed in a heated organ bath, where intravesical pressures were measured. In acute experiments, PIP was added directly to the bathing solution. For prolonged effects, animals were pre-treated intravesically with vehicle (ethanol 5%) or PIP (10(-4) M) and sacrificed 72 hr later. The effects of selective (capsazepine (CZP)) and non-selective (ruthenium red (RR)) TRPV1 antagonists on volume-evoked contractile parameters were also studied. RESULTS: Acute administration of PIP 10(-4) M significantly increased amplitude of bladder contractions (P < 0.05). These effects were significantly antagonized (P < 0.05) by the TRPV1-selective antagonist CZP (10(-5) M) and the non-selective TRP-antagonist RR (10(-5) M). Intravesical pre-treatment with PIP induced shorter contractions with more periods of non-activity (P < 0.05) compared to controls. Inhibition of TRPV1 with CZP and RR significantly reduced the volume-evoked rise in contractile amplitude in isolated bladders (P < 0.05). CONCLUSION: We found evidence for acute and prolonged effects of PIP on bladder contractility, which seem to be mediated through TRPV1. Furthermore, we found evidence for involvement of TRPV1 in afferent signaling of mechanical stimuli.

Autonomous contractile activity in the isolated rat bladder is modulated by a TRPV1 dependent mechanism.

AIMS: Resiniferatoxin (RTX), a vanilloid compound and agonist of the transient receptor potential channel 1 (TRPV1), is known for its beneficial effects on neurogenic detrusor overactivity. The mainstream rationale for its use is the desensitization of TRPV1 on sensory bladder afferents. However, recent findings showed that TRPV1 is present in other cell types in the bladder. To eliminate the effects of RTX on spinal and central neural circuits, we investigated autonomous contractility in normal and neurogenic rat bladders after treatment with RTX. METHODS: Female Wistar rats were made paraplegic at vertebral level T8-T9. Animals were intravesically pre-treated with vehicle (ethanol 5%) or RTX (100 nM) and sacrificed after 72 hr. Each bladder was excised and placed in a heated organ bath, where intravesical pressures were measured. Effects on contractile parameters of intravesical volume load, the non-selective muscarinic receptor agonist carbachol (CA) and electrical stimulation (ES) of nerves were studied in both groups. RESULTS: In RTX-treated normal bladders we found shorter contractions with higher amplitude than in control bladders (P < 0.05). In RTX-treated neurogenic bladders the amplitude and duration of autonomous contractions were increased compared with controls (P < 0.05). Furthermore RTX induced an increased response to CA and to ES (P < 0.05). CONCLUSIONS: RTX significantly affected the properties of autonomous bladder contractile activity. This provides evidence for local effects of RTX on bladder contractile activity, which are not mediated by afferent neural pathways and which may contribute to the beneficial effects on detrusor overactivity. TRPV1 and TRPV1(+) cells seem to play an important role in (autonomous) bladder contractility.