Determination of hypericin in human plasma by high-performance liquid chromatography after intravesical administration in patients with transitional cell carcinoma of the bladder.

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 of transitional cell carcinoma rat bladder tumors using intravesically administered 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.

Microscopic quantification of hypercin fluorescence in an orthotopic rat bladder tumor model after intravesical instillation.

We have previously investigated the possibility of using hypericin as a diagnostic tool for the fluorescence detection of flat bladder carcinoma. In these clinical studies, it was shown that following intravesical application in humans, hypericin becomes selectively localized in transitional papillary carcinoma and carcinoma in situ (CIS). In the present study, we characterized the biodistribution of hypericin in rat bladder tumor and normal bladder layers after intravesical administration. The biodistribution was evaluated using fluorescence microscopy with computerized image analysis to image and quantify the fluorescence of hypericin across the urothelial tumor and normal bladder wall. The results show that the photosensitizer intravesical administration route provides selective labelling of both the tumor and normal urothelium. A hypericin dose and instillation time-dependent increase in the hypericin fluorescence intensity in both the tumor and urothelium was observed, without significant hypericin fluorescence in the submucosa and muscle layers. The highest fluorescence ratios in hypericin accumulation in the tumor and normal urothelium to the muscle layer were achieved at 4 h with 30 micro M hypericin (20:1 for the urothelium to muscle layer and 30:1 for the tumor to muscle layer). The difference in fluorescence intensity in tumor tissue to the muscle layer following instillation of 8 micro M hypericin was 11:1, 25:1 and 28:1 at 1, 2 and 4 h, respectively. The difference in fluorescence intensity in tumor tissue to the muscle layer using 30 micro M hypericin was 17:1, 27:1 and 31:1 at 1, 2 and 4 h, respectively. The highest absolute fluorescence levels were observed in the tumor at 4 h with 30 micro M hypericin instillation. The results suggest that under these conditions, PDT with hypericin is likely to produce uniform urothelial tumor eradication, without causing damage to the underlying muscle layers.

Overexpression of fatty acid synthase is an early and common event in the development of prostate cancer.

The expression of fatty acid synthase (FAS), a key lipogenic enzyme and potential target for antineoplastic therapy, was analyzed in 87 frozen needle biopsies of prostate cancer using a highly sensitive immunohistochemical detection technique (Envision). In comparison to normal or benign, hyperplastic glandular structures, which were all negative for FAS staining, immunohistochemical signal was evident in 24/25 low grade prostatic epithelial neoplasia (PIN) lesions, in 26/26 high grade PIN lesions and in 82/87 invasive carcinomas. Staining intensity tended to increase from low grade to high grade PIN to invasive carcinoma. Cancers with a high FAS expression had an overall high proliferative index. No correlation was found between FAS expression and lipid accumulation. These findings indicate that increased FAS expression is one of the earliest and most common events in the development of prostate cancer, suggesting that FAS may be used as a general prostate cancer marker and that antineoplastic therapy based on FAS inhibition may be an option for chemoprevention or curative treatment for nearly all prostate cancers.

Biodistribution of hypericin in orthotopic transitional cell carcinoma bladder tumors: implication for whole bladder wall photodynamic therapy.

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.