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Purpose: To study whether formulation influences biodistribution, necrosis avidity and tumoricidal effects of the radioiodinated hypericin, a necrosis avid agent for a dual-targeting anticancer radiotherapy. Methods: Iodine-123- and 131-labeled hypericin (I-Hyp and I-Hyp) were prepared with Iodogen as oxidant, and formulated in dimethyl sulfoxide (DMSO)/PEG400 (polyethylene glycol 400)/water (25/60/15, v/v/v) or DMSO/saline (20:80, v/v). The formulations with excessive Hyp were optically characterized. Biodistribution, necrosis avidity and tumoricidal effects were studied in rats ( n = 42) without and with reperfused liver infarction and implanted rhabdomyosarcomas (R1). To induce tumor necrosis, R1-rats were pre-treated with a vascular disrupting agent. Magnetic resonance imaging, tissue-gamma counting, autoradiography and histology were used. Results: The two formulations differed significantly in fluorescence and precipitation. I-Hyp/Hyp in DMSO/PEG400/water exhibited high uptake in necrosis but lower concentration in the lung, spleen and liver ( p < 0.01). Tumor volumes of 0.9 ± 0.3 cm with high radioactivity (3.1 ± 0.3% ID/g) were detected 6 days post-treatment. By contrast, I-Hyp/Hypin DMSO/saline showed low uptake in necrosis but high retention in the spleen and liver ( p < 0.01). Tumor volumes reached 2.6 ± 0.7 cm with low tracer accumulation (0.1 ± 0.04%ID/g). Conclusions: The formulation of radioiodinated hypericin/hypericin appears crucial for its physical property, biodistribution, necrosis avidity and tumoricidal effects. [ABSTRACT FROM AUTHOR]

1. Iodogen (tetrachloro-diphenyl glycoluril) dissolved in DMSO (dimethyl sulphoxide) appears indispensable in radioiodination of hypericin for a new anticancer strategy. We studied the safety of intravenously administered iodogen/DMSO in mice ( n = 132). 2. Median lethal dose (LD50) of iodogen/DMSO was determined with doses of 40.0, 50.0, 55.0, 60.0, 65.0 and 70.0 mg/kg. Next, toxicity of iodogen/DMSO at 30.0 mg/kg was evaluated using saline and DMSO as controls. Changes in behaviour, body weight and serum biochemistry were evaluated. Histopathology of lungs, heart, liver and kidney was performed. 3. LD50 values of iodogen/DMSO were 59.5 mg/kg (95% confidence limits (CI): 54.1-65.4 mg/kg) and 61.0 mg/kg (95%CI: 56.2-66.2 mg/kg) for female and male mice, respectively. Similar to that of control groups, no animal deaths were encountered after iodogen/DMSO administration at 30.0 mg/kg. Body weights over 24 h were not altered in all groups, but significantly higher in iodogen/DMSO and DMSO groups ( p < 0.05) 14 d post-injection. Blood urea nitrogen and alkaline phosphatase increased ( p < 0.05) in iodogen/DMSO group without clinical symptoms. No pathologies were found by gross and microscopic inspection. 4. A single dose of iodogen/DMSO up to 30.0 mg/kg, over 3000 times the dose in potential human applications, appears safe, with an LD50 doubling that dose in mice. [ABSTRACT FROM AUTHOR]

Purpose: To obtain unambiguous evidence for the putative role of E-cadherin in the selective accumulation of hypericin after intravesical instillation in humans we investigated the accumulation of hypericin in spheroids from 3 clones of the human bladder carcinoma cell line T-24 that express different levels of E-cadherin, as determined by immunohistochemistry and reverse transcriptase-polymerase chain reaction. Materials and Methods: Clones of T-24 cells transfected with the E-cadherin gene were analyzed for E-cadherin expression and 3 cell lines with different expression levels were selected. Spheroids of these cell lines were incubated with 10 μM hypericin in cell culture medium supplemented or not with fetal calf serum for 2 hours. After the incubation period centrally cut sections were examined by fluorescence microscopy. An imaging software system was used to measure average fluorescence in concentric layers from rim to center. Results: Data showed that in the presence of serum the accumulation of hypericin in spheroids was inversely associated with the level of E-cadherin expressed by the T-24 transfectants used, whereas in the absence of serum differential accumulation of the compound was completely abolished. Conclusions: Spheroids composed of cancer cell lines expressing variable levels of E-cadherin represent an excellent model in which to study the role of intercellular adhesion in bladder cancer. The outcome of this study strongly suggests that E-cadherin is the key mediator in the selective accumulation of hypericin in superficial bladder cancer after intravesical instillation in humans. [Copyright &y& Elsevier]

Rationale and Objectives: In this preclinical proof-of-principle study, the necrosis avid agent hypericin was investigated as a potential early indicator for therapeutic response after ethanol-mediated chemical ablation in murine liver tumors. Materials and Methods: Seven mice bearing intrahepatic radiation-induced fibrosarcoma-1 tumors were intravenously injected with hypericin 1 hour before (n = 3) or 24 hours after (n = 4) intratumoral ethanol injection. Mice were euthanized 24 hours after hypericin injection and, taking advantage of the fluorescent property of the compound, the excised livers were investigated qualitatively and quantitatively by means of fluoromacroscopic and fluoromicroscopic examinations, colocalized with conventional histomorphology. Results: Significant differences in hypericin fluorescence were found in necrosis, viable tumor and normal liver tissue in decreasing order (P < .05) (ie, in necrosis, mean fluorescence densities were about 4.5 times higher than in viable tumor and approximately 14 times higher than in normal liver). When hypericin was injected 1 hour before, maximal blood concentrations were achieved at the time of ethanol treatment, so that on ablation an outstanding extravasation took place in the entire necrotic area in comparison with accumulation of hypericin only at the peripheral zone of necrosis when it was injected 24 hours after ablation. Conclusions: Hypericin specifically enhanced the imaging contrast between necrotic and viable tissues and nonspecifically distinguished viable tumor from normal liver. Injection of hypericin shortly before ablation is more favorable than after ablation, because it circumvents difficulties with no-entry zones for hypericin and requires shorter intervals between ethanol ablation and imaging. [Copyright &y& Elsevier]

OBJECTIVE To investigate the influence of a glycosaminoglycan (GAG) layer on the specific location of hypericin in superficial urothelial carcinoma lesions of the bladder after intravesical instillation. MATERIALS AND METHODS Fisher rat bladders were incubated with 15 or 30 µm hypericin for 2 h. To examine the influence of the GAG layer on the permeation of hypericin, bladders were pre-treated with chondroitinase ABC, n-dodecyl-β-d-maltoside (DDM) or sodium dodecyl sulphate (SDS) to disrupt, or protamine to neutralise the GAG layer before incubating with hypericin. After incubation, the photosensitizer permeation was examined quantitatively in cryostat sections of the bladders, using fluorescence microscopy and image analysis. RESULTS Disrupting or neutralising the GAG layer in the bladder had no influence on the permeation of hypericin. Pre-treatment of the bladder with chondroitinase, DDM or SDS resulted in a significantly lower accumulation of hypericin, whereas neutralising the GAG layer in rats with protamine had no significant effect on the biodistribution of hypericin. CONCLUSION The GAG matrix causes no obstacle to the permeation of hypericin in the urothelium of the bladder, and modification of this GAG layer cannot explain the enhanced accumulation of hypericin in superficial bladder tumours. [ABSTRACT FROM AUTHOR]

Summary: To study the in vivo penetration and skin distribution of hypericin, the compound (0.1%) was formulated in 10 different vehicles that are commonly used in pharmaceutical compounding, and applied on the skin of hairless mice for 4h. After application of hypericin in PEG ointment, white petrolatum or unguentum emulsificans, fluomicroscopic analysis of skin sections revealed penetration to be confined to the stratum corneum. On the contrary, Beeler base, unguentum sorbatis 100 and cremor non ionicus caused penetration of hypericin in the viable epidermis. To reduce the prominent depot formation in the stratum corneum, which was observed irrespectively of the formulation applied, hypericin was esterified into its hydrolyzable acetate derivative. The influence of esterification proved to be substantial when hypericin acetate (0.15%) was incorporated in unguentum sorbatis 100, as hypericin-related fluorescence could be detected deeply within the dermis. Moreover, accumulation in the sebaceous glands was found to be prominent. These results indicate the value of further studies regarding the application of hypericin and hypericin acetate as topical photosensitizers for photodynamic therapy. [Copyright &y& Elsevier]

Purpose: We have labelled hypericin, a polyphenolic polycyclic quinone found in St. John's wort (Hypericum perforatum), with 123I and evaluated mono-[123I]iodohypericin (MIH) as a potential necrosisavid diagnostic tracer agent. Methods: MIH was prepared by an electrophilic radioiodination method. The new tracer agent was evaluated in animal models of liver infarction in the rat and heart infarction in the rabbit using single-photon emission computed tomography (SPECT), triphenyltetrazolium chloride (TTC) histochemical staining, serial sectional autoradiography and microscopy, and radioactivity counting techniques. Results: Using in vivo SPECT imaging, hepatic and cardiac infarctions were persistently visualised as well-defined hot spots over 48 h. Preferential uptake of the tracer agent in necrotic tissue was confirmed by perfect match of images from post-mortem TTC staining, autoradiography (ARX) and histology. Radioactivity concentration in infarcted tissues was over 10 times (liver; 3.51% ID/g in necrotic tissue vs 0.38% ID/g in normal tissue at 60 h p.i.) and over 6 times (myocardium; 0.36% ID/g in necrotic tissue vs 0.054% ID/g in normal tissue; ratios up to 18 for selected parts on ARX images) higher than in normal tissues. Conclusion: The results suggest that hypericin derivatives may serve as powerful necrosis-avid diagnostic agents for assessment of tissue viability. [ABSTRACT FROM AUTHOR]

To optimise the diagnostic and phototherapeutic efficacy of hypericin in superficial bladder cancer, by developing a bladder instillation fluid that does not depend on the presence of plasma proteins for an appropriate and reliable urothelial uptake of hypericin.Sodium hypericinate (in distilled water, in sodium phosphate buffer, or in polyethylene glycol) and several other hypericinate salts (potassium, lysine, TRIS or hexylamine) were instilled with no plasma constituents into the rat bladder. The accumulation of hypericin was assessed with fluorescence microscopy.The diagnostic and phototherapeutic efficacy of hypericin depends on its ability to penetrate the tumour lesions sufficiently to show a fluorescent signal or elicit a photodynamic response. Several instillation fluids meet the purpose, as the urothelial accumulation of hypericin was similar to that obtained with the instillation fluid supplemented with plasma proteins, used in clinical practice. The highest concentrations of hypericin in the urothelium of the rat bladder were obtained with hypericin instillation solutions prepared with distilled water or 20% polyethylene glycol 400 in distilled water. Fluorescence microscopy showed that hypericin was selectively localized in the urothelium. Furthermore, all variables investigated (hydrophilic/lipophilic balance, pH, saline, presence of organic solvent) can dramatically influence thein vivoaccumulation of hypericin.An appropriate and reliable urothelial uptake of hypericin does not depend on the presence of plasma protein supplements in the bladder instillation fluid. [ABSTRACT FROM AUTHOR]

Summary: Hypericin is a photo-active dye originating from the St. John''s wort. Two patients with disseminated superficial actinic porokeratosis (DSAP) were treated with photodynamic therapy (PDT) using topical hypericin. Although a partial response was obtained in one patient topical hypericin-PDT does not emerge as a promising treatment for DSAP. [Copyright &y& Elsevier]

Abstract: Hypericin, a naturally occurring photosensitizer, is currently being investigated for topical use in photodynamic therapy (PDT). In a previous study, it was found that hypericin can be delivered in the epidermis of hairless mouse skin after a 4-h application in Beeler base. With the intention to further optimize the penetration conditions, the present study examines the effect of the concentration of hypericin in the cream, the application time, the presence of penetration enhancers and occlusion on the penetration of hypericin in the skin of hairless mice. Experiments with different hypericin concentrations and application times indicated that application of 0.1% hypericin for 12–24h maximizes the accumulation of hypericin-related fluorescence in the skin, as estimated by fluorescence microscopy with image analysis. Depending on the formulation, the use of an occlusive dressing did not alter, or even reduced the accumulation of hypericin in the viable layers. Also, the addition of propylene glycol (30%) and oleic acid (5%) did not change hypericin fluorescence levels in the epidermis. Conversely, incorporation of ethanol (40%, integrated in a gel, and added to Beeler base) increased dramatically the fluorescence levels in all skin layers. Consequently, the optimized conditions were used to investigate the penetration of hypericin into UV induced skin tumors. It was found that application under occlusion of hypericin, formulated in the gelcream containing ethanol, during 24h enabled the penetration of hypericin in the entire skin lesions with a relatively homogenous distribution. In conclusion, our results suggest the possible use of 0.1% hypericin in a gelcream containing ethanol for PDT of skin lesions. [Copyright &y& Elsevier]

Abstract: Hypericin, solubilized in an instillation fluid consisting of an aqueous buffer supplemented with 1% plasma proteins, is currently used as a clinical diagnostic tool for the detection of superficial TCC (transitional cell carcinoma) tumors. However, the development of a sterile and stable hypericin stock formulation, excluding the presence of plasma constituents, would be an important factor in a more general clinical application of the method. Therefore, we investigated the stability of several heat sterilized hypericin formulations and ion pairs. Besides sodium hypericinate (in distilled water, in phosphate buffer, in polyethyleneglycol (PEG) 400), several other hypericinate salts (potassium, lysine, TRIS or hexylamine) were investigated. As to that, the physical appearance of different hypericin concentrates stored at 4 and 37°C was investigated. Besides, after dilution into cell culture medium, the ability of hypericin remaining to accumulate in tumor cells and demonstrating photocytotoxic effects upon light irradiation was assessed. These findings suggest that PEG 400 is an excellent hypericin formulation, since it maintained the stability of the compound for at least 120d when stored at either 4 or 37°C. PEG 400 therefore is a suitable vehicle for the storage of hypericin prior to preparation of the bladder instillation solution. [Copyright &y& Elsevier]

Highlights: [•] Hypericin-photodynamic therapy induces phox-ER stress both in vitro and in vivo. [•] Phox-ER stress activates PERK and CHOP. [•] Phox-ER stress causes up-regulation of BH3-only proteins, Bim and Noxa. [•] Noxa, but not Bim, contributes toward phox-ER stress induced apoptosis. [•] PERK regulates Noxa in a CHOP-independent, temporally-defined manner. [ABSTRACT FROM AUTHOR]

Abstract: Photodynamic therapy (PDT) is an established anticancer modality and hypericin is a promising photosensitizer for the treatment of bladder tumors. We show that exposure of bladder cancer cells to hypericin PDT leads to a rapid rise in the cytosolic calcium concentration which is followed by the generation of arachidonic acid by phospholipase A2 (PLA2). PLA2 inhibition significantly protects cells from the PDT-induced intrinsic apoptosis and attenuates the activation of p38 MAPK, a survival signal mediating the up-regulation of cyclooxygenase-2 that converts arachidonic acid into prostanoids. Importantly, inhibition of p38α MAPK blocks the release of vascular endothelial growth factor and suppresses tumor-promoted endothelial cell migration, a key step in angiogenesis. Hence, targeted inhibition of p38α MAPK could be therapeutically beneficial to PDT, since it would prevent COX-2 expression, the inducible release of growth and angiogenic factors by the cancer cells, and cause an increase in the levels of free arachidonic acid, which promotes apoptosis. [Copyright &y& Elsevier]

Abstract: 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 (8μM) was instilled in the bladder for 2–3h before photodynamic diagnosis of bladder tumors. Blood was then collected from a peripheral vein 1h 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 20nM, respectively. Extraction and analysis of the plasma of patients after intravesical administration showed hypericin concentrations below the detection limit (<6nM). In addition, photodynamic treatment of in vitro cultured HeLa cells incubated with 1–100nM hypericin concentrations showed that lower concentrations (1–20nM) 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. [Copyright &y& Elsevier]

Photodynamic therapy (PDT) has been described as a promising new modality for the treatment of cancer. PDT involves the combination of a photosensitizing agent (photosensitizer), which is preferentially taken up and retained by tumor cells, and visible light of a wavelength matching the absorption spectrum of the drug. Each of these factors is harmless by itself, but when combined they ultimately produce, in the presence of oxygen, cytotoxic products that cause irreversible cellular damage and tumor destruction. Hypericin, a powerful naturally occurring photosensitizer, is found in Hypericum perforatum plants, commonly known as St. John’s wort. In recent years increased interest in hypericin as a potential clinical anticancer agent has arisen since several studies established its powerful in vivo and in vitro antineoplastic activity upon irradiation. Investigations of the molecular mechanisms underlying hypericin photocytotoxicity in cancer cells have revealed that this photosensitizer can induce both apoptosis and necrosis in a concentration and light dose-dependent fashion. Moreover, PDT with hypericin results in the activation of multiple pathways that can either promote or counteract the cell death program. This review focuses on the more recent advances in the use of hypericin as a photodynamic agent and discusses the current knowledge on the signaling pathways underlying its photocytotoxic action. [Copyright &y& Elsevier]