UNLABELLED:Hypericin, isolated from Hypericum perforatum, is an effective photodynamic substance as demonstrated by various studies. Practical forms of applications of hypericin solutions for systemic use and introduction into body cavities are, however, lacking. We developed an aqueous solution of hypericin non-covalently bound to polyvinylpyrrolidone (PVP). PVP is a poly-N-vinylamide of various degrees of polymerization and forms of intermolecular crosslinks suitable for diagnostic and therapeutic applications. We used PVP (molecular weights of PVP between 10 kD and 40 kD) as a complex forming agent to prepare hypericin for photodynamic therapy and diagnostics. In pure water, hypericin forms aggregates which are non-soluble and non-fluorescent. The hypericin-PVP complex binds more than 1000 mg of hypericin in presence of 100 g PVP or less and is soluble in 1 liter of pure water. Aqueous complex solutions of hypericin-PVP display a characteristic absorption spectrum and fluorescence emission band around 600 nm wavelength. Varying concentrations of hypericin do not cause a blue- or red-shift in the absorption maximum at 595 nm. Excitation at 200 nm to 500 nm leads to emission at 590 nm; a property conducive to diagnostic investigations both in vitro and in vivo. Furthermore, hypericin-PVP exhibits high photostability in the presence of oxygen and broad band light which ensures reproducible photodynamic therapy and diagnosis. CONCLUSION:Hypericin forms liquid molecular chromophore complexes in water when bound to PVP thus allowing investigations in biological media.

Hypericin is a naturally occurring substance found in the common St. Johns Wort (Hypericum species) and can also be synthesized from the anthraquinone derivative emodin. As the main component of Hypericum perforatum, it has traditionally been used throughout the history of folk medicine. In the last three decades, hypericin has also become the subject of intensive biochemical research and is proving to be a multifunctional agent in drug and medicinal applications. Recent studies report antidepressive, antineoplastic, antitumor and antiviral (human immunodeficiency and hepatitis C virus) activities of hypericin; intriguing information even if confirmation of data is incomplete and mechanisms of these activities still remain largely unexplained. In other contemporary studies, screening hypericin for inhibitory effects on various pharmaceutically important enzymes such as MAO (monoaminoxidase), PKC (protein kinase Q, dopamine-beta-hydroxylase, reverse transcriptase, telomerase and CYP (cytochrome P450), has yielded results supporting therapeutic potential. Research of hypericin and its effect on GABA-activated (gamma amino butyric acid) currents and NMDA (Nmethyl-D-aspartat) receptors also indicate the therapeutic potential of this substance whereby new insights in stroke research (apoplexy) are expected. Also in the relatively newly established fields of medical photochemistry and photobiology, intensive research reveals hypericin to be a promizing novel therapeutic and diagnostic agent in treatment and detection of cancer (photodynamic activation of free radical production). Hypericin is not new to the research community, but it is achieving a new and promizing status as an effective agent in medical diagnostic and therapeutic applications. New, although controversial data, over the recent years dictate further research, re-evaluation and discussion of this substance. Our up-to-date summary of hypericin, its activities and potentials, is aimed to contribute to this process.

Background/Aim:L-Ascorbic acid (AA) is the predominant circulating form of vitamin C found in human blood. It has been hypothesized that surgical stress increases the vitamin C metabolite dehydroascorbic acid (DHAA). Vitamin C is mainly excreted through the kidneys. In this study, the ratio of AA to DHAA excreted in urine was determined in patients who had undergone total hip joint endoprosthesis surgery (n = 12), and the results were compared with data obtained from healthy controls (n = 12). Methods: All subjects received 1,000 mg sodium ascorbate intravenously three times a day (every 8 h) for 8 days, starting 2 days prior to surgery. Total urine was collected daily while subsequent determinations of AA and DHAA were performed photometrically. Results: Administration of vitamin C led to average daily excretions of the combined products AA + DHAA of 2,343 ± 438 mg/day (mean value ± confidence intervals). The initial average ratio DHAA/AA of all 24 probands was 0.064 (6% DHAA; 153 ± 76 mg/day). One day after surgery, an increase in the DHAA/AA ratio to 0.165 (15% DHAA; 332 ± 107 mg/day) was measured in the patients. The ratio decreased 2 days after surgery and returned to normal within 5 days. Conclusion: Our data indicate that surgery increases the oxidation of AA and urinary excretion of DHAA, as a result of the enhanced formation of free radicals.

Hypericin (Hyp) has been proposed as a fluorochrome for fluorescence diagnostics and as a photosensitizer for photodynamic therapy of cancer. However, its insolubility in water is a serious drawback. A novel water-soluble hypericin derivative (Hyp-S) has been constructed, using polyvinylpyrrolidone as a carrier. We used the crayfish stretch receptor, consisting of receptor neuron and satellite glial cells, for comparison of the photodynamic effects of Hyp and Hyp-S. Hyp-S was more toxic in the dark than Hyp and inactivated the neurons at concentrations exceeding 4 μM while Hyp was toxic to the neurons only at the concentrations larger than 20 μM. Electrophysiological investigations revealed polyphasic neuron responses to photosensitization with Hyp as well as with Hyp-S (1 μM concentration, 30 min incubation; irradiation with filtered light from a lamp with an emission maximum near 600 nm and an intensity of 0.2 W/cm2). In the concentration range 1–4 μM Hyp-S was more phototoxic than Hyp. Fluorescence microscopy showed that both sensitizers were predominately localized in the glial envelope surrounding the neuron. A minor fraction of hypericin was found in the neuron perinuclear area rich in cytoplasm organelles. This suggests the potential application of Hyp and Hyp-S for visualization and selective photodynamic treatment of malignant gliomas.

The subject of this survey is Jindra's use of photodiagnosis, a new method for investigating conditions of the female breast. Light, produced by a laser (670 nm, 0.8 mW) and backscattered from tissue is converted into an electrical signal, amplified and displayed as a curve on an oscilloscope. The curve's fractal dimension (FD), which indicates the metabolism of the tissue under investigation, is calculated using specially developed software, allowing conclusions about the tissues structure to be drawn. By means of 96 painless measurements on 12 healthy premenopausal women, the average FD of healthy tissue was established as 2.37 +/- 0.18. No statistically significant differences were detected between paramamillary measurements taken on the glandular body and those made on the breast parenchyma. Tissue was defined pathological if the FD value was equal to or more than one standard deviation below than this mean (FD less than or equal to 2.19). This procedure was applied to seventeen patients with various breast conditions (previeously diagnosed by imaging techniques and palpation and afterwards verfied by histology), yielding a sensitivity of 83.3 % and a specifity of 72.7 %.We have found that the metabolism of biological tissue acts in a chaotic mode visible by fractal response and that differentiation of healthy from malignant tissue is possible by the fractal dimension.

The subject of this survey is Jindra's use of photodiagnosis, a new method for investigating conditions of the female breast. Light, produced by a laser (670 nm, 0.8 mW) and backscattered from tissue is converted into an electrical signal, amplified and displayed as a curve on an oscilloscope. The curve's fractal dimension (FD), which indicates the metabolism of the tissue under investigation, is calculated using specially developed software, allowing conclusions about the tissues structure to be drawn. By means of 96 painless measurements on 12 healthy premenopausal women, the average FD of healthy tissue was established as 2.37 ± 0.18. No statistically significant differences were detected between paramamillary measurements taken on the glandular body and those made on the breast parenchyma. Tissue was defined pathological if the FD value was equal to or more than one standard deviation below than this mean (FD ≤2.19). This procedure was applied to seventeen patients with various breast conditions (previeously diagnosed by imaging techniques and palpation and afterwards verfied by histology), yielding a sensitivity of 83.3% and a specifity of 72.7%. We have found that the metabolism of biological tissue acts in a chaotic mode visible by fractal response and that differentiation of healthy from malignant tissue is possible by the fractal dimension.

Meta-tetrahydroxyphenylchlorin (mTHPC) exhibits significant cytotoxicity against a variety of human cells in culture in combination with light, but also in dark reaction. The ovarian cancer cell line SK-OV3 was incubated with various concentrations of mTHPC and in comparison with Taxol and Cisplatin: then the effect on cell growth was determined. mTHPC exhibited an IC50 of 0.9 muM after 24 hours incubation (IC50 of 1.25 after 2 hours), whereas Cisplatin and Taxol, which, have been used as first line agents for the treatment of ovarian carcinomas, inhibited cell proliferation with an IC50 concentration of 4.6 muM and 78 nM after 24 hours incubation, respectively. Incubation of SK-OV3 cells with mTHPC for 5 days resulted in cytostatic cytotoxicity at a concentration of 0.5 muM. The photodynamic effect of mTHPC depends/among other parameters/on the concentration of the dye present. In combination with light (approximately 15 J/cm2) a linear relationship between the dose of mTHPC and the amount of necrotic cells was observable. Higher concentrations of mTHPC caused necrosis of the ovarian tumor cells. The intracellular concentration of mTHPC showed a linear increase up to 28.6 nM (incubation concentration). In summary, these studies demonstrated that mTHPC exhibits potent antiproliferative activity by inducing necrosis after application of light. MTHPC might be a promising agent with cytostatic and photodynamic properties for the treatment of metastasing ovarian carcinomas. A sensitive PCR method was not able to show the induction of apoptosis in the SK-OV3 ovarian cell line. Using propidium staining, it could be proved that the cell death was caused by necrosis and not through apoptosis after irradiation with light.

Photodynamic therapy involves the application of a photosensitizer activated by visible light to generate cytotoxic reactive oxygen. In addition to clinical investigations, in vitro studies concerning photodynamic potency of sensitizers as well as quantification of illumination procedures are necessary. In our investigation, the objective was to evaluate not only the effects of photosensitizer and light on Gram-positive Staphylococcus aureus, but also to investigate possible synergistic or antagonistic effects of these sensitizers. Therefore, we used hypericin, Photofrin II, porfimer sodium and meso-tetrahydroxyphenylchlorin (mTHPC) alone, as well as in combination. Log-phase cells of S. aureus exhibited a marked sensitivity to white thermal light irradiation in the presence of Photofrin II and mTHPC. However, hypericin caused a rather stimulated growth expressed in increased optical density (OD) and increase of total cell count (TCC) of the culture. Combination sensitization of S. aureus by Photofrin II and mTHPC with hypericin likewise caused a stimulation of bacterial growth. No synergistic effects were obtained by combination of Photofrin II and mTHPC; photoresponse of S. aureus was rather decreased by using combined porphyrins. In comparison, TCC and colony-forming units (CFU) were suppressed in the presence of mTHPC after an illumination procedure as well as in dark reactions. These effects were also obtained in the combination photosensitization by mTHPC and Photofrin II. In the presence the of hypericin, photodynamic effects of mTHPC and Photofrin II were inhibited. It was finally concluded that hypericin in our model is not a proper sensitizer for combination photo-sensitization due to antagonistic effects on photodynamic activity of mTHPC and Photofrin II.

This study reports our first results of ambulant photodynamic treatment with 5-aminolevulinic acid (5-ALA) in combination with folic acid and subsequent illumination with a noncoherent light source. The compound was topically applied to avoid total body skin sensitivity which occurs in the case of systemic administration. If no therapeutic response could be proved, we added folic acid to 5-ALA for a further treatment attempt. Illumination was performed by broad band red thermic light to also excitate reaction products with absorption bands located near to that of the sensitizer. As a result, we observed a response in all cases, however, in some cases only after the addition of folic acid.

The aim of this study was to treat patients for ectocervical dysplasia [cervical intraepithelial neoplasia (CIN) grades 1 and 2] and associated human papilloma virus (HPV) infections with photodynamic therapy (PDT). In 20 patients, 5-aminolevulinic acid (5-ALA, 12% w/v) was applied topically with a cervical cap 8 h prior to illumination. A thermal light source (150 W halogen lamp) emitting a broadband red light (total energy: 100 J/cm2, fluence rate: 90 mW/cm2) was used for superficial illumination of the portio. In addition, an Nd:YAG pumped dye laser (652 nm) was used to illuminate the cervical canal (total energy: 50 J/cm2, fluence rate: 300 mW/cm2). Preliminary results of follow-ups at 1, 3, 6, and 9 months posttherapy showed a cytological improvement in the grading of the PAP smears in 19 patients and the eradication of cervical HPV in 80%. These results demonstrate that ectocervical dysplasia and associated HPV infections can be treated by PDT.

In order to measure inotropic influences of physiologically occurring substances and drugs we used a newly developed guinea pig papillary muscle (GPPM) bioassay. GPPM were suspended in air and surface coated with buffer (Krebs-Henseleit solution). The muscles were stimulated (pulsating direct current, 1.5 V; 0.5 Hz, 20 ms duration) which led to contraction. This method enables measurements of inotropic effects up to 5 days, contrary to previous studies (1 day), in which immersions of GPPM in buffer were performed. In order to investigate the comparability of the new method we measured the effect of metabolites (citric acid cycle), lactic acid, lactate, and extracellular pH on muscle contractility. The H(+)-dependent decrease of the contractile force of the GPPM can be compensated by an increased Ca(2+)-concentration. Further, the influence of catecholamines (isoproterenol) on the contractility was investigated. As a result, isoproterenol caused arrhythmias and extrasystoles as it was observed in clinical studies. Several pharmaceutical substances were tested to show the reproducibility and repeatability of the bioassay.

Photodynamic eradication of tumour cells in vivo depends on the presence of a photosensitizer, light delivery to the cells, and an oxygen supply. Hypericin, a polycyclic quinone with absorption maxima in the ultraviolet and visible ranges, was prepared for clinical use as a photosensitizer. Due to antitumoral and antineoplastic activities as well as the generation of singlet oxygen after photoexcitation, hypericin was applied in clinical oncology and photodynamic therapy. Hypericin was administered subcutaneously (20 micrograms hypericin in 200 microliters Nacl/pyridine solution) into the ante brachium (forearm) of two volunteers. After the diffusion and equilibration of 120 min phototesting was carried out using outdoor light exposure, halogen lamp, laser 514 nm (argon), laser 632 nm (argon dye) and laser 670 nm (diode laser), from 60 to 120 J cm-2. Positive phototests to outdoor light exposure, halogen lamp and laser 514 nm were characterized by rubescence, oozing, vesiculation and darting pain. Phototests with laser 632 nm and 670 nm showed no effects after irradiation. When hypericin was administered topically on skin, erythema and flaring could not be induced by any irradiation. These results suggest that hypericin is a potent photosensitizer only within the UV and green light ranges. This characteristic photoresponse could also be obtained in guinea pig papillary muscle (GPPM) bioassay, which may be established as a model for photosensitizer testing. Irradiation of hypericin-incubated GPPM with 514 nm (20 J cm-2) led to a decrease of the contractile force of about 31%. However, excitation with 632 nm and 670 nm did not cause inotropic effects on GPPM. In addition, hypericin and Photosan 3 were shown to be capable of sensitizing the photo-oxidation of sodium linoleate. This assay should be established for testing interactions between photosensitizers and light sources in vitro.

Chlorophyll and some of its synthetically produced derivatives are important sensitizers in photodynamic cancer therapy. Other natural products from plants with light dependent activity include quinones like hypericin and fagopyrin. These compounds have extended π-electron systems which upon photoexcitation with visible light are responsible for singlet oxygen production.

We describe the first local use of hypericin as photosensitizer for photodynamic therapy in a patient with recurrent malignant mesothelioma. Hypericin is a polycyclic quinone, which has been shown to possess in vivo and in vitro antiretroviral and photosensitizing activity; moreover, it is used in depressive disorders. The semiquinone radical, singlet oxygen, and superoxide anion radical are reported to be the toxic agents in hypericin phototherapy. Our first experience with locally applied hypericin in a superficial tumor-plate was performed 8 weeks after the systemic administration of hematoporphyrin derivatives. For tumor light illumination we used an argon pumped dye laser tuned to 632 nm. Owing to satisfactory results, we repeated the same therapy 4 weeks later — and no therapeutic effect was noted. Following this, we proved the interstitial application of HPD and the combination of interstitial HDP and superficially applied hypericin. The subsequent light illumination 6 hours later had no efficacy in the HDP-photosensitized area but there was tumor destruction in the field with both administered photosensitizers. Our first experience suggests a potentiation of two photosensitizers: hematoporphyrin derivatives and hypericin.

*Ludwig Bottzmann Institut für Onkologie und Photodynamische Therapie A-1130WIEN, Wolkersbergenstrasse 1 **Department of Chemistry University of Bodenkultur, A - 1190WIEN. Peter-Jordanstrasse 70. ***Department of Cardiothoracic-Anaesthesia and Intensive Care. University of Vienna Austria A - 1090 WIEN Wähnnger Gürtel AKH

*Department of Chemistry, University of Bodenkultur Vienna, Austria. **Department of Cardiothoracic-Anaesthesia and Intensive Care, University of Vienna, Austria