P. de Witte

A comparative study of the photosensitizing characteristics of some cyanine dyes

The present work has been carried out to explore the potential application of cyanines in photodynamic therapy. After photosensitization, the in vitro cytotoxic and antiproliferative activity on HeLa cells of a total of 35 cyanines belonging to several chemical subgroups is explored. Most of these cyanines have never been used before in similar experimental work. From a first set of experiments, it is found that none of the krypto-, oxa- and imidacyanines is photobiologically active on HeLa cells. Conversely, five thiacyanines (Thiacl-5), one rhodacyanine (Rhodac) and four indocyanines (Indoc2, Indoc4, Indoc5, Indoc7) show photodependent cytotoxicity or antiproliferative effects. A more detailed study shows that out of the ten selected compounds, eight cyanines feature significant photodependent cytotoxic and antiproliferative effects. All possess maximum absorption ranges between 545 and 824 nm. In particular, Rhodac, a tetramethinemeromonomethine rhodacyanine dye with an absorption maximum of 655 nm (ethanol) and a molar absorption coefficient epsilon = 108000 shows very promising photo-dependent biological activity. In general, the measured singlet oxygen quantum yield of the selected cyanines is low (< 0.08) and does not correlate with the degree of photosensitization. Furthermore, the present study shows that cyanines with a partition coefficient close to 1.5 accumulate to the highest extent in HeLa cells, while the more hydrophobic compounds (e.g., indocyanines) concentrate less intracellularly.

Photosensitized inhibition of growth factor-regulated protein kinases by hypericin

The naphthodianthrone hypericin causes a photosensitized inhibition of protein kinases involved in growth factor signalling pathways. Nanomolar concentrations of hypericin inhibit the protein tyrosine kinase activities (PTK) of the epidermal growth factor receptor and the insulin receptor, while being ineffective towards the cytosolic protein tyrosine kinases Lyn, Fgr, TPK-IIB and CSK. Photosensitized inhibition by hypericin is not restricted to receptor-PTKs since the Ser/Thr protein kinases (protein kinase CK-2, protein kinase C and mitogen-activated kinase) are also extremely sensitive to inhibition (IC50 value for protein kinase CK-2 = 6 nM). A comparison of the hypericin-mediated inhibition of the epidermal growth factor-receptor PTK and protein kinase CK-2 revealed that the inhibition is irreversible, strictly dependent upon irradiation of the enzyme-inhibitor complex with fluorescent light and likely mediated by the formation of radical intermediates (type I mechanism). Although the exact molecular basis for the selectivity of enzyme inhibition by hypericin remains unknown, our results suggest that distantly related protein kinases could still share common reactive domains for the interaction with hypericin.

Evidence That Total Extract of Hypericum perforatum Affects Exploratory Behavior and Exerts Anxiolytic Effects in Rats

Clinical trials have extensively reported the ability of Hypericum perforatum extracts to exert a significant antidepressant activity. Hypericin, the main constituent of H. perforatum extract, is no more regarded as the active principle of the antidepressant activity of the drug. Hence, the question of which constituents are involved in the basic activity of the total extract, is still waiting for an answer. In the present study we focused our attention on the potential anxiolytic activity of H. perforatum total extract, and of some pure components such as protohypericin and a fraction containing hypericin and pseudohypericin. Herein we report that the total extract of H. perforatum increases the locomotor activity in the open field and exerts anxiolytic activity in the light-dark test, whereas the single components did not show any effect. Interestingly, the anxiolytic activity of the total extract was blocked by pretreatment of rats with the benzodiazepine antagonist Flumazenil, hence suggesting an implication of benzodiazepine receptor activation in the anxiolytic effect of H. perforatum extract. Electrophysiological studies, performed to gain more information on the mechanism of action, showed that hypericin reduced the GABA-activated chloride currents, while pseudohypericin did an opposite effect. Furthermore, both hypericin and pseudohypericin inhibited the activation of NMDA receptors.

Metabolism and Pharmacokinetics of Anthranoids

Anthranoid derivatives are used all over the world as a treatment for constipation. These compounds are present in several drugs of plant origin, especially as O- or C-glycosides. Besides featuring different substituents, the aglycone might consist of an anthraquinone, an anthrone or a dianthrone. So far, detailed information concerning their metabolism and pharmacokinetic characteristics is available only in a few cases. The best characterized compounds are sennoside, a dianthrone O-glycoside present in senna leaves and senna pods, and its aglycone (rhein anthrone). After oral administration, sennoside is degraded only in the lower parts of the gastrointestinal tract, releasing its active metabolite rhein anthrone. Nowadays, this process is understood at the molecular level. A study with 14C-labelled rhein anthrone administered intracecally to rats, revealed that the compound is scarcely absorbed. Since on the contrary its anthraquinone equivalent is absorbed to a much larger extent, it is inferred that dianthrone- or anthrone-glycosides exhibit a lower systemic availability than anthraquinone O-glycosides.

In Vitro Study of the Photocytotoxicity of Some Hypericin Analogs on Different Cell Lines

In the present study, hypericin analogs with an increased hydrophilic character were synthesized. As chemical modifications alter the lipophilicity/hydrophilicity balance together with the photophysical/chemical background of the molecule the influence of these structural changes on the cellular uptake, retention and subcellular localization in HeLa cells was investigated. Besides, their photocytotoxic effects using three cell lines (HeLa, MCF‐7, A431), as well as their plasma protein binding were also assessed. To assess the relative hydrophilic/lipophilic character of hypericin and analogs their retention times were determined on a reversed phase high performance liquid chromatography (C‐18) column. The retention time of all the hypericin analogs was <46 min, except for dibenzyltetramethylhypericin (118 min), while the retention time of hypericin was >200 min (solvent system: methanol/citrate buffer 30 mM pH 7; 70/30). Hypericin, hexa‐, penta‐ and dibenzyltetramethylhypericin displayed a potent antiproliferative effect at the nanomolar range after photosensitization (3.6 J/cm2). On the contrary, photoactivated tetrasulfonhypericin and fringelite D had no antiproliferative effect on the three cell lines, whereas hypericin polyethylene glycol showed only an intermediate cytotoxic effect on A431 cells. In dark conditions no antiproliferative effect was observed for any photosensitizer. The antiproliferative photoeffect correlated well with the intracellular accumulation as measured using HeLa cells. In general, the photocytotoxic hypericin analogs concentrated to a large extent, while the noncytotoxic compounds were not taken up by the HeLa cells. Furthermore, confocal laser microscopy revealed that all photosensitizers mainly concentrated in the perinuclear region, probably corresponding with Golgi apparatus and the endoplasmic reticulum, except for tetrasulfonhypericin which located at the plasma membrane. In addition, the plasma protein binding studies illustrated that hypericin bind extensively to the low‐density lipoproteins, while the other hypericin analogs were mainly bound to heavy proteins (mostly albumin) and to a small extent to low‐density lipoproteins.

Photocytotoxicity of hypericin in normoxic and hypoxic conditions

The normoxic and hypoxic photocytotoxicity of hypericin has been examined on A431 cells as assessed by the Neutral Red method, using cell-culture flasks made of polystyrene and glass, different hypericin concentrations and light fluences. Using polystyrene flasks, lower hypoxic photoactivities of hypericin than those in normoxic conditions are seen under low fluence. In these conditions the hypoxic photocytotoxic effect can be (partially) rescued by increasing the fluence. However, a completely different outcome is observed when using glass flasks, since most of the hypoxic photocytotoxicity is lost under these conditions. The differences can be explained in terms of efficiency of deoxygenation of the medium present in polystyrene or glass flasks. Polystyrene holds large amounts of oxygen that effuses very slowly. Glass, on the other hand, does not cause this inconvenience. Therefore the type of material of the container used to investigate the oxygen dependency of the photobiological activity of photosensitizers dramatically influences the outcome of the hypoxic experiments. Our results unequivocally prove that the cytotoxic effect induced by photoactivated hypericin is completely oxygen dependent. Hence hypericin does not differ from other phototherapeutics used in photodynamic therapy of cancer, since haematoporphyrin derivative and the second-generation photosensitizers used all seem to depend on the presence of oxygen for their antitumour activity.

Photosensitizing activity of hypericin and hypericin acetate after topical application on normal mouse skin

Background  Hypericin, originating from Hypericum perforatum, is a potent photosensitizer known to induce skin phototoxicity when given systemically. Previously, we have examined the penetration and distribution of hypericin and its acetate ester in the skin of hairless mice after topical application.

In vitro transport and uptake of protohypericin and hypericin in the Caco-2 model

The intestinal absorption characteristics of protohypericin, a protonaphthodianthrone present in Hypericum extract, were studied and compared with those of hypericin. The Caco-2 model was used as a model of the intestinal mucosa to assess transepithelial transport and cell uptake. The experimental work was performed in specific light conditions that prevented both the photoconversion of protohypericin into hypericin and the photosensitization of the cells. Following application of the individual compounds (80-200 microM) to the apical side of the monolayers, the appearance in the basolateral compartment was found to be very low (<0.5%/5 h), but was comparable for both compounds. A lag-time of 2-3 h was observed, suggesting gradual saturation of binding sites on the membrane or inside the cells. Uptake experiments of protohypericin and hypericin by Caco-2 cells revealed a very significant cellular accumulation (4-8%); uptake was characterised by saturation after 3 h. The findings of this study suggest that protohypericin has comparable absorption characteristics as hypericin and may contribute to the beneficial effect of Hypericum extract after oral dosing.

The effect of rhein and rhein anthrone on intestinal fluid transport and on large intestine transit in germ-free rats

The effect of rhein and rhein anthrone on the transit and the transport of water and electrolytes in the large intestine was investigated in germ-free rats. After intracaecal administration, neither of the two compounds was found to accelerate the transit of a colour marker through the large intestine. Both drugs reduced the net absorption of sodium and chloride in the colon and enhanced net potassium secretion. Net water absorption was decreased by rhein and even reversed into net secretion by rhein anthrone. Our results show that the secretagogue activity of the compounds is not sufficient to induce laxation in germ-free rats. Furthermore rhein and rhein anthrone had no laxative properties under our experimental conditions.

Photodynamic therapy using topically applied hypericin: Comparative effect with methyl-aminolevulinic acid on UV induced skin tumours

Photodynamic therapy (PDT) is a treatment option particularly well-suited for superficial (pre)malignant skin lesions due to the skins accessibility to light. In the present study, the efficacy of topical hypericin-PDT was evaluated using a mouse model for actinic keratosis. For comparison, similar experiments were conducted with methyl-aminolevulinic acid (Me-ALA). Small skin tumours (1-2 mm) were induced in hairless mice by chronic UV irradiation. After topical application of hypericin (0.1% in gelcream for 24 h) or Me-ALA (Metvix® for 4 h), the lesional/non-lesional skin surface fluorescence ratio was determined and fluorescence microscopy was used to study the skin penetration of the photosensitizers. The antitumour activity of topical PDT (20 mW cm(-2), 40 J cm(-2)) was evaluated by measurement of the lesional diameters. Moreover, biopsies were taken at various time points after PDT for histological evaluation of the therapy. Our results demonstrate that after topical application of hypericin and Me-ALA, tumour selectivity is limited in mouse skin. The microscopic distribution of hypericin fluorescence showed an accumulation in the stratum corneum and low fluorescence levels in the rest of the lesions, whereas the distribution of PpIX in the skin was more homogenous. Topical hypericin-PDT was found to be less efficient (44% total lesional clearance) as compared to Me-ALA-PDT (80% total lesional clearance). Full lesional necrosis was observed in responsive lesions, and the atypical cells of actinic keratosis were replaced by normal keratinocytes 3 weeks later, both after hypericin-PDT and Me-ALA-PDT.