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 skin's 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.

In vitro and in vivo multidrug resistance reversal activity by a Betti-base derivative of tylosin.

BACKGROUND: The multidrug resistance (MDR) proteins are present in a majority of human tumours. Their activity is important to understand the chemotherapeutic failure. A search for MDR-reversing compounds was conducted among various Betti-base derivatives of tylosin. METHODS: Here, we evaluate the in vitro and in vivo P-glycoprotein (P-gp)-modulating activity of the most promising compound N-tylosil-1-alpha-amino-(3-bromophenyl)-methyl-2-naphthol (TBN) using human MDR1 gene-transfected and parental L5178 mouse lymphoma cell lines. RESULTS: In vitro experiments showed that TBN dramatically increased the P-gp-mediated cellular uptake of the fluorescent substrate rhodamine 123. Similarly, TBN was found to act as a very potent enhancer of the cytotoxicity of doxorubicin on the resistant cell line. We also provide in vivo evidence using DBA/2 mice in support for an increased tumoural accumulation of doxorubicin, without affecting its tissue distribution, resulting in an enhanced antitumoural effect. CONCLUSION: Our results suggest that TBN is a potent modulator of the P-gp membrane pump and that the compound could be of clinical relevance to improve the efficacy of chemotherapy in MDR cancers.

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. OBJECTIVES: In this study, we assessed the time course and skin histopathology of the phototoxic response after a single topical application of hypericin and hypericin acetate, and subsequent irradiation. The amount of blood-borne photosensitizer and the skin clearance, as well the remaining photosensitizing capacity as a function of time, were evaluated. Furthermore, elicited phototoxic responses were compared with those after application of methyl aminolaevulinic acid (Me-ALA). METHODS: At different time points after topical application of hypericin (0.1-1%) and hypericin acetate (0.015-1.5%) onto mouse ears, penetration and retention of hypericin were assessed by fluorescence microscopy. After definite application times, the ears were irradiated (10 J cm(-2), 20 mW cm(-2)). Ear thickness measurements were conducted daily, and frequently ear samples were taken for histological analysis. RESULTS: Application of hypericin on mouse ears resulted only in limited phototoxicity, probably due to confined penetration into the epidermal layers. Extended penetration achieved by administration of hypericin acetate did give rise to a more severe and prolonged response after irradiation, characterized by intense erythema and ear swelling. Skin damage induced by 0.15% hypericin acetate application completely healed in 14 days without scar formation. After a single application of hypericin acetate, the residual photosensitizing capacity was found to decline quickly and was hardly detectable after 7 days. Under the experimental conditions used, hypericin acetate induced equal or more severe phototoxic responses compared with Me-ALA, depending on the concentration. CONCLUSIONS: Our results indicate that hypericin is an effective photosensitizer not only after systemic administration, but also after topical application, especially when applied as its precursor acetate ester. Moreover, our data provide some insights on safety limits and the time course of skin phototoxicity following hypericin and hypericin acetate application. These data will aid in developing protocols for future photodynamic therapy in the dermatological clinic.

Molecular effectors and modulators of hypericin-mediated cell death in bladder cancer cells.

Photodynamic therapy (PDT) is an anticancer approach utilizing a light-absorbing molecule and visible light irradiation to generate, in the presence of O(2), cytotoxic reactive oxygen species, which cause tumor ablation. Given that the photosensitizer hypericin is under consideration for PDT treatment of bladder cancer we used oligonucleotide microarrays in the T24 bladder cancer cell line to identify differentially expressed genes with therapeutic potential. This study reveals that the expression of several genes involved in various metabolic processes, stress-induced cell death, autophagy, proliferation, inflammation and carcinogenesis is strongly affected by PDT and pinpoints the coordinated induction of a cluster of genes involved in the unfolded protein response pathway after endoplasmic reticulum stress and in antioxidant response. Analysis of PDT-treated cells after p38(MAPK) inhibition or silencing unraveled that the induction of an important subset of differentially expressed genes regulating growth and invasion, as well as adaptive mechanisms against oxidative stress, is governed by this stress-activated kinase. Moreover, p38(MAPK) inhibition blocked autonomous regrowth and migration of cancer cells escaping PDT-induced cell death. This analysis identifies new molecular effectors of the cancer cell response to PDT opening attractive avenues to improve the therapeutic efficacy of hypericin-based PDT of bladder cancer.

Elucidation of the tumoritropic principle of hypericin.

Hypericin is a potent agent in the photodynamic therapy of cancers. To better understand its tumoritropic behaviour, we evaluated the major determinants of the accumulation and dispersion of hypericin in subcutaneously growing mouse tumours. A rapid exponential decay in tumour accumulation of hypericin as a function of tumour weight was observed for each of the six tumour models investigated, and a similar relationship was found between tumour blood flow and tumour weight. Moreover, there was a close correlation between the higher hypericin uptake in RIF-1 tumours compared to R1 tumours and tumour vessel permeability. To define the role of lipoproteins in the transport of hypericin through the interstitial space, we performed a visual and quantitative analysis of the colocalization of hypericin and DiOC18-labelled lipoproteins in microscopic fluorescent overlay images. A coupled dynamic behaviour was found early after injection (normalised fluorescence intensity differences were on the whole less than 10%), while a shifted pattern in localisation of hypericin and DiOC18 was seen after 24 h, suggesting that during its migration through the tumour mass, hypericin is released from the lipoprotein complex. In conclusion, we were able to show that the tumour accumulation of hypericin is critically determined by a combination of biological (blood flow, vessel permeability) and physicochemical elements (affinity for interstitial constituents).

Investigation of fractions present in the stem bark of Annickia kummeriae on their P-glycoprotein inhibitory pump activity.

Using MCF-7R cells and rhodamine 6G as the fluorescent probe, a bioassay-targeted purification process was pursued in order to isolate the active P-gp inhibitory fractions from Annickia kummeriae. Of 24 fractions obtained in the first preparative liquid chromatography (p-LC) run, only fraction 1 exhibited activity. Further p-LC fractionation led to the separation of fraction 1 into fractions 1.1-1.8. Fractions 1.4, 1.5 and 1.6 proved to be active by inducing a significant accumulation of rhodamine 6G by 3.3-, 4.5- and 4.9-fold at 10 microg/mL, and by 5.3-, 6.3- and 6.8-fold at 100 microg/mL, respectively. Fraction 1.6 was separated into several fractions by using an analytical liquid chromatography (a-LC) system. Fractions 1.6.18, 1.6.19 and 1.6.20 were active and they induced an accumulation of rhodamine 6G by 3.0-, 1.8- and 3.5-fold at 1x microg/mL and by 4.8-, 6.7- and 6.8-fold at 10x microg/mL, respectively. Afterwards, 28.3 mg of fraction 1.6 was processed by a-LC, and fractions 1.6.18, 1.6.19 and 1.6.20 were collected separately and dried. The amounts of materials recovered were 6.2, 7.4 and <1 mg, corresponding to 21.9%, 26.1% and <3.5% of fraction 1.6, respectively. From the total amount injected and the relative masses represented by these fractions, it can be calculated that the 1x microg/mL level corresponded to ca. 35, 42 and <5 microg/mL, respectively. Fluorescence microscopy revealed that incubation of the cells with rhodamine 6G alone did not show any fluorescence, whereas cells which were incubated in medium containing rhodamine 6G together with fraction 1.4, 1.6 or reserpine, clearly indicated accumulation of the dye intracellularly. This is an indication that the active compounds effected high intracellular fluorescence by inducing accumulation of the dye in the cells through inhibition of the P-gp pump.

Hypericin-based fluorescence diagnosis of bladder carcinoma.

OBJECTIVE: To determine the use of hypericin instillation for the fluorescent detection of papillary bladder cancer and carcinoma in situ. PATIENTS AND METHODS: Eighty-seven patients with papillary bladder cancer and/or carcinoma in situ received instillations with 40 mL of an 8 micromol/L hypericin solution for at least 2 h. Fluorescent excitation with blue light was effective for up to 16 h, and biopsies were examined by fluorescence microscopy. RESULTS: There were no side-effects reported, no photobleaching and all papillary lesions fluoresced red. The sensitivity and specificity for detecting carcinoma in situ was 94% and 95%, respectively. An interval of 4 months is recommended after BCG instillations before using this test. Fluorescence microscopy showed that hypericin was selectively localized in the epithelium. CONCLUSIONS: Hypericin-induced fluorescence has a high sensitivity and specificity for detecting bladder cancer. After 4 months there are few false-positive results in patients treated with BCG.

Cellular photodestruction induced by hypericin in AY-27 rat bladder carcinoma cells.

In a recent clinical study we showed that hypericin accumulates selectively in urothelial lesions following intravesical administration of the compound to patients. In the present study the efficacy of hypericin as a photochemotherapeutic tool against urinary bladder carcinoma was investigated using the AY-27 cells (chemically induced rat bladder carcinoma cells). The uptake of hypericin by the cells increased by prolonging the incubation time and increasing the extracellular hypericin concentration. Photodynamic treatment of the cells incubated with 0.8 and 1.6 microM hypericin concentrations resulted in remarkable cytotoxic effects the extent of which depended on the fluence rates. Photoactivation of 1.6 microM hypericin by 0.5, 1.0 or 2.0 mW/cm2 for 15 min resulted in 3, 30 and 95% of the antiproliferative effect, respectively. Increasing the photoactivating light dose from 0.45 to 3.6 J/cm2 resulted in a five-fold increase in hypericin photodynamic activity. Irrespective of the fluence rates and irradiation times incubation of the cells with 10 microM hypericin induced rapid and extensive cell death in all conditions. The type of cell death (apoptosis or necrosis) induced by photoactivated hypericin depended largely on the hypericin concentration and the postirradiation time. At lower hypericin concentrations and shorter postirradiation times apoptosis was the prominent mode of cell death; increasing the hypericin concentration and/or prolonging the postirradiation time resulted in increased necrotic cell death. Cell pretreatment with the singlet oxygen quencher histidine, but not with the free-radical quenchers, significantly protected the cells from photoactivated hypericin-induced apoptosis, at least when a relatively low concentration (1.25 microM) was used. This result suggests the involvement of a Type-II photosensitization process. However, cells treated with higher hypericin concentrations (2.5-5 microM) were inadequately protected by histidine. Since hypericin is thus shown to be a potent and efficient photosensitizer, and since the conditions used were the same as when hypericin is used clinically to locate early-stage urothelial carcinoma lesions, hypericin may well become very important for the photodynamic treatment of superficial bladder carcinoma.

Different pathways mediate cytochrome c release after photodynamic therapy with hypericin.

In this study we show that overexpression of Bcl-2 in PC60R1R2 cells reveals a caspase-dependent mechanism of cytochrome c release following photodynamic therapy (PDT) with hypericin. Bcl-2 overexpression remarkably delayed cytochrome c release, procaspase-3 activation and poly(adenosine diphosphate-ribose)polymerase cleavage during PDT-induced apoptosis while it did not protect against PDT-induced necrosis. PDT-treated cells showed a reduction in the mitochondrial membrane potential which occurred with similar kinetics in PC60R1R2 and PC60R1R2/Bcl-2 cells, and was affected neither by the permeability transition pore inhibitor cyclosporin A nor by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Hypericin-induced mitochondrial depolarization coincided with cytochrome c release in PC60R1R2 cells while it precedes massive cytochrome c efflux in PC60R1R2/Bcl-2 cells. Preincubation of PC60R1R2 cells with zVAD-fmk or cyclosporin A did not prevent the mitochondrial efflux of cytochrome c, and caspase inhibition only partially protected the cells from PDT-induced apoptosis. In contrast, in PC60R1R2/Bcl-2 cells cytochrome c release and apoptosis were suppressed by addition of zVAD-fmk or cyclosporin A. These observations suggest that the progression of the PDT-induced apoptotic process in Bcl-2-overexpressing cells involves a caspase-dependent feed-forward amplification loop for the release of cytochrome c.

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 photo-effect 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.

In vitro photobiological evaluation of Rhodac, a new rhodacyanine photosensitizer.

We have previously shown that the rhodacyanine dye, Rhodac, exhibits a potent photocytotoxic activity in HeLa cells. In this study several aspects of the photobiological activity of Rhodac were further examined. Rhodac displayed no selective cytotoxicity toward several malignant cell lines after photosensitization (3.6 J/cm2), although HeLa cells were found to be the most sensitive. Interestingly, MCF-7/Adr cells, a multidrug-resistant subline, were less sensitive to the antiproliferative effect of photoactivated Rhodac. The subcellular localization, as revealed by confocal laser microscopy, demonstrated that the dye was mainly concentrated in the cytosolic membranes of the perinuclear region. The Rhodac-induced inhibition of HeLa cell proliferation after light exposure was found to be strictly oxygen dependent. In addition, photoactivated Rhodac induced poly(adenosine 5' diphosphate-ribose)polymerase cleavage, caspase-3 activation and apoptosis in HeLa cells. In the current work it was further demonstrated that Rhodac binds specifically to high-density lipoproteins and low-density lipoproteins, while no binding was observed to very low-density and heavy proteins. To sum up, our results show that Rhodac is an interesting and potent photosensitizer. Further in vivo experiments are required to elucidate whether the lipoprotein binding leads to a selective uptake of Rhodac in tumor cells and to address its efficacy in photodynamic therapy.

Synergistic effect of photodynamic therapy with hypericin in combination with hyperthermia on loss of clonogenicity of RIF-1 cells.

Hypericin is a natural photosensitizer produced in plants of the genus Hypericum. The compound exhibits a potent phototoxicity both in vitro and in vivo. In the present study we investigated the effect of hypericin-mediated PDT on hyperthermia (43 degrees C) in RIF-1 cell line. Our results demonstrated a synergistic effect on loss of cell clonogenicity when PDT exposure was followed immediately by hyperthermia. This synergistic effect was diminished by introducing an interval (at 37 degrees C) between the two treatments. Furthermore, it was found that combining PDT treatment with hyperthermia could significantly enhance the cell death by necrosis as indicated by morphological examination and significant loss of membrane integrity. Our data suggest that the common cell membrane damage by both PDT and hyperthermia is likely to be responsible for this synergistic effect.

In vivo photodynamic activity of hypericin in transitional cell carcinoma bladder tumors.

In a recent clinical study, we showed that hypericin accumulates selectively in urothelial lesions of the bladder following intravesical administration of the compound in patients. This observation infers that hypericin, a potent photosensitizer, could be used as a selective photodynamic therapy (PDT) tool against superficial bladder cancer. In the present study we investigated the in vivo PDT activity of hypericin in transition cell carcinoma (TCC) tumors of the bladder. Both the distribution and tumor PDT response were carried out using subcutaneous heterotopic AY-27 TCC tumors in syngeneic rats. For both PDT and distribution studies, hypericin (1 or 5 mg/kg) was injected intravenously 0.5, 6 or 24 h before PDT or distribution evaluation. The data show that hypericin is a potent photosensitizer in the treatment of TCC tumors in vivo and that the interval between drug administration and photo-irradiation has a dramatic effect on the PDT outcome. Using a 0.5 h interval between drug administration and photo-irradiation the tumor regrowth study indicated that no tumor mass could me measured 9-10 days after PDT. On the contrary, lengthening the time interval between drug administration and photo-irradiation resulted in a gradual loss of PDT efficiency in these tumors. For instance, while the 6 h drug interval protocol produced a moderate PDT activity in which the tumor sizes decreased to about 50% of their original sizes 11-16 days after photo-irradiation, the 24 h interval protocol was even less effective. The distribution data indicate that the PDT efficiency of hypericin in TCC tumors corresponded to the plasma concentrations rather than to the over all concentrations in the tumor. It is therefore conceivable that the mechanism of PDT efficacy of hypericin in TCC tumors is through indirect (vascular effects) rather than through direct effects (cellular destruction) of hypericin in these tumors. In conclusion, our data indicate that hypericin is a potent photosensitizer against AY-27 TCC tumors and that the PDT efficacy of hypericin is largely determined by photosensitizer distribution in the tumor at the time of photo-irradiation.

Photodynamic therapy with hypericin in a mouse P388 tumor model: vascular effects determine the efficacy.

Hypericin, a polycyclic quinone obtained from plants of the Hypericum genus, exhibits strong photodynamic antitumor effects. In the present study, PDT efficacy of hypericin under different conditions was compared in a P388 mouse tumor model. Plasma and tumor drug measurements and assessment of vascular damage by fluorescein dye exclusion were performed to determine the relative contributions of vascular effects and direct tumor cytotoxicity. Furthermore, the influence of modifying tumor oxygenation on PDT effect was also evaluated. Study of PDT efficacy and tissue distribution revealed that PDT efficacy was more dependent on plasma concentration than tumor drug level. Fluorescein dye exclusion indicated the complete microvascular occlusion in the tumor and surrounding skin immediately after effective PDT treatments, while only a limited vascular occulation was observed after non-effective PDT treatment. It was found that neither tumor hypoxia induced by hydralazine nor increasing tumor oxygenation achieved by nicotinamide could significantly affect the effectiveness of various PDT protocols. These results suggest that tumor vasculature damage might be the primary mechanism of hypericin-mediated PDT effect. The existence of this potent secondary vascular effect is likely to account for the inability of tumor oxygenation modifiers to affect tumor response after PDT with hypericin.

Photodynamic activity of hypericin in human urinary bladder carcinoma cells.

Recently, we reported the selective accumulation of hypericin in transitional cell carcinoma cells following intravesical instillation of hypericin in humans. This observation infers that hypericin, a potent photosensitizer, could be used as a selective PDT (photodynamic therapy) tool against superficial bladder cancer. The aim of the present study was to investigate in vitro whether hypericin exhibits specific affinity for TCC transitional cell carcinoma) bladder cells and to assess its photocytotoxic effect. Three human TCC cell lines (J-82, T-24 and RT-4), a chemically induced rat TCC cell line (NBT-II), but also non-bladder carcinoma cells (HeLa, A431, MCF-7 and MCF-***ADR) and normal cells (HEL229, RPE and PHK), were used in this comparative study. Flow cytometric analysis of cells treated with different hypericin-containing vehicles for various incubation times (2 hours or 24 hours) indicated that short exposure of the cells (2 hours) to hypericin in the absence of serum results in the highest intracellular accumulation of the compound. As expected, prolonging the incubation time increased both the cellular accumulation and photocytoxicity of hypericia. With the exception of the RT-4 and MCF-7 cells (which were less sensitive to hypericin), all the other carcinoma cell lines examined showed equal sensitivity to the photoactivated hypericia, independently of their histological origin (bladder or non-bladder). Moreover, normal cells exhibited the same pattern of hypericin photosensitivity as shown by the cancer cells, indicating that, in cultured cells, hypericin cellular uptake and subsequent photokilling is not selective. This suggests that in vivo factors other than the cancer cells themselves are responsible for the specific accumulation of hypericin in urothelial carcinoma lesions.

Fluorescence detection of flat bladder carcinoma in situ after intravesical instillation of hypericin.

PURPOSE: We determined the sensitivity and specificity of detecting flat bladder carcinoma in situ through fluorescent detection after intravesical hypericin instillations. MATERIALS AND METHODS: The study included 40 patients, of whom 26 presented with macroscopic visible tumor, 9 had a positive cytology without visible tumor and 5 underwent cystoscopy after bacillus Calmette-Guerin instillations (4) or radiotherapy (1). We instilled 40 ml. of a 8 microM. solution of hypericin intravesically for at least 2 hours. Fluorescence excitation with blue light was effective up to 16 hours after termination of the instillation. RESULTS: All visible papillary tumors showed red fluorescence. In addition, 134 flat fluorescent areas were detected. Analysis of 281 biopsies from flat bladder wall indicated 93% sensitivity and 98.5% specificity for detecting carcinoma in situ. Visible lesions resulting from radiotherapy, chemotherapy or immunotherapy did not show any fluorescent signs and, therefore, did not induce false-positive readings. There were no signs of photobleaching during inspection and resection. CONCLUSIONS: We report a simple yet comprehensive endoscopic method for early detection of bladder cancer, including carcinoma in situ. Hypericin induced fluorescence has a high sensitivity and specificity for detection of bladder transitional cell carcinoma, papillary and flat carcinoma in situ. When carcinoma in situ is suspected, this technique is highly recommended.

Investigation of the absorption of hypericin into the skin of hairless mice.

The skin absorption of hypericin was evaluated in hairless mice to develop an optimised hypericin topical formulation that could be used in the clinical study of psoriasis. Hypericin (0.01-1.0%) in Beeler basis, polyethylene glycol ointment, carbopol gel, cetomacrogol cream, petrolatum or emulsifying ointment, with and without skin-absorption enhancers (isopropylidene glycerol and diethylene glycol monoethyl ether), was tested in-vivo on hairless mice skin. Using a skin-stripping technique and the intrinsic fluorescence of hypericin under standardised UV365 irradiation, it was demonstrated that the absorption of hypericin very much depended on the vehicle used. The concentrations of hypericin in the skin were then estimated by HPLC analysis. For this purpose, two vehicles were employed, with which hypericin penetrated the skin of hairless mice well (emulsifying ointment with isopropylidene glycerol) or very poorly (polyethylene glycol ointment). In the case of emulsifying ointment with isopropylidene glycerol (0.05% hypericin), a substantial concentration of hypericin (8.6+/-3.2 microg g(-1)) (mean +/- s.d., n = 5) was found in the skin. With polyethylene glycol ointment, however, only a limited hypericin skin concentration (0.38+/-0-34 microg g(-1), n = 5) was achieved. These results show that emulsifying ointment with polyethylene glycol holds promise as an effective topical vehicle for the treatment of skin diseases, such as psoriasis, with hypericin.

Photodynamic therapy efficacy and tissue distribution of hypericin in a mouse P388 lymphoma tumor model.

The phototherapeutic properties and tissue distribution of hypericin were investigated in DBA/2 mice bearing subcutaneously transplanted P388 lymphoma cells. The efficacy of the photodynamic therapy (PDT) 2 h after administration of hypericin (2, 5, or 20 mg/kg, i.p., 120 J/cm2, 595 nm) was substantially greater than the efficacy after a 24 h interval. PDT with Photofrin (5 mg/kg, i.p., 24-h interval, 120 J/cm2, 630 nm) showed no significant antitumoral effect. The hypericin uptake in some tissues was measured after administration of hypericin (5 or 20 mg/kg, i.p.) up to 168 h. A comparison of the distribution data and the PDT efficacy at various intervals suggests that the plasma concentration of hypericin, and to a lesser extent the tumor uptake, determines the tumor response to PDT with hypericin.

Bromohypericins are potent photoactive antiviral agents.

Several hypericin derivatives, previously shown to have interesting light-mediated biological activities, were evaluated for antiviral activities against herpes simplex virus and influenza virus. Three brominated hypericins, the dibromo- and tetrabromo-derivatives and the natural compound gymnochrome B were all very active against both viruses, particularly herpes simplex virus, although light was required in all cases for maximum activity. The dibromohypericin was the most potent, under standard assay conditions, gymnochrome B was approximately as active as hypericin itself and tetrabromohypericin significantly less so. Surprisingly, hexamethylhypericin, which is known to have potent anti-protein kinase (PK) C activity, as well as anticell proliferation properties, showed no antiviral activity at all. The compounds were also evaluated in different serum concentrations. All the active compounds were inhibited by increasing concentrations of serum, but to different degrees, such that their relative antiviral potencies changed to some extent. Thus, in summary, there was no correlation between antiviral and anti-PK or anticellular activities, and consequently it is not possible at present to define those structural features of hypericin-type molecules that are required for their various biological activities.

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.