Yehuda Mazur

Hypericin as an inactivator of infectious viruses in blood components

BACKGROUND: Hypericin is a potent virucidal agent with activity against a broad range of enveloped viruses and retroviruses. The effective virucidal activity emanates from a combination of photodynamic and lipophilic properties. Hypericin binds cell membranes (and, by inference, virus membranes) and crosslinks virus capsid proteins. This action results in a loss of infectivity and an inability to retrieve the reverse transcriptase enzymatic activity from the virion.

A method for the quantitation of hypericin, an antiviral agent, in biological fluids by high-performance liquid chromatography

Hypericin, a polycyclic aromatic dianthroquinone, is a natural plant product with antiviral properties. We report here the development of a methodology for the extraction and quantitation of hypericin from plasma and biological fluids and the adaptation of a sensitive and selective method for detection of the compound by high-performance liquid chromatography. The methodology offers a rapid and specific means of monitoring drug blood levels in clinical and pharmacokinetic studies. The chromatographic procedure utilizes the substantial retentive properties of hypericin on reverse-phase media and detection by the strong visible absorbance maximum at 590 nm. Verification by the fluorescence spectral properties of hypericin in organic media can also be utilized. The assay is linear over a 3 log concentration range and hypericin is consistently recovered from murine, simian, and human plasma. The methodology was applied to assess the pharmacokinetic properties of hypericin in mice receiving a single bolus injection of 350 micrograms. A distribution half-life of 2.0 h and an elimination half-life of 38.5 h were calculated. We also discuss the limitations of direct analysis of hypericin by absorbance or fluorescence measurements.

Liposome binding constants and singlet oxygen quantum yields of hypericin, tetrahydroxy helianthrone and their derivatives: studies in organic solutions and in liposomes

The spectroscopy and photophysics of several hypericin and helianthrone derivatives were studied in methanol and when bound to liposomes. The singlet oxygen quantum yields (phi(delta)) were measured indirectly relative to Rose Bengal and hematoporphyrin IX, employing 9,10-dimethylanthracene as a singlet oxygen trap. Hypericin was found to have a phi(delta) of 0.39+/-0.01 in methanol, and 0.35+/-0.05 in lecithin vesicles, in agreement with literature values. A heavy atom effect was evident upon bromination, resulting in phi(delta) for tetrabromohypericin of 0.72+/-0.02, presumably due to enhanced intersystem crossing. Elimination of the anionic hydroxyls by methylation also enhanced phi(delta) to 0.81+/-0.01. Conversely, addition of anionic sulfate groups drastically reduced phi(delta) resulting in phi(delta)s of 0.12+/-0.01, 0.052+/-0.003 and 0.40+/-0.01 for hypericin disulfonate, hypericin tetrasulfonate and hexamethyl hypericin tetrasulfonate, respectively. The non-sulfonated helianthrones exhibited low phi(delta)s in solution. The liposome binding constants, Kb, were measured using a spectroscopic assay. Except for hexamethyl hypericin, all non-sulfonated compounds bound well with Kbs ranging from 15.5+/-0.1 to 48.7+/-3.9 (mg/ml)(-1). None of the tetrasulfonated compounds bound, however the hypericin disulfonate had a Kb of 4.1+/-0.2 (mg/ml)(-1). The phi(delta)s of the compounds capable of binding were measured and, in the case of the hypericin derivatives, were found not to vary dramatically from those in the free state. Liposome-bound helianthrone and dimethyl tetrahydroxy helianthrone both exhibited high phi(delta)s, i.e. >0.5. The variations in binding constant and sensitization efficiencies are explained in conjunction with the molecular structure. The relevance of the above data to photodynamic therapy is briefly discussed.

The correlation between hydrophilicity of hypericins and helianthrone: internalization mechanisms, subcellular distribution and photodynamic action in colon carcinoma cells

The internalization mechanism and subcellular distribution of hypericin (Hyp), hypericin tetrasulfonic acid (HypS4) and 1,3,4,6-tetrahydroxyhelianthrone (Hel) were studied in murine colon carcinoma CT26 cells, in protein-free medium or in the presence of serum proteins. The correlation between the extent of uptake of the sensitizers by cells that were incubated in the presence of different serum components, and the internalization mechanisms, was studied. The results indicate that sensitizer internalization may be a result of three mechanisms: partitioning, pinocytosis and endocytosis, and as a direct consequence is targeted to specific subcellular sites. While Hyp and Hel, the two lipophilic sensitizers, were localized in the endoplasmic reticulum after protein-free internalization, the hydrophilic HypS4 was localized in the cytoplasmic membrane and in lysosomes. An endolysosomal internalization route was revealed for Hyp and Hel under serum-enriched conditions showing lysosomal localization, as for HypS4. The lysosomal accumulation of Hyp-serum and specifically Hyp-LDL points to an endocytotic mechanism which is supported by its higher uptake parameter in an LDL-enriched medium, compared to the medium with 10% serum. The different uptake parameters of Hyp to cells, with or without serum, reflect the different mechanisms. Smaller differences in the uptake parameter for HypS4 reflect the distinction between partitioning and endocytosis, which, in this case, are both targeted to the lysosomes. The same uptake parameter of Hel to cells incubated in media with or without serum indicates the absence of the endocytotic mechanism. The interrelationship between subcellular targeting and photodynamic treatment was shown for the three sensitizers Hyp was found to be the most efficient sensitizer for PDT under our illumination protocol and it was dependent on internalization and localization sites.

Strategies for Evaluation of Enveloped Virus Inactivation in Red Cell Concentrates Using Hypericin

Abstract Photodynamically induced virus inactivation appears promising in preventing transmission of enveloped virus infections in transfusible blood products. The potential for utilizing hypericin as a photosensitizer to inactivate key enveloped viruses in packed red cell concentrates (PRC) was evaluated. In addition to inactivating effectively ≥106 TCID50 of human immunodeficiency virus (HIV), inactivation of bovine viral diarrhea virus (BVDV) in PRC was used as a model for hepatitis C virus to overcome the deficiency in reliable experimental systems for hepatitis C virus (HCV) inactivation. BVDV was two orders of magnitude more sensitive to inactivation by hypericin than HIV. As part of the virucidal efficacy analyses, the effects of photosensitization on hemopoietic cell lines carrying quiescent integrated HIV provirus were studied as models for evaluating virus inactivation in latently infected cells. Phorbol ester-induced virus production by these cells was effectively prevented by photosensitization with hypericin. A refinement of the illumination conditions, incorporating a monochromatic sodium light source with an emission spectrum coinciding with the absorption peak of hypericin, was highly virucidal, however, caused unacceptable levels of hemolysis. Red blood cells could be protected from phototoxic cellular damage by complexing hypericin with human serum albumin (albumin–hypericin), but the decrease in hemolysis was at the expense of virucidal efficacy. Thus, excitation of hypericin with a fluorescent source appears to be useful potentially for virus inactivation in PRC.

EPR studies of hypericin. Photogeneration of free radicals and superoxide

Hypericin, a potent antiviral agent, displays, in the absence of light and electron donors, an EPR signal which is attributed to a semiquinone-like radical, formed by intermolecular electron transfer. On irradiation with visible light the amplitude of the EPR signal increases significantly. This increase is highest (ca. 20 fold) in aqueous dispersions of the lysine salt of hypericin. Irradiation of hypericin water aggregates in the presence of oxygen generates superoxide radicals which may be registered by the spin trap technique.This finding implies that the free radicals of hypericin and superoxide radicals formed on photoirradiation of hypericin may play a hitherto unrecognized role in the biological activities elicited by hypericin both in vivo and in vitro.

Reactions in dry media: reactions of cholesterol and cholestanes on silica bound ferric chloride : Cholestane—diacholestene rearrangement

Abstract Reaction of cholesterol with silica bound FeCl3 resulted in a mixture of 3β-cholesteryl chloride and dicholesteryl ether. 5-Cholestene and hydroxy- and halogeno-substituted cholestane derivatives gave on heating at 100° with this reagent a 1:1 mixture of 20-epimeric diacholestenes. The 20(R)-isomer gave with meta-chloroperbenzoic acid 20(R)-α-epoxide, while the 20(S)-gave a mixture of 20(S)-α- and 20(S)-β-epoxides. 5α,6β-Dihydrocholestane reacted with the FeCl3/SiO2 under milder conditions (50°) to give 6β-hydroxy-20(R)-diacholestene, which was converted to the 20(R)-diacholestene.

Solvatochromic Effects in the Electronic Absorption and Nuclear Magnetic Resonance Spectra of Hypericin in Organic Solvents and in Lipid Bilayers

Abstract The natural product hypericin was tested in recent years as a biological photosensitizer with a potential for viral and cellular photodamage. We thus studied extensively its spectroscopy and membrane partitioning. Absorption, fluorescence excitation and emission spectra of the sodium salt (HyNa) were measured in 36 protic and aprotic, polar and apolar, solvents. Electronic transition bands as well as vibrational progressions were identified. Aggregation in some nonpolar solvents and protonation in organic acids were demonstrated. Modeling solvatochromism was done by Lippert equation, by the ET(30) parameter and by the Taft multiparameter approach. In all cases, separation into protic and aprotic solvents gave much better fits to the models. 13C chemical shift data could also be correlated with solvent polarity. They correlated best with Lipperts Δf polarity measure, but tended to fall into two distinct solvent groups—each along different lines—corresponding to protic and aprotic media, respectively. This interesting phenomenon suggests that in the case of the charged and slightly water soluble HyNa, two mechanisms of solvation are involved, each resulting in its own line equation. In aprotic media, dipole–dipole interaction is the predominant solvation mechanism. In protic solvents, the most effective means of solvation is likely to be hydrogen bonding. When intercalated into the liposomal phospholipid bilayer, HyNa is oriented at an angle to the interface, thus experiencing a gradient of solvent polarities: a highly polar environment (similar to methanol) for C-2/5, suggesting that they lie not far from the interface; a moderately polar environment (similar to that of n-propanol) for C-6a/14a, which are somewhat deeper within the bilayer; and a more lipophilic environment (akin to n-hexanol) for C-10/11. The fluorescence excitation peak in liposomes also correlates with an aprotic medium of relatively high polarity, as might be excepted from a molecule in a shallow position in the bilayer.

(7) Glucosylation and (1-6) rhamnosylation of exogenous flavanones by undifferentiated Citrus cell cultures

Abstract The ability of citrus cultures to biotransform flavanones was tested. Only one undifferentiated sour orange ( Citrus aurantium L.) ovule derived cell culture glucosylated exogenous naringenin at position 7 and further rhamnosylated the product at position 6 of the glucose, synthesizing narirutin. The products were verified by 1 H-NMR. Other ovule-derived cell lines, one from sour orange ( Citrus aurantium L.) and one from a Poorman × Poncirus trifoliata (L.) Raf. hybrid were only able to glucosylate exogenous naringenin and hesperetin at position 7, as previously shown in C. paradisi Macf. cv. Duncan cell cultures. TLC analyses indicated that cultures of ‘Ponderosa’ lemon ( C. limon (L.) Burm. f. cv. Ponderosa) and grapefruit ( C. paradisi Macf. cv. White Marsh) also glucosylated exogenous hesperetin at position 7. None of the cell-lines tested accumulated flavanone-glycosides without exogenously supplying aglycones.

Reactions of acid anhydrides—I: Reactions of ketones with trichloroacetic anhydride

Abstract The reactions of saturated and unsaturated ketones with trichloroacetic anhydride both alone and in the presence of trichloroacetic and p -toluenesulphonic acids were studied. Treatment of pentan-3-one ( 1 ), cyclobutanone ( 2 ), cyclohexanone ( 3 ), nor-camphor ( 4 ), 17β-acetoxy-5α-androstan-3-one ( 5a ) 17β-acetoxy-5β-androstan-3-one ( 6a ) and cyclohexen-2-one ( 25 ) with trichloroacetic anhydride resulted in the corresponding geminal bis-trichloroacetates 7, 8, 9, 10, 11, 12 and 26 . The presence of trichloroacetic acid decreases the rate of the conversion of these ketones to the geminal bis-trichloroacetates. Testosterone acetate ( 28a ), acetophenone ( 31 ) and the steroidal dimethylketone 21a , gave, on treatment with trichloroacetic anhydride, the corresponding enol trichloroacetates 30, 33 and 23 . Camphor ( 17 ) resulted in a rearrangement product, the 1-trichloroacetoxy-camphene 19a . In the presence of trichloroacetic acid, the dimethylketone 21a gave in addition to 23 the aromatization product, 24a and camphor 17 gave in addition to 19a the 2β,4-bis-trichloroacetoxy-bornane 20a . Trichloroacetic anhydride in the presence of p -toluenesulphonic acid converted ketones 1, 3, 5a and 6a , as well as the geminal bis-trichloroacetates 7,9,11 and 12 , to the corresponding enol trichloroacetates 13, 14, 15 and 16 . The spectral properties of the geminal bis-trichloroacetates and enol trichloroacetates are discussed. The reactivities of the various ketones towards trichloroacetic anhydride were compared, and the mechanism of formation of gem diesters and enol esters is proposed. It is suggested that the gem diesters are invariably intermediates in the conversion of the ketones to the enol esters.