Antiviral properties of photosensitizers

Abstract— We have studied the antiviral properties of three different groups of photo-sensitizers, viz. (i) various furyl compounds; (ii) β-carboline alkaloids; (iii) thiophenes and their acetylene derivatives. In general the antiviral potency of the furyl compounds correlated with their ability to produce DNA photoadducts. Among the naturally occurring β-carboline alkaloids, harmine was considerably more potent (in the presence of long wavelength UV radiation, UVA) than several other harmane-related compounds. Slight alterations in chemical structure had profound effects on their antiviral activities. Harmine was shown to inactivate the DNA-virus murine cytomegalovirus (MCMV) by inhibiting viral gene expression, although other targets may also exist. Several eudistomins, carboline derivatives isolated from a tunicate, were also photoactive against viruses. Various plant thiophenes and polyacetylenes were studied in detail. These compounds also required UVA for antiviral activity, and some of them were extremely potent against viruses with membranes, e.g. α-terthienyl, which showed significant activity at only 10^-5μg/ml. When MCMV had been treated with α-terthienyl plus UVA, the virus retained its integrity and penetrated cells normally; but the virus did not replicate. More than 30 additional thiophenes have recently been evaluated, including many synthetic ones, and some of these are even more potent than a-terthienyl. We believe that certain thiophenes possess potential therapeutic value and should be tested against model virus infections in animals.     

ANTIVIRAL ACTIVITY OF THE PHOTOACTIVE THIOPHENE α-TERTHIENYL

The naturally occurring thiophene, α-terthienyl, was investigated for phototoxicity against several viruses and a line of mouse cells. The compound was extremely phototoxic to the two-membrane-containing animal viruses, murine cytomegalovirus (MCMV) and Sindbis virus (SV). Antiviral activity was detected at 10⁵μg/m in the presence of UVA. However, no effect was seen in the absence of UV-A, even at 0.1 μg/m of αT. Mouse cells were much more resistant to αT, as was the bacterial virus T4, which does not contain a membrane. Murine CMV, which had been inactivated by αT and UVA, penetrated mouse cells efficiently; but the viral DNA could not replicate, and late viral proteins were not made. Thus viral gene expression was inhibited in the photoinactivated virus. In order to account for all these data we suggest that αT may interact with viral proteins in addition to membrane lipids.     

INDUCTION OF CROSS-LINKS IN VIRAL DNA BY NATURALLY OCCURRING PHOTOSENSITIZERS

Abstract— Six different photosensitizers were compared for their ability to form cross-links in murine cellular DNA and murine cytomegalovirus DNA, in the presence of long wave UV radiation. The viral DNA was in the form of free DNA or intact virions. The compounds consisted of the linear furanocoumarins 8-methoxypsoralen (8-MOP) and isopimpinellin; the angular furanocoumarin, angelicin; the two furanochromones, visnagin and khellin; and the β-carboline alkaloid, harmine. Cross-linking was assessed by alkaline agarose gel electrophoresis and hydroxyapatite chromatography. 8-MOP produced extensive cross-linking (as expected), as did isopimpinellin. Visnagin produced less cross-linking, such that not all DNA molecules were affected at the concentrations used. Khellin, angelicin and harmine produced no detectable cross-linking. The same result was obtained for DNA which was treated in situ in the virion. To some degree there was a correlation between the amount of cross-linking and the relative potency of anti-MCMV infectivity. But other factors evidently contribute to the phototoxic effect of these compounds.     

ANTIVIRAL EFFECT OF HARMINE, A PHOTOACTIVE β-CARBOLINE ALKALOID

Abstract— Harmine, a β-carboline alkaloid, was found to be an efficient photoactive agent against the DNA-containing murine cytomegalovirus (MCMV) and the RNA containing Sindbis virus, a togavirus, both of which contain membranes. In contrast the host cells themselves were relatively resistant. The MCMV was investigated in more detail. Virus which had been inactivated by harmine plus UVA retained its structural integrity and was fully capable of penetrating host cells, following which the viral genome entered the cell nucleus. The viral genes which normally code for the switch-off in thymidine kinase activity arid for the normal cytopathic effects were not expressed; however the viral geneis responsible for the cytotoxic effect due to high multiplicities of infection was still expressed, indicating that there may be a differential block of some viral genes     

ALPHA-TERTHIENYL, A NON-PHOTODYNAMIC PHOTOTOXIC COMPOUND

Abstract— Alpha-terthienyl (αT), a natural product first isolated in the plant Tagetes erecta (family Compositae), is the first member of a new class of phototoxic compounds, whose activity is not based on the generation of singlet oxygen. Its maximum activity against Candida utilis was at 370 nm, and was not affected by the presence of sodium azide. Furthermore, its phototoxicity against E. coli B cells was expressed under nitrogen, but not under oxygen. In the presence of UVA, radioactive αT formed covalent bonds with calf thymus DNA, and was also incorporated into the DNA of C. utilis. Two different methods proved that the photochemical interaction of αT and DNA did not involve cross linking of double strands.     

In vitro phototoxicity of nifedipine: sequential induction of toxic and non-toxic photoproducts with UVA radiation.

Anecdotal reports suggest that the dihydropyridine calcium antagonist, nifedipine (NIF), may be phototoxic in human skin. We have studied NIF phototoxicity in vitro using UVA fluorescent tubes (Sylvania PUVA). NIF was phototoxic to Candida albicans and induced photohaemolysis both with NIF present during irradiation and with pre-irradiated drug. In V79 hamster fibroblasts, NIF (10 micrograms ml-1) was phototoxic MTT assay) 24 h after irradiation (0-112 kJ m-2); at 7.5 kJ m-2, about 70% of cells were damaged whilst at 37.5 kJ m-2, only about 45% of cells were damaged. A similar pattern was seen with pre-irradiated NIF. Absorption spectroscopy showed that the NIF absorption maximum (Amax approximately 340 nm) blue-shifted to 314 nm at low UVA doses (7.5 kJ m-2 or less) and red-shifted to 345 nm at higher doses (isosbestic point, 325 nm). Thin layer chromatography of irradiated NIF showed a single photoproduct (PP1; Amax approximately 314 nm) formed at 7.5 kJ m-2 or less which disappeared at higher UVA doses to give further photoproducts. PP1 was highly dark toxic to V79 cells (50% damage at about 5 micrograms ml-1) but PP1 pre-irradiated with UVA was non-toxic. Preliminary gas chromatography-mass spectroscopy studies suggest that PP1 is the nitroso derivative of NIF. These results indicate that NIF phototoxicity in vitro is partially mediated by initial formation of a toxic photoproduct (PP1) but, paradoxically, subsequent UVA irradiation may reduce phototoxicity. The NIF concentrations required to induce in vitro phototoxicity are much greater than therapeutic plasma levels. Unless there is skin accumulation of NIF or PP1, our in vitro results suggest that NIF may not be an important skin-photosensitizing agent in vivo.     

RESEARCH NOTE PHOTOTOXICITY OF CITRAL TO Trichoplusia ni (LEPIDOPTERA: NOCTUIDAE) AND ITS AMELIORATION BY VITAMIN A

Abstract Citral, a monoterpene aldehyde synthesized by several plant families, was recently shown to exhibit ultraviolet A light (320–400 nm) (UVA)-enhanced oxygen-dependent toxicity against bacteria and fungi ( Escherichia coli and Fusarium species). In this study, we report for the first time that citral is phototoxic to insects; at concentrations of 300 ppm in artificial diet, mortality of first instar Trichoplusia ni (cabbage loopers) approached 30% after 120 h of UVA exposure, approximately double the mortality level in the absence of UVA. At 300 ppm, the antioxidant vitamin A reduces citral phototoxicity by about 50% in this species, suggesting that citral phototoxicity against insects is oxygen dependent.     

β-CARBOLINE ALKALOIDS: MECHANISMS OF PHOTOTOXICITY TO BACTERIA AND INSECTS

Abstract— Phototoxicity of five naturally occurring (3-carboline alkaloids was assayed against a series of Escherichia coli strains and against the insect Trichoplusia ni (Lepidoptera: Noctuidae). Rank order efficacy of the compounds was comparable in both organisms. Although the bacterial assay demonstrated oxygen dependence, the degree of phototoxicity did not correlate with the relative efficiency of in vitro singlet oxygen or hydrogen peroxide photoproduction by the alkaloids. A better correlation was observed between chromatographic migration distances (lipophilicity) and phototoxicity. Therefore, hydrophobic mechanisms in which the alkaloids diffuse with varying efficiencies into cells or into the vicinity of target molecules may be important modes of phototoxicity for these compounds.     

Quinolone Antibiotic Photodynamic Production of 8-Oxo-7,8-dihydro-2'-deoxyguanosine in Cultured Liver Epithelial Cells

To study the basis for the phototoxicity of quinolones, a class of synthetic antibacterials, the photodynamic ability to mediate 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) formation in cultured cells was measured for lome-floxacin (LMX), which is strongly associated with clinical phototoxicity in humans, and ciprofloxacin (CFX), which has few reports of phototoxicity. Adult rat liver (ARL-18) cells were exposed to the quinolones in the presence of UVA and DNA was extracted and analyzed by HPLC with electrochemical detection. Low levels of 8-oxo-dG were found in the DNA of nonirradiated ARL-18 cells and this was increased up to 6-fold in the presence of either LMX (50–400 uAf) or up to 3.6-fold in the presence of CFX (50–400 µM) and UVA (20 J/cm²) when compared to the UVA control. Comparing separate experiments with LMX and CFX, LMX produced greater levels of 8-oxo-dG either after dark exposure or after UVA exposure at 20 J/cm². Also, LMX and CFX were both shown to photodegrade in the presence of UVA, and it was determined that UVA photoinstability alone does not reflect phototoxic potential. These data suggest that the photodynamic potential of LMX and CFX to produce 8-oxo-dG may relate to their human clinical phototoxicity profile. We suggest that the observed clinical phototoxicity is mediated through a UVA photodynamic effect on the quinolone to form reactive oxygen species in the presence of molecular oxygen. The findings indicate that 8-oxo-dG formation can serve as a marker for the potential phototoxicity of new quinolones.     

FURTHER STUDIES ON THE ANTIVIRAL ACTIVITY OF HARMINE, A PHOTOACTIVE β-CARBOLINE ALKALOID

The UV-A mediated antiviral effect of harmine was investigated using murine cytomegalovirus (MCMV) as the target. Virus, which had been inactivated by harmine + UVA, was used to infect cultured mouse cells, and various stages in the viral replication cycle were examined. No viral protein synthesis or RNA synthesis (as measured by polyacrylamide gel electrophoresis or DNA-RNA hybridization) could be detected, and the viral DNA did not replicate (measured by DNA-DNA hybridization). In contrast virus which had been treated with harmine in the dark promoted a complete growth cycle in mouse cells.
An attempt was made to identify the primary target of harmine + UVA activity by examining the bacteriophages T4 and M13, which unlike MCMV do not possess membranes. Both bacteriophages were sensitive, but the single-stranded DNA phage M13 was considerably more so. These results, together with others discussed in the text, suggest that DNA and possibly other macromolecules can serve as targets for harmine photoactivity.     

Synthesis of bioactive nitrogen heterocycles from marine organisms

Recently many bioactive indole alkaloids have been isolated from marine organisms. Many of them have novel ring systems which are not found in the indole alkaloids isolated from higher plants and molds on the land. We have chosen β-carboline alkaloids, eudistomins and manzamines, as targets for total synthesis.     

Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.) 3: effect on human lens and retinal pigment epithelial cells

The dried root or rhizome of Goldenseal (Hydrastis canadensis L.) contains several alkaloids including berberine, hydrastine, palmatine and lesser amounts of canadine and hydrastinine. Preparations derived from Goldenseal have been used to treat skin and eye ailments. Berberine, the major alkaloid in Goldenseal root powder, has been used in eye drops to treat trachoma, a disease characterized by keratoconjunctivitis. Berberine and palmatine are also present in extracts from Berberis amurensis Ruprecht (Berberidaceae) which are used to treat ocular disorders. We have previously shown that Goldenseal alkaloids are phototoxic to keratinocytes (Chem Res Toxicol. 14, 1529, 2001; ibid 19, 739, 2006) and now report their effect on human lens and retinal pigment epithelial cells. Human lens epithelial cells (HLE-B3) were severely damaged when incubated with berberine (25 microM) and exposed to UVA (5 J cm(-2)). Under the same conditions, palmatine was less phototoxic and hydrastine, canadine and hydrastinine were inactive. Moderate protection against berberine phototoxicity was afforded by the antioxidants ascorbate (2 mM) and N-acetylcysteine (5 mM). When exposed to UVA (5 J cm(-2)) both berberine (10 microM) and palmatine (10 microM) caused mild DNA damage as determined by the alkaline comet assay which measures single strand breaks. Berberine and palmatine are the only Goldenseal alkaloids with appreciable absorption above 400 nm. Because light at wavelengths below 400 nm is cut off by the anterior portion of the adult human eye only berberine and palmatine were tested for phototoxicity to human retinal pigment epithelial (hRPE) cells. Although berberine did damage hRPE cells when irradiated with visible light (lambda > 400 nm) approximately 10 times higher concentrations were required to produce the same amount of damage as seen in lens cells. Palmatine was not phototoxic to hRPE cells. Neither berberine nor palmatine photodamaged DNA in hRPE. Infusions of Goldenseal are estimated to contain approximately 1 mM berberine, while in tinctures the alkaloid concentration may be more than 10 times higher. Our findings show that eyewashes and lotions derived from Goldenseal or containing berberine must be used with caution when the eyes are exposed to bright sunlight but that oral preparations are not likely to cause ocular phototoxicity.     

1-氨基-7-甲氧基-β-咔啉及其1-烷氧羰基氨基衍生物的合成和 初步抗肿瘤活性研究

以1-甲氧羰基-7-甲氧基-β-咔啉为原料经肼解、重氮化反应得1-叠氮酰基-7-甲氧基-β-咔啉(6), 再经Curtius重排、碱解反应得1-氨基-7-甲氧基-β-咔啉(2). 化合物6的Curtius重排产物与各种醇反应得1-烷氧羰基氨基-7-甲氧基-β-咔啉(3). 所得到的10个化合物结构经1H NMR, 13C NMR, MS和元素分析确证. 采用MTT法对合成的化合物进行了体外抗肿瘤活性测定, 结果表明, 在10－5 mol/L浓度下目标化合物具有一定的抗肿瘤活性, 其中化合物3e, 3g和3h对HepG2和SGC-7901抑制率均高于阳性对照物骆驼蓬碱(1).     

Amino acid derivatives of pyropheophorbide-α ethers as photosensitizer: Synthesis and photodynamic activity

Ten new water-soluble amino acid conjugates of pyropheophorbide-α ethers 4a-4j were synthesized and investigated for their in vitro photodynamic antitumor activity. The results showed that all compounds exhibited higher phototoxicity and lower dark toxicity against three kinds of tumor cell lines than BPD-MA. In particular, themost phototoxic compound 4d and 4j individually showed IC₅₀ values of 41 nmol/L and 33 nmol/L against HCT116 cell, which represented 7.8- and 9.7-fold increase of antitumor potency compared to BPD-MA, respectively, suggesting that they were promising photosensitizers for PDT applications because of their strong absorption at long wavelength (λ_max>650 nm), high phototoxicity, low dark cytotoxicity and good water-solubility.     

Design and synthesis of novel water-soluble amino acid derivatives of chlorin p6 ethers as photosensitizer

Eight new water-soluble amino acid derivatives of chlorin p₆ ethers 6a-h were designed and synthesized using purpurin-18 (2) as key intermediate. All target compounds exhibited better phototoxicity than talaporfin and the most phototoxic compound 6d showed IC₅₀ values of 0.20 μmol/L against A549 cell and 0.41 μmol/L against B16-F10 cell, which represented 31- and 24-fold increase of PDT antitumor efficacy compared to talaporfin.     

Cytotoxic and insecticidal activities of derivatives of harmine, a natural insecticidal component isolated from Peganum harmala

In a continuing effort to develop novel β-carbolines endowed with better insecticidal activity, a simple high-yielding method for the synthesis of harmine compounds starting from L-tryptophan has been developed and a series of 1,3-substituted β-carboline derivatives have been synthesized and evaluated for their cytotoxicity against insect cultured Sf9 cell line in vitro and insecticidal activities against 4th instar larvae of mosquitos, Culex pipiens quinquefasciatus and mustard aphid, Lipaphis erysimi. The results demonstrated that 1-phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (compound 2) and methyl 1-phenyl-β-carboline-3-carboxylate (compound 13) represented the best potential compounds, with Sf9 cells inhibition rates of 71.55% and 60.21% after 24 h treatment at concentrations of 50-200 mg/L, respectively. Both compounds 2 and 13 also showed strong insecticidal activity towards 4th instar larvae of mosquitos with LC(50) values of 20.82 mg/L and 23.98 mg/L, and their LC(90) values were 88.29 mg/L and 295.13 mg/L, respectively. Furthermore, the LC(50) values of compounds 2 and 13 against mustard aphids were 53.16 mg/L and 68.05 mg/L, and their LC(90) values were 240.10 mg/L and 418.63 mg/L after 48 h treatment. The in vitro cytotoxicity of these compounds was consistent with the insecticidal activity in vivo. The results indicated that the 1- and 3-positions of the β-carboline ring deserve further investigation to develop biorational insecticides based on the natural compound harmine as a lead compound.     

THE EFFECT OF PROTOPORPHYRIN ON HISTAMINE SECRETION BY RAT PERITONEAL MAST CELLS: A DUAL PHOTOTOXIC REACTION

Abstract— Protoporphyrin-induced phototoxicity in rat peritoneal mast cells was manifested either by inhibition of 48/80-stimulated histamine secretion or by cell lysis. At a protoporphyrin concentration of 100ng/m/ (0.17 μM), histamine secretion was completely inhibited after 30min illumination. After initiation, the inhibited state progressed in the dark, and was irreversible, however, it did not develop into cell lysis. More severe phototoxic reactions in mast cells could not be produced by increasing the PP concentration or the incubation time; however, cell lysis was evoked by increasing the light intensity between 180–950W/m², using a light source with emission maxima in the 350–470nm region. Dual phototoxic effects could also be demonstrated in erythrocytes by manipulating the illumination conditions. Increased resistance to osmotic lysis was seen under moderate conditions, and decreased resistance and cell lysis were seen under severe conditions. In the absence of protoporphyrin, the effect of light alone on mast cells was similar to protoporphyrin-phototoxicity, although the light intensities required were higher both for inhibition (60–130W/m²) and lysis (280–950W/m²). The data therefore indicate that certain cell functions can be specifically disrupted by phototoxic reactions that are not cytotoxic; however, phototoxic reactions that lead to severe membrane protein denaturation and cell lysis also occur. The manifestation of these dual effects depends on the intensity of illumination in the 350–470nm region.     

COAL TAR PHOTOTOXICITY: ACTIVE COMPOUNDS AND ACTION SPECTRA*

Abstract Eight compounds present in crude coal tar were tested for phototoxicity on guinea-pig skin. Four concentrations (5 μM to 5 mM) of each compound in ethanol were applied to the dorsal surface of 6 guinea-pigs. Each animal received 1.0 x 10⁵ j/m² of UVA radiation. The erythematous (phototoxic) response was evaluated after 20 h. Pyrene, anthracene and fluoranthene were strongly phototoxic. Acridine was markedly less phototoxic. Action spectra based on erythema as an endpoint were determined in guinea-pigs for anthracene, pyrene and fluoranthene. Each compound was applied to 5 animals which received irradiations from 274 to 502 nm in 12 nm bands for 3, 6, 9, 12 and 15 min. The maximum erythema response to anthracene was observed between 346 and 382 nm, to fluoranthene between 322 and 382 nm and to pyrene between 322 and 358 nm.     

Impairment of eye lens cell physiology and optics by broadband ultraviolet A-ultraviolet B radiation

The phototoxicity of ultraviolet A (UVA) alone and UVA plus ultraviolet B (UVB) combined on cultured porcine lenses was investigated by analyzing cellular function as measured with a fluorescence bioassay approach and optical integrity, in terms of sharpness of the lens focus as measured with a scanning laser system. The bioassay consisted of carboxyfluorescein diacetate-acetoxymethyl ester and alamarBlue fluorescent dyes. Aseptically dissected porcine lenses were maintained in modified medium 199 without phenol red supplemented with 1% penicillin-streptomycin and 4% porcine serum. At 1 week of preincubation, baseline measurements were obtained. Then the lenses were treated with single exposures of different UVA and UVB energy levels. The lenses treated with 86 J/cm2 UVA alone showed a significant (P < 0.05) decrease in cellular and optical integrity at 48 h after exposure, whereas those treated with 43 J/cm2 UVA alone did not show significant phototoxic effect. Lenses treated with 15.63 J/cm2 UVA plus 0.019 J/cm2 UVB combined showed significant adverse effects beginning from 48 h after exposure. Also, there was no recovery. These findings show that a high UVA dose alone and relatively low UVA in combination with low UVB radiant exposure can impair lens cellular and optical functions, respectively.