Pharmac. Ther. Vol. 49, pp. 181-222, 1991

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THERAPEUTIC POTENTIAL OF PLANT PHOTOSENSITIZERS J. B. HUDSON*and G. H. N. TOWERS1" *Division of Medical Microbiology and t Department of Botany, University of British Columbia, Vancouver, Canada Abstract--Many bioactive phytochemicals have been shown in recent years to be photosensitizers, i.e. their toxic activities against viruses, micro-organisms, insects or cells are dependent on or are augmented by light of certain wavelengths. These activities are often selective, and this has led to the concept of therapeutic prospects in the control of infectious diseases, pests and cancer. Reaction mechanisms commonly involve singlet oxygen and radicals, which are thought to cause photodamage to membranes or macromolecules. The main classes of plant photosensitizers reviewed here are polyyines (acetylenes, thiophenes and related compounds); furanyl compounds;/~-carbolines and other alkaloids; and complex quinones. We propose that within each group of phytochemicals there are several representatives that merit further study for therapeutic abilities in appropriate animal models.

CONTENTS 1. Introduction 1.1. Medicinal plants--phytochemicals 1.2. Photosensitizers 1.2.1. Photodynamic inactivation of viruses---eariy studies 1.2.2. Photodynamic therapy of virus infections 1.3. Distribution of photosensitizers in the plant kingdom 1.4. Test systems 1.4.1. Action spectra and lamps 1.4.2. Bioassays 2. Polyyines 2.1. Nonsulfur containing polyyines 2.1.1. Viruses 2.1.2. Bacteria, fungi and algae 2.1.3. Cytotoxicity 2.1.4. Invertebrates 2.1.5. Vertebrates and plants 2.1.6. Mechanisms of action 2.1.7. Therapeutic prospects 2.2. Thiophenes 2.2.1. Viruses 2.2.2. Bacteria and fungi 2.2.3. Cytotoxic activities 2.2.4. Invertebrates 2.2.5. Vertebrates 2.2.6. Mechanism of action 2.3. Dithiins (thiarubrines) 2.3.1. Phototoxic effects of thiarubrine A 2.3.2. Activity in mice 2.3.3. Therapeutic potential of thiarubrines 3. Furanyl Compounds 3.1. Viruses 3.2. Bacteria and fungi 3.3. Cytotoxic effects 3.4. Invertebrates 3.5. Animals 3.6. Mechanisms of action 3.7. Therapeutic prospects 4. Alkaloids 4.1. Antiviral activities 4.2. Antimicrobial activities 4.3. Cytotoxicity 4.4. Mechanism of action 4.5. Therapeutic prospects 181

182 182 183 183 183 184 185 185 185 189 190 190 190 191 191 192 192 192 193 193 194 194 197 197 197 197 198 198 199 199 200 202 202 203 203 203 204 205 205 208 208 209 209

182

J. B. HUDSONand G. H. N. ToweRs 5. Quinones 5.1. Viruses 5.2. Bacteria and fungi 5.3. Cytoxicity 5.4. Invertebrates 5.5. Animals 5.6. Mechanism of action 5.7. Therapeutic prospects 6. Miscellaneous Compounds 6.1. Sesquiterpenoids 6.2. Isoflavonoids 6.3. Cinnamates 6.4. Gilvocarcins 7. Porphyrin Derivatives 8. Cyanins 9. Concluding Remarks Acknowledgements References Notes added in proof

1. I N T R O D U C T I O N 1.1. MEDICINALPLANTS--PI-IYTOCHEMICALS A substantial fraction of the world's population continues to use natural products, especially medicinal plant extracts, in helping to control infectious diseases and to combat pests. In many cases, however, the knowledge of the correct preparation and use of these materials is disappearing, and the resources themselves are being depleted. The incorrect preparation, application, or dosage of the plant material can lead to undesirable side effects and toxicity, and possibly destruction of the active ingredients. Many of the more exotic plants are not as readily available as they once were; consequently it is imperative to characterize the active ingredients before too many useful plant species, and the knowledge of how to use them, disappear forever. In some countries (India and China, for example) pharmaceutical companies are already marketing preparations of tablets, capsules, etc. made directly from the appropriate plant extracts, for the treatment of specific diseases e.g. hepatitis. The expansion of these markets, while being a commendable goal, may accelerate the depletion of the resources. We anticipate that, on the basis of historical experience with the many pharmacologically useful compounds that have been obtained from plants, as well as experimental tests, there are probably innumerable potentially useful antiviral, antimicrobial, antiparasitic and insecticidal phytochemicals awaiting characterization. When the active ingredients have been chemically identified, they can sometimes be synthesized economically and made into pharmaceutical or agricultural preparations; or they can be chemically modified to produce more potent analogs. Alternatively the plant extracts, which may contain two or more compounds acting in synergy, might be more beneficial. In addition the plant source itself could be cultivated. There are precedents for believing that plant materials contain specific bioactive ingredients. For example, in the Ayurvedic, Siddha and Unani Schools of Medicine in India, various extracts have

209 209 211 212 212 212 212 212 213 213 213 213 213 213 215 215 216 216 222

been shown to be beneficial in the control of hepatitis, and in some cases specific antiviral and immunemodulating factors have been implicated, but not identified. Recent studies have revealed that some of these plants are rich in thiophenes, which are very potent antivirals (discussed below in Section 2.2). Another example is the recent discovery that Hypericum species, which have had traditional applications to various diseases in Ayurvedic medicine, contain certain quinones, hypericins, that are capable of controlling retrovirus infections in mice. This discovery may be relevant to AIDS. These quinones have been known for some time as photosensitizers, although they had not been tested previously for antiviral activity (for more details, see Section 5). Included in the lists of phytochemicals that have been investigated more recently are those which are only biologically active in the presence of UVA (long wavelength ultraviolet) or visible light and which are known as photosensitizers. The importance of light in the use of certain medicinal plant extracts has been appreciated, if not understood, for centuries. Certain skin diseases such as psoriasis and vitiligo have been treated by extracts of Ammi majus and related species, which contain psoralens and furanochromones as active ingredients. These compounds only exert their desired biological activities in the presence of long wavelength ultraviolet UVA (320-400 nm); providing a reason for the traditional use of sunshine in some folk remedies. The reason for the prevalence of photosensitizers is not known, and their significance or function in the plant is not entirely understood, although they have been implicated as defence mechanisms against insect pests (Heitz and Downum, 1987; Arnason et al., 1990). Nevertheless this gives us a means of manipulating these chemicals in their applications since, in the absence of UVA or visible light, many of them are not biologically active. In many of the countries that practise traditional medicine, local scientists and practitioners have expressed a strong desire to analyze their medicinal plants, in order to learn more about their active ingredients, how they work, and for what medical or agricultural purposes they could be developed.

Therapeutic potential of plant photosensitizers

183

animal viruses with membranes were more sensitive than those without membranes. In fact the enteroviruses such as poliovirus appeared to be quite resistant. However, Wallis and Melnick showed that poliovirus could be rendered equally photosensitive by propagating the virus in the presence of the dye (in this case proflavin), whereupon the dye was intercalated between nucleotides of the viral RNA. Furthermore they showed that when normal poliovirus was subjected to higher pH (up to 9) or higher temperature (up to 37°), it became susceptible to photoinac1.2. PHOTOSL~SITIZERS tivation by dyes (Wallis and Melnick, 1965). Essentially a photosensitizer is a chemical which Consequently it is believed that probably all viruses can be excited to a new electronic state by the can be inactivated by these dyes, provided that the absorption of a photon and which, in the excited viruses are permeable. This may also explain why the state, reacts with itself or a second type of molecule. spectrum of susceptibility is different for each dye. The rate of photo-oxidation by these kinds of dyes In biological systems this second molecule is frequently oxygen, which is thereby converted to a is generally governed by several parameters: light reactive oxygen species, such as ringlet-oxygen (~O~), intensity; concentrations of substrate and sensitizer; which then reacts with a nearby target molecule, such 02 concentration (not usually limiting in biological as the various membrane components or macromol- systems); pH; and of course accessibility of the target ecules of a micro-organism, in the so called photody- groups to the photoactive species (singlet oxygen or namic reaction (Towers, 1984; Spikes, 1989). The other reactive species; Spikes, 1989). As far as viruses are concerned, the most important result is usually damage to the target molecule and hence the organism. The precise chemical details and oxidizable targets are guanosine nucleotides in DNA nature of the damage involving micro-organisms has and RNA (which can lead to single and doublestrand missions); the amino acids histidine, tryptoseldom been elucidated. Traditionally photosensitizers and photosensitized phan, tyrosine, methionine and cysteine in viral proreactions have been classified as type I reactions, teins (which can lead to cross-linking); and various which do not require oxygen, but occur through the unsaturated lipid moieties. Obviously the relative mediation of radicals of various kinds; or type II importance of these reactions will be dictated by their (photodynamic) reactions, in which molecular oxy- accessibility to the reactive species generated and the gen participates, usually through singlet oxygen ~O2. relative efficiency of each oxidation reaction. The Type II reactions are more commonly encountered quantum yields of many of these reactions with the with plant photosensitizers. However it should be heterotricyclic dyes are quite high (Spikes, 1989). emphasized that these reactions are not necessarily mutually exclusive. In fact recent studies have indi- 1.2.2. Photodynamic Therapy of Virus Infections cated that furanocoumarins, for example, traditionally viewed as type I photosensitizers, can also elicit The results on virus photoinactivation led to studtype II reactions in the presence of oxygen (see ies in vivo on animal models, particularly herpes Section 3.6 for details). simplex and vaccinia infections of rabbit eyes. Several In either type of reaction, the resulting radicals or of the dyes were shown to be successful in controlling singlet oxygen can then cause damage to macromol- such infections; consequently clinical trials were esecules in close proximity. It should also be noted that tablished in various centers with a view to therapy for some phytochemicals (e.g. carotenoids) can act as cutaneous lesions (corneal, oral and genital) of herpes quenchers of singlet oxygen or radicals, and accord- simplex types 1 and 2, and papilloma warts. Many ingly have the capacity to protect organisms from such studies reported successful therapy (by intralephotosensitized reactions. Similarly the presence of sion inoculation of dye plus exposure to florescent high levels of catalases can be protective against lamps); but some reported no significant benefits potential photodamage from peroxides (Daub, 1987; (Bockstahler et al., 1979, 1984). Tuveson et al., 1989). In the meantime however, Rapp and colleagues had demonstrated that various procedures that inac1.2.1. Photodynamic Inactivation of Viruses--Early tivated herpes viruses, including photo-dye treatment, could lead to subsequent transformation of Studies cells by the noninfectious viruses. In other words, if Several decades ago investigators found that a the replication capacity of a herpes virus could be number of heterotricyclic dyes, notably acridine abrogated, its oncogenic potential could then be orange, proflavine, neutral red, methylene blue, and realized (Rapp and Li, 1982). toluidine blue, could inactivate viruses in the presence These results forced investigators to question the of visible light. These studies have been reviewed by ethics of photodynamic therapy. In addition other Wallis and Melnick (1965), Bockstahler et al. (1979, potential disadvantages were also raised, such as the 1984) and Spikes (1989). The initial work was done possible induction of latent endogenous retroviruses with bacteriophages, and this was followed by studies (or other viruses) by the inactivated virus or by the with various animal viruses and plant viruses. treatment protocol itself; mutagenesis of the virus or At first it appeared that T-even phages were signifi- adjacent cells in the lesion area; and increased transcantly more sensitive than T-odd phages, and that formation susceptibility of these cells (Bockstahler This article attempts to review what is known about the biological effects of specific plant photosensitizers in regard to possible therapeutic uses against infectious diseases and possible insecticidal uses. The emphasis is on antimicrobial and antiviral applications. Anticancer aspects are outside the scope of this review however, and little attention is given to work with crude extracts in which the active ingredients have not yet been chemically characterized.

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184

J.B. HUDSONand G. H. N. TOWERS

et al., 1979, 1984). As a result of these considerations, interest in photodynamic therapy of virus infections waned. A renewed interest has emerged recently however, in view of the applications of other classes of photosensitizers, such as porphyrins and cyanins, to cancer chemotherapy. Theoretically these reagents are less likely to be mutagenic or oncogenic (see Section 7). In addition some of the newly discovered plant photosensitizers discussed in this review are also potentially useful in controlling virus infections.

pounds which are characteristic of the largest plant family, the Asteraceae, but which are also found in several related families including the Apiaceae, Campanulaceae, Pittosporaceae, Olacaceae, Euphorbiaceae, Valerianaceae, Annonaceae, Opiliceae, Sapindaceae, Araliaceae and certain basidiomycetous fungi (Bohlmann et al., 1973; Hansen and Boll, 1986; Bohlmann, 1988; Christensen and Lam, 1990). The highly conjugated compounds of the Asteraceae and the fungi are often phototoxic; but so far polyyines with phototoxicities have not been recorded from the other families, although extremely 1.3. DISTRIBUTIONOFPHOTOSENSITIZERSIN THEPLANT toxic acetylenes have been isolated from the Apiaceae, e.g. cicutoxin from Cicuta sp. A number of KINGDOM fungal polyyines are also phototoxic (Arnason et al., Naturally occurring phototoxins (i.e. photosensi- 1990). Polyyines are derived by desaturation and chain tizers with toxic effects on some organisms) include polyketides (polyyines, thiophenes, quinones, and shortening of fatty acids. Sulfur derivatives include chromenes); cinnamate derivatives (coumarins and thiophenes, a number of which have been well furanocoumarins); alkaloids based on tryptamine studied. Alpha-terthienyl (alpha-T, 2,2': 5',2" (harmane), on phenylalanine and tyrosine (berberine, terthiophene) and many bithiophenes, for example sanguinarine) or anthranilic acid (furanoquinolines); 5-(4-hydroxy- l-butenyl)-2,2'-bithiophene (BBTOAc), and porphyrins (precursors and degradation products and 5-(3-buten-l-ynyl)-2,2'-bithiophene (BBT), are of chlorophylls). Undoubtedly, many others remain widely distributed in the subtribe Pectidinae of the to be discovered (see Towers, 1984; Knox and Dodge, Asteraceae (Downum et al., 1985). They can be found in flowers, leaves, stems and roots. In species of the 1985a). It is of considerable interest that biogenetically genus Porophyllum, they are located in prominent unrelated photosensitizers, with the same mode of marginal glands in the leaves in the same way that action, can co-occur in a given plant species. For hypericin occurs in Hypericum. A second type of example, skimmianine (a furanoquinoline) and xan- sulfur derivative of the polyyines are the unusual thotoxin (a furanocoumarin) occur in the leaves of thiarubrines which contain two sulfur atoms in a Skimmia japonica (Rutaceae) (Towers et al., 1981); ring, and are also known to be phototoxic (Towers while visnagin and khellin (furanochromones), and et al., 1985; Balza and Towers, 1990). Widespread phototoxic alkaloids include the beta psoralen, occur together in Psoralea coryifoloia (Fabaceae). A third example is the co-occurrence of carbolines, which are common in the Rutaceae and polyyines and chromenes in some asteraceous species, Simarubaceae, and can also be found in Cyperaceae, Fabaceae, Polygonaceae, Rubiaceae, Rutaceae, e.g. Encelia. The oldest known plant photosensitizers are the Sapindaceae, Passifloraceae, Zygophyllaceae, and red anthraquinone derivatives, hypericin and pseudo- Solanaceae. These photogenotoxic compounds (Mchypericin, which are found in the glands of leaves, Kenna and Towers, 1981) are also found in marine flowers and stems of St John's wort, Hypericum organisms and in animal materials such as urine and perforaturn (Guttiferae) and many other species of the charred meats. Furanoquinolines, such as dictamnine same genus (Giese, 1980). However, medicinal plants, and skimmianine, are found in species of the such as Ammi visnaga, in which the active phyto- Rutaceae including the skin sensitizing gas plant, chemicals are furanocoumarins (xanthotoxin) and Dictamnus alba (Towers et al., 1981). Phototoxic furanochromones (khellin), have also been used for isoquinoline alkaloids such as berberine (Berberis thousands of years in India and the Middle East for spp.) and sanguinarine (found in the red sap of the treatment of vitiligo and other skin diseases. Sanguinaria canadensis) occur in at least nine families Xanthotoxin (8-methoxypsoralen) is still used in (Anonaceae, Papaveraceae, Berberidaceae, JuglanPUVA therapy for the treatment of psoriasis and daceae, Magnoliaceae, Menispermaceae, Ranuncuin emerging phototoxin technologies for the treat- laceae, Rubiaceae and Rutaceae) (Philogene et al., ment of T-cell lymphoma (Edelson, 1988). Fura- 1984). Finally, there are a number of photosensitizers, e.g. nocoumarins are found in oil ducts and cuticles of species of the Apiaceae (carrot family), Rutaceae quinolines, chromenes and lachnanocarpones, that (citrus family), Fabaceae (bean family), Moraceae have not yet been examined for insecticidal or antimi(fig family), Solanaceae (tobacco family), Pitto- crobial activities (Towers, 1986). It is evident from the above discussion that the sporaceae, Thymeleaceae, and Orchidaceae (Towers, 1984). Common compounds are bergapten phototoxins of the plant kingdom are biosyntheti(5-methoxypsoralen), xanthotoxin, psoralen, and cally unrelated for the most part. Apparently, the advantages of photochemical defenses are sufficient angelicin. The phototoxicity of the polyyines (polyacetylenes) for phototoxicity to have arisen independently in such as phenylheptatriyne, which is found in the evolution several times. These advantages are probleaves of Bidens spp. and Coreopsis spp., has been ably due to the use of light in the environment to known for less than twenty years (Carom et al., 1975; produce exceptionally toxic photochemical reactions Gommers, 1972). Together with the related sulfur that are not normally possible in the ground state of derivatives, they comprise over 700 known corn- these chemicals.

185

Therapeutic potential of plant photosensitizers

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FIG. 1. Subdivisions of the UV spectrum. Reprinted from Hudson (1989) with permission from the copyright holder, CRC Press Inc., Boca Raton, FL. 1.4. TESTSYSTEMS 1.4.1. Action Spectra and Lamps The extent of photoinduced damage to organisms is dependent on the wavelength of the incident light, since photosensitizers are generally excited over a fairly narrow band of radiation. For many of the photosensitizers discussed here, the effective radiation peaks in the UVA region (320--400 nm wavelength), while others require longer wavelengths of visible light from blue to red (~400-800 nm). The UV spectrum is normally subdivided into three distinct parts---UVA, UVB, and UVC, which have characteristic biological activities (Fig. 1). The spectral dependency of these biological attributes are described by the action spectrum of a photosensitizer, as exemplified by Fig. 2, which

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Another interesting feature is the observation that aT and a very small number of related thiophenes show cytotoxicity (to the test cell line) in the dark, although the shapes of the cell-killing curves suggest that the mechanism of this effect is different from the

therapeutic aspect, especially since aT itself is not particularly toxic to animals kept in normal room light. Furthermore we believe that aT is much less cytotoxic to cultured fibroblasts than to the cell line used for the toxicity tests.

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Therapeutic potential of plant photosensitizers

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197

sensitive larvae better, and to be cleared slower, than in resistant larvae (Arnason et al., 1987). The high potency of s T to mosquito larvae, with an LCs0 of 19 ppb (approximately 50 riM), has led to field trials, in which various formulations of the compound have been tested for their ability to control Aedes larvae (Arnason et al., 1988). In addition to some insects, nematodes and Daphnia, but not snails, were killed by ctT in light (Gommers et al., 1980). The nature of the photodamage to insects is not clear, although it is generally assumed that cell membranes in various tissues are involved. In addition inhibition of specific enzyme activities have been described. Alpha-terthienyl was also phototoxic to cercariae (Graham et al., 1980), in contrast to PHT which only possessed dark activity. This activity of ~tT was extended to field trials in ponds, where the presence of sunlight augmented the effect of the compound. 2.2.5. Vertebrates

phototoxic effects (Fig. 10). In contrast most of the thiophenes, including those with potent antiviral or antibiotic activities, show little or no dark cytotoxicity. This leads us to expect that many of these compounds should be safer to use in animals than ~tT.

Fish seem to be able to tolerate larvicidal doses of ~tT, and mice and rats survive substantial doses, depending on the route of inoculation. However sT produces photodermatitis in animal skin as well as allergic contact dermatitis (Rampone et al., 1986). Since many of the other thiophenes are considerably less cytotoxic, some of them may be safer to use in vivo than sT; thus animal tests need to be done with the potentially therapeutic derivatives.

2.2.4. Invertebrates

2.2.6. Mechanism o f Action

The most detailed studies have been done by Arnason and collaborators (Downum et al., 1984; Arnason et al., 1986, 1987, 1988; Champagne et al., 1986), who compared many thiophenes (including some of those tested for antiviral activity) for phototoxicity to mosquito larvae (Aedes sp.). There was fairly good correlation with antiviral potency (Table 5). Thus the monomethyl, aldehyde, alcohol, cyano, and silyl derivatives of ~tT had good larvicidal activity, whereas the presence of bulky groups, two side chains, phenyl groups, or pyridine groups, tended to decrease activity. There appeared to be a correlation of phototoxicity with the octanol:water partition coefficient of a compound, possibly an indication of the relative ease of insertion of the compound into membranes (Marles et al., 1990). This overall similarity between larvicidal, antiviral and cytotoxic activities could be a reflection of a common mechanism involving eukaryotic membranes. There are other factors however. Thus adult mosquitos were much more resistant to the thiophenes, and other insects had markedly different responses. For example many fly larvae were resistant, whereas midges were susceptible. This selectivity is probably due to different degrees of penetration or different types of handling, metabolism, or clearance of the compounds. It is well known that phytochemicals suffer quite different fates in different species of insect (Arnason et al., 1983, 1987, 1990). This concept was supported by studies with 3Hlabelled sT, which was shown to penetrate the gut of

In contrast to PHT, the mechanism of action of~tT is thought to be a relatively simple type II mechanism which requires singlet oxygen exclusively, as indicated by various experiments involving the use of inhibitors and aerobic/anaerobic atmospheres (Evans et al., 1986; Scalano et al., 1989). In liposome models it appears that s T inserts into membrane regions rich in unsaturated fatty acids, and in the presence of UVA generates ~O2 and hence membrane damage. This may also explain phototoxicity in insects and vertebrates in which vital tissues that have accumulated ~tT are exposed to UVA (McRae et al., 1985; Towers and Hudson, 1987). Alternative targets such as proteins or nucleic acids have been suggested (Hudson, 1989), and recently it has been shown that ctT can produce photodamage in DNA molecules in vitro i.e. single strand breaks, which can be repaired in cells (Wang et al., 1991). 2 . 3 . DITHIINS (THIARUBRINES)

A naturally occurring dithiin, or dithiacyclohexadiene, thiarubrine A (see Figs 4 and 11), was first described many years ago in several species of Asteraceae (Bohlmann et al., 1973). Our studies on its potent antifungal activity (Towers et al., 1985; Constabel and Towers, 1989) were further stimulated by the observations that chimpanzees in Tanzania ritualistically and daily consume leaves of Aspilia plants, which are rich in thiarubrine A (Wrangham and Nishida, 1983; Rodriguez et al., 1985).

198

J.B. HUDSONand G. H. N. TOWERS

CHs--C==C-~

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FIG. 11. Photolysis of thiarubrine A to thiophene A. Reprinted from Constabel and Towers (1989) with permission of the copyright holder, Georg Thieme Verlag, Stuttgart. Thiarubrines can be isolated directly from appropriate plants, or in higher yields from Crown gall tumor lines or 'hairy root' cultures of Rudbeckia hirta (Cosio et al., 1986; Constabel and Towers, 1989). Additional thiarubrines were characterized recently (Baiza and Towers, 1990). 2.3.1. Phototoxic Effects o f Thiarubrine A Thiarubrine A absorbs well in the UVA and in the visible region; consequently it can be excited by both UVA lamps and incandescent lamps. The photochemistry is somewhat complicated however, because the initial photodegradation is accompanied by the elimination of one sulfur atom (see Fig. 11), with the result that thiophene A is produced, and this product itself is also a photosensitizer. This scheme, as suggested recently by Constabel and Towers (1989), could explain some of the diverse activities of thiarubrine A against different organisms. Thus the compound is a very potent antifungal agent (Towers et al., 1985) but this property does not require light, although UVA enhances the activity. Likewise the antibacterial activity is manifest in the dark, but is enhanced in UVA. In contrast thiophene A has antifungal and antibiotic activity only in the presence of UVA, and in this respect resembles other biologically active thiophenes (described above). Therefore the activity of thiarubrine A in UVA is thought to arise from the photoconversion to thiophene A, which then becomes photoactive itself. This is illustrated in Fig. 12 (Cosio et al., 1986; Constabel and Towers, 1989).

Thlarubrlne dark

Antlfungal

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FIG. 12. Biological activities of thiarubrine A in relation to light. Reprinted from Constabel and Towers (1989) with permission of the copyright holder, Georg Thieme Verlag, Stuttgart.

Thiarubrine A was also found to be active against membrane-containing viruses, but only in UVA; there was no comparable dark activity (Hudson et al., 1986a; Table 6). Viruses without membranes were also affected at much higher concentrations. The mechanism of the antiviral activity appeared to be similar to that caused by thiophenes, although it was surprising to find that the isolated thiophene A had very weak antiviral activity in UVA (Hudson et al., 1987a). However our studies on thiophenes had indicated that potent antiviral activity required two or three conjugated thiophene rings (described above). Therefore the thiophene A may have had significant though relatively weak antiviral activity. More recently we have shown that thiarubrine A has potent antiviral activity against HIV-I, but only in the presence of UVA (Table 6; Hudson, Baiza and Towers, unpublished results). Thiarubrine A also showed cytotoxicity equally in the presence or absence of UVA. This is in marked contrast to the thiophenes, which generally showed low cytotoxic effects in the dark (although ~tterthienyl is somewhat exceptional, see Fig. 10). Nevertheless the shapes of the concentration-dependent killing curves were quite different for the dark and UVA-dependent effects of thiarubrine A. This suggests that different mechanisms may be in operation, and this would be in accord with the hypothesis of Constabel and Towers (1989), who suggested that the dark activities of thiarubrine A could be attributed to an unknown mechanism, whereas in the presence of UVA a type ;I reaction involving singlet oxygen (or possibly 'singlet sulfur') could operate similar to thiophenes. This concept was supported by the observation that thiarubrine A caused leakage of glucose or K + from liposomes in UVA but not in the dark (MacRae, Abramowski and Towers, unpublished data). Therefore it is important to emphasize that these compounds, and also other polyyines that demonstrate dark activities, may work by different mechanisms depending on the presence or absence of light. Accordingly their therapeutic activities in vivo may likewise differ. More recently, other thiarubrines, with similar structures to thiarubrine A, have been shown to possess antifungal and antibiotic activities that are even more potent (Towers, unpublished data). 2.3.2. Activity in Mice Preliminary experiments showed that thiarubrine A, administered intraperitoneally or orally to mice infected 24 hr previously with C. albicans, reduced the titers of the organism in several visceral tissues. Larger doses of the compound however rendered the mice more susceptible to the yeast (Shim, Cheung, Hudson and Towers, unpublished results). More detailed analysis will be required to determine the optimal dose of thiarubrine A and the best mode and timing of the application. Nevertheless these results are promising, especially since the mice were only exposed to normal room lights; consequently the effects observed probably reflected the dark effect of the compound against the organism.

199

Therapeutic potential of plant photosensitizers

Compound

TABLE7. Mechanism of Antiviral Action of Polyyines Treated MCMV Phototoxicity vs viruses Entry into Early protein Viral DNA cell nucleus s y n t h e s i s replication + membranes -membranes

Late protein synthesis

Normal Normal Normal Normal

+ -

Phenylhepatriyne ++ Thiarubrine-A +++ 5Thiophene-A + -Terthienyl ++++ - to + Synthetic thiophenes - to 5 +* * - to + + + + indicates increasing phototoxicity.

2.3.3. Therapeutic Potential o f Thiarubrines Thiarubrine A and the other natural dithiins have very potent antifungal activity which does not require light, although this activity is also displayed in UVA and visible light. They are very active against C. albicans and other pathogenic fungi. In the in vitro tests these compounds were more effective than amphotericin B. In contrast the antibiotic and antiviral activities of thiarubrine A, though significant, were not as impressive as the most active thiophenes. Thiarubrine A also has significant cytotoxicity in the dark, and this seems to be reflected by toxicity in the mouse, which was found to be more pronounced than with g-terthienyl. More work needs to be done on structurally modified thiarubrines, and hopefully synthetic dithiins may become available in the near future. Table 7 summarizes the relative antiviral potencies and mechanisms of action of polyyines, thiophenes and thiarubrines.

3. F U R A N Y L COMPOUNDS Furocoumarins are common constituents of many members of the Rutaceae and Apiaceae (Umbelliferae), although they have also been found in several other families of plants, and in fact are important components of a number of known medicinal plants. They can occur in concentrations of more than 1% by dry weight in various tissues of the plant. Biosynthetically they are derived from phenylalanine (Towers, 1984). Many types of coumarins have been synthesized in recent years (e.g see Averbeck, 1989), and compared with psoralens for biological activities. Novel compounds are still occasionally isolated from plants however. For example the furanoisocoumatin, coriandrin, of polyketide origin, was isolated recently from coriander (cilantro, chinese parsley) by Ashwood-Smith and colleagues (Ceska et al., 1988; Ashwood-Smith et al., 1989). Its biological activities are under study. The multiple biological activities of furanocoumarins (in UVA) have been recognized for some time. Figure 13 shows some structural formulae. Thus they are commonly phototoxic to some insects, cells, bacteria, fungi and viruses; the underlying cause usually being attributed to their capacity to intercalate in DNA followed by light-activated production of monoadducts or biadducts with pyrimidine bases, especially thymidine, although other mechanisms can

+ -

+ -

also operate (Pathak et al., 1977; Song and Tapley, 1979; Ashwood-Smith et al., 1982; Cimino et al., 1985; Averbeck, 1989). Some of them are used therapeutically in the treatment of skin disorders such as psoriasis and vitiligo (the so-called PUVA therapy; Pathak et al., 1977; Gupta and Anderson, 1987), and more recently in the treatment of cutaneous lymphomas, by photophoresis (Edelson et al., 1987). Other supposedly beneficial applications include suntan creams and lotions, in spite of the suspected carcinogenetic risks (Ashwood-Smith et al., 1980; Grekin and Epstein, 1981). In addition, plants containing furocoumarins are known to be responsible for a significant number of photodermatoses in livestock, which occur when the animals consume foliage containing such compounds and are subsequently exposed to sunlight (Ivie, 1982). The furanochromones visnagin and khellin often occur in association with the coumarins, e.g. in species of Ammi (Apiaceae). Extracts of these plants and their purified compounds have long standing medicinal applications (Abdel-Fattah et al., 1982), and they are also phototoxic to cells and microorganisms. The similarity of their structures to furocoumatins is illustrated in Fig. 13. The furanoquinolines will be discussed in the section on alkaloids, although

OCH 3

OCH 3

8 - mop (M)

vlsnagln ( V )

angellcin(A ]

khellln i x )

dlctarnnlne ( n )

FIG. 13. Structural formulae of representative furyl photosensitizers.

200

J. B. HuDsor~ and G. H. N. TOWERS TAaLE 8. LD~ Values for Antiviral Activities of Furyl Compounds Compounds ( I 0/t g/ml) MCMV SV T4 M 13 8-methoxypsoralen Angelicin Visnagin Khellin Dictamnine

1,200 6,600 3,150 10,500 1,800

7,500 > 20,000 10,200 > 20,000 6,900

700 2,150 1,550 15,000 1,850

2,000 2,000 8,500 > 20,000 8,500

LD99 = dose of UVA radiation required (exposure in seconds x 5 W/m 2 to decrease infectivity by 2 log~0 PFU). Data from Hudson et al., (1985, 1988a and unpublished). their chemical and biological similarity to furocoumatins must be borne in mind (see Section 4). 3.1 .VIRUSES

Early work indicated that DNA-containing animal viruses and bacteriophages were sensitive to the combined effects of psoralen + UVA, whereas RNAviruses at first appeared to be resistant. However subsequent studies have clearly shown that RNAviruses are also inactivated by various furocoumarins + UVA, but that higher doses of radiation and/or higher concentrations of chemicals are required (Hearst and Thiry, 1977; Hanson et aL, 1978; Redfield e t a / . , 1981). The results of these antiviral studies can be summatized as follows: (i) Both D N A and RNA viruses 1.0

J x\

"'--,

\

." ..........

10 a

~ 10 -8 M e.. Z

\

\

\\

\

•

\ "--..

v ",\, "--?

_._.?

"%'%., n 10 -4

10 -e*

I 5

/

10

MINUTE

I

16

i

SO

I

85

I

~0

B

FIG. 14. Survival curves for murine cytomegalovirus in the presence of various furyl compounds (each at 10 ,ug/ml) plus variable UVA exposure. M, 8-methoxypsoralen; D, dictamnine; V, visnagin; A, angelicin; K, khellin. Reprinted from Hudson et al. (1985) with permission of the copyright holder, Pergamon Press, Oxford.

are susceptible to photoinactivation by psoralens; the D N A viruses generally being more sensitive; (ii) a variety of substitutions in the psoralens are compatible with antiviral activity, and in fact many derivatives, such as trimethyl psoralen (TMP), 4' aminomethyl TMP, 4'hydroxymethyl TMP, are more potent antivirals than psoralen itself; (iii) virusinfected cells are also susceptible to these psoralens. These latter observations led to the suggestion that psoralen-inactivated viruses or infected cells might constitute useful vaccines (Redfield et al., 1981). The results were generally ascribed to the formation of D N A - D N A or R N A - R N A cross-links in the viral genomes. In more recent comparative studies, in which several viruses were treated under identical conditions with different furyl compounds (Hudson et al., 1985, 1987a; Fig. 14), 8-MOP (8-methoxypsoralen M) was found to be considerably more phototoxic than angelicin (A) to the D N A viruses MCMV and phage T4. This was determined by kinetic survival curves, from which LD99 values were constructed for Table 8. Furthermore, while 8-MOP was moderately active against the R N A virus Sindbis, the angular compound angelicin had very little activity. But in contrast, when single-stranded D N A phage M 13 was the target, angelicin and 8-MOP were equally potent (Table 8). It is difficult to draw definitive conclusions from these kinds of study concerning relative potencies; but it is clear that angelicin can effectively inactivate viruses without forming 'conventional' cross-links characteristic of linear furanocoumarins (Song and Tapley, 1979; Cimino et al., 1985; Averbeck, 1989). This could be due to monoadducts that render the viral D N A incapable of replicating, or to the presence of D N A - D N A or DNA-protein links produced within the virion, which somehow prevent the initiation of the replication cycle (Kittler et al., 1980; see below). Dictamnine (D) is included in Table 8 for comparison; it was also quite active (see Section 4.1.). In additional studies it was shown that when MCMV had been inactivated by 8-MOP or angelicin, the viral genome penetrated into the cell nucleus, as usual, but no viral RNA, D N A or protein synthesis could be detected, as shown by nucleic acid hybridization techniques and polyacrylamide gel electrophoresis (Hudson et al., 1988a, and unpublished results). This is summarized in Table 9. An interesting application of psoralens, which represents an extension of the work on virus-infected cells (referred to above, Redfield et al., 1981), was reported by Watson et al. (1990~, who showed that at

Therapeutic potential of plant photosensitizers

201

TABLE9. Mechanism of Antiviral Activity of Furyl Compounds Viral replication cycle Compound ( + UVA) 8-Methoxypsoralen Angelicin Visnagin Khellin Isopimpinellin All compounds - UVA

Cross-links in DNA

Entry into cells

Early proteins

DNA

proteins

+

Normal Normal Normal Normal Normal Normal

__. + +

__. + +

_ + +

+ +

TABLE 10. Effect of lsopimpinellin on Viruses and Cells % infectivity (viability) remaining Organism

Isopimpinellin

8-MOP

MCMV 64*