Natural products as antiparasitic drugs
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- Kayser, O., Kiderlen, A.F. & Croft, S.L. Parasitol Res (2003) 90: S55. doi:10.1007/s00436-002-0768-3
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Natural products are not only the basis for traditional or ethnic medicine. Only recently, they have provided highly successful new drugs such as Artemisinin. Furthermore, screening natural products found in all sorts of environments such as the deep sea, rain forests and hot springs, and produced by all sorts of organisms ranging from bacteria, fungi and plants to protozoa, sponges and invertebrates, is a highly competitive field where all of the major pharmaceutical companies are encountered. Already, many new natural product groups have revealed antiparasitic properties of surprising efficacy and selectivity, as will be shown in this review for plant-derived alkaloids, terpenes and phenolics. Many novel lead structures, however, have severe chemico-physical drawbacks such as poor solubility. Here, innovative drug formulations and carrier systems might help, as discussed by the authors in another article of this series.
Higher plants and microbial organisms are used as natural sources for the discovery of new drug leads. Artemisinin, quinine and licochalcone A are examples for plant-derived products, and amphothericin B and ivermectin are important antiparasitics isolated from microorganisms like Streptomyces. Many other natural products of diverse molecular structure have revealed antiparasitic potency in the laboratory and represent interesting lead structures for the development of new and urgently needed antiparasitics.
The search for innovative antiparasitics must consider, among other things, the following problems: at present, there are only a few drugs on the market for the treatment of many different parasitic diseases. Target specificity, making better use of the biochemical and biological characteristics of individual parasite species, should enhance drug efficacy. Further, most drugs in clinical use – also natural products like quinine and emetine – have variable efficacy, toxic side effects and require long courses of administration (reviewed by Phillipson and Wright 1991; Rayo Camacho Corona et al. 2000; Kayser et al. 2002,). Screening natural products provides the chance to discover new molecules of unique structure with high activity and selectivity which can be further optimized by semi- or fully synthetic procedures (Holzgrabe and Bechthold 1999).
The molecular diversity and efficacy of antiparasitic plants, extracts and herbal preparations have been discussed intensively in recent reviews (Phillipson 1991). Here, only those compounds or chemical groups are considered which have already shown a potential as new drug leads or may have an impact on future drug development. All compounds mentioned in this review have been extensively investigated, covering at least first in vivo and toxicity studies.
Alkaloids are one of the most fascinating natural products already providing many drugs for human use. Despite the fact that alkaloids may be seriously toxic for the host, they are of major interest in finding new antiparasitics (Phillipson et al. 1993). Here, only the most important sub-classes and some specific structures will be covered, as alkaloids have already been intensively discussed in the recent literature.
Many antiprotozoal isoquinolines have been isolated from the families Annonaceae, Berberidaceae, Menispaermaceae and Hernandiaceae (Akendengue et al. 1999). In particular berberine (structure 8, Fig. 1), as a representative of the benzylisoquinoline alkaloids, is well known for its antiparasitic activity (Vennerstrom et al. 1990). Berberine is active at an EC50 of 10 µg ml–1 against intracellular Leishmania amastigotes in murine peritoneal macrophages. Vennerstrom and colleagues (Vennerstrom et al. 1990) also tested berberine and several of its derivatives for antileishmanial activity against L. donovani and Leishmania panamensis in vivo in golden hamsters. Tetrahydroberberine (structure 9, Fig. 1) is less toxic and more potent than berberine against L. donovani but not as potent as meglumine antimonate (Glucantime) as a therapy standard. Against the New World pathogen L. panamensis, only berberine, the natural product, showed significant in vivo activity (greater than 50% suppression of lesion size). Although berberine has an ethnomedicinal history in India for cutaneous leishmaniasis, topical application was ineffective in in vivo tests (El-On et al. 1988).
Eight further berberine-related analogs were tested against Leishmania and Trypanosoma parasites (Vennerstrom et al. 1990). In a model for cutaneous leishmaniasis all compounds were as effective as the standard drug Glucantime. However, when tested against visceral leishmaniasis the analogs revealed only limited efficacy compared to berberine. For example, cyanodihydroberberine given s.c. over 4 days at 208 mg kg–1 day–1 caused a 54% reduction of the L. donovani parasite load in the liver (Vennerstrom et al. 1990).
Another interesting group of antiprotozoal alkaloids are the bisbenzylisoquinolines like (+)-gyrocarpine (structure 10, Fig. 1) and (–)-cycleanine (structure 11, Fig. 1). In vitro, most bisbenzylisoquinolines exhibit activities well below 1 µg, close to the EC50 value of chloroquine (EC50≈0.2 µM). Angerhoffer et al. (1999) discussed some structure activity relationships in a series of 53 structurally distinct bisisoquinolines. Considering antiplasmodial activity, quaternization of the nitrogen, N-2′, and N-oxide formation, decrease in lipophilicity, and a change of configuration of the chiral center are parameters of prime importance for antiplasmodial activity.
Benzyl- and naphthylisoquinoline alkaloids
Naphthylisoquinoline alkaloids, isolated from tropical lianas, have only recently been discovered as a group of alkaloids with high antiparasitic potential. When tested in vitro and in vivo against Plasmodium falciparum they show remarkable activity. This was also the case against Leishmania and Trypanosoma species (Francois et al. 1997a, 1997b). Extracts from Triphophyllum peltatum (Dioncophyllaceae) led to the isolation of dioncophylline B (structure 12, Fig. 1) and dioncophylline C (structure 13, Fig. 1), both exhibiting high antiplasmodial activity (Francois et al. 1997b). Dioncophylline C cured malaria-infected mice completely after a 4-day oral treatment with 50 mg kg–1 day–1 without noticeable toxic effects. Recently, a novel dimeric naphthylisoquinoline alkaloid heterodimer with antiplasmodial activity, korundamine A (structure 14, Fig. 1), was isolated from Ancistrocladus korupensis (Ancistrocladaceae), which is biogenetically related to the Dioncophyllaceae (Hallock et al. 1998).
A number of indolquinoline alkaloids have been isolated from Cryptolepsissangunolenta (Periplocaceae) and tested for antiplasmodial activity. Of this series, cryptolepin (structure 15, Fig. 1) showed variable effects ranging from an only moderate (34–46%) (Kirby et al. 1995) to a high (80%) reduction of parasitemia (Grellier et al. 1996). The reasons for these differences in efficacy are unclear and need to be resolved. Possibly, an innate sensitivity of Plasmodium vinckei petteri, which was used by the latter authors provides an explanation.
Classically, emetine is used for the treatment of Entamoeba infections but it also shows antiplasmodial activity. Several other indole alkaloids with antiplasmodial activity have been isolated from Strychnos including usambarensine (structure 16, Fig. 1) [IC50(Entamoeba histolytica)=0.023 µM] and usambarine (structure 17, Fig. 1) [IC50(E. histolytica)=1.13 µM] which did not show any significant cytotoxicity for KB cells (Wright et al. 1994). When tested against multidrug resistant P. falciparum K1, 3,4-dihydrousamberine (structure 18, Fig. 1) was five times more active (IC50=0.023 µM) than chloroquine (IC50=0.156 µM).
Among the group of terpenes, even simple monoterpenes (e.g. espintanol, piquerol A) and iridoids (e.g. arbortristosides A, C, amarogentin) show some activity against protozoan parasites (Hocquemiller et al. 1991;Tandon et al. 1991; Kayser et al. 2002), but will not be discussed here because of their limited value for drug development. The group of quassinoids like cedronin or different chaparrinones will also not be discussed in detail because of their high toxicity which should inhibit further development.
More important from a clinical viewpoint are the unwanted side-effects, such as neurotoxicity in the case of artemisinin derivatives. The application of the established principles of modern drug design should allow the creation of the first truly rationally designed endoperoxides.
Other sesquiterpene peroxides are yingzhaosu A (structure 20, Fig. 2) and yingzhaosu C (structure 21, Fig. 2) which show activity in Plasmodium berghei-infected mice comparable to that of mefloquine and quinine but tenfold less than that of artemisinin (Posner 1998). Further synthetic compounds are in preclinical trials, such as Fenozan-50F (structure 22, Fig. 2) (Pharma Mar) and the recently discontinued arteflene (structure 23, Fig. 2) (Roche Holding). In the development of new endoperoxide-based antimalarials, a series of 3-aryl-1,2,4-trioxan (structures 24–26, Fig. 2) has been tested against CQ-sensitive P. falciparum NF54 (Posner et al. 1998). Detected in vitro IC50 values ranged between 42 and 76 nM and in vivo after subcutaneous injection between 2.8 and 6.8 mg–1 kg–1 (Posner et al. 1998).
Diterpenes are among the most widely distributed terpenes in the plant kingdom and are well known for their biological activity. However, most of them combine both high antiparasitic activity as well as high toxicity for mammalian cells. Nevertheless, axisonitrile (structure 27, Fig. 2), a sequiterpene derivative isolated from the sponge Acanthella klethra, shows potent antiplasmodial activity with no detectable cytotoxicity; it might therefore be attractive for further development (Angerhofer et al. 1992).
Over 100 terpenes have been isolated from marine organisms. Due to their unique structural features, they are of considerable interest in antiparasitic drug research. In contrast to plant metabolites, these marine products contain isonitrile, isothiocyanate, and thiocyanate substituents. To date, novel compounds with EC50 values below 1 µg ml–1 with high selective indices (SI>50) have been identified as reviewed by König and Wright (1996). The potent and selective biological activities of these compounds represent an exciting advance, especially in the search of novel antiplasmodial agents. In vivo studies are now needed to validate this potential.
Recently, following ethnomedicinal analysis, cryptotanshinone (structure 28, Fig. 2), a quinoid diterpene with a nor-abietane skeleton, and three derivatives, 1β-hydroxycryptotanshinone (structure 29, Fig. 2), 1-oxocryptotanshinone (structure 30, Fig. 2), and 1-oxomiltirone (structure 31, Fig. 2) were isolated from the roots of the Iranian plant Perovskia abrotanoides (Sairafianpour et al. 2001). These compounds exhibited in vitro leishmanicidal activity (IC50 values 18–47 mM). The isolated tanshinones also inhibited the growth of cultured malaria parasites (3D7 strain of P. falciparum), the drug-sensitive KB-3-1 human carcinoma cell line, multi drug-resistant KB V1 cell line, and human lymphocytes activated with phytohemagglutinin A (IC50 values 5–45 mM) (Sairafianpour et al. 2001).
Limonoids are biosynthetically related to quassinoids and mainly found in the species of Meliacea (MacKinnon et al. 1997). The neem tree, Azadirachata indica (Meliaceae), is widely used in Asian ethnomedicine for the treatment of malaria. Nimbolide (structure 32, Fig. 2) was identified as an antiplasmodial compound (IC50=0.95 ng ml–1, P. falciparum K1). The derivatives nimbinin and geduine (structure 33, Fig. 2) and its dihydro-derivative were also found to be active in vitro against Plasmodium parasites with EC50 values from 0.72 to 1.74 µg ml–1 (MacKinnon et al. 1997).
Chalcones and aurones
Licochalcone A (structure 36, Fig. 3), from Glycyrrhiza inflata, is the prominent representative of this group (Chen et al. 1993, 1994a). It has been identified as a potent inhibitor of different mitochondrial functions that are essential for the vitality of Leishmania species. In vivo, licochalcone A reduced the parasite load in the liver by >96% when given i.p. at 20 mg kg–1 day–1 for 6 days (Zhai et al. 1995). Apart from its antileishmanial activity, licochalcone A has been successfully tested against P. falciparum in vitro and in vivo (Chen et al. 1994b) and synthetic strategies have been developed to optimize drug action (Li et al. 1995).
Aurones (structure 37, Fig. 3) share structural similarities with chalcones which explain their similar antiparasitic activities. Aurones show strong activity against Leishmania parasites (Kayser et al. 1999) and against P. falciparum (Kayser et al. 2001a). Recent studies indicate that aurones are also promising drug candidates for the treatment of Cryptosporidium parvum infections (Kayser et al. 2001b). With the exception of paromomycin, no natural drug has yet been identified for the treatment of cryptosporidiosis, an opportunistic infection in AIDS patients or otherwise immunodeficient persons.
Flavonoids are widely distributed in the plant kingdom and have long been under investigation for antiparasitic activity without significant prospects for therapeutic value. With intensive studies on A. annua (Asteraceae) the situation has changed dramatically. Artemetin (structure 38, Fig. 3) and casticin (structure 39, Fig. 3) act synergistically with artemisinin (Elford et al. 1987). Both are methoxylated flavonones and are discussed as inhibitors of L-glutamine uptake by infected macrophages. From a second Artemisia species, Artemisia indica, the flavonoids sakuranetin (structure 40, Fig. 3) and 7-methoxyaromadendrin (structure 41, Fig. 3) also show high antiprotozoal activity (Ribeiro et al. 1997). The mode of action of antiprotozoal flavonoids remains unclear. The effect on the generation of reactive oxygen species (Ribeiro et al. 1997) has been discussed, but so have sophisticated biochemical mechanisms like the inhibition of the P-glycoprotein-like transporter (Perez-Victoria et al. 1999) or modulation of protein phosphorylation on the SPK89 protein kinase in trypanosomes (Gale et al. 1994).
Naphthoquinones and related quinones
Quinones have been tested intensively in the past against different parasites including Plasmodium (Weiss et al. 2000; Bezabih et al. 2001), Leishmania (Kayser et al. 2000; Teixeira et al. 2001), Toxoplasma gondii (Khan et al., 1998) and Trypanosoma sp. (Lopes et al. 1978; Pinto et al. 2000). Most tested compounds can be classified as monomeric and dimeric naphthoquinones like plumbagin (structure 42, Fig. 3) and diospyrin (structure 43, Fig. 3), respectively. Plumbagin, isolated from Pera benensis (Euphorbiaceae), is active at IC50=0.42 µg ml–1 against L. donovani (Fournet et al. 1992) and at IC50=0.27 µg ml–1 when tested against P. falciparum (Likhitwitayawuid et al. 1998). Semi-synthetic tetramethoxy- and tetraacetoxy derivatives of diospyrin, isolated from Diospyros montana (Ebenaceae), show a 100-fold increase in activity in comparison to the genuine compound and are thus as effective against P. falciparum as chloroquine at IC50=0.21 µg ml–1 (Hazra et al. 1995). The major disadvantage of natural naphthoquinones is their high cytotoxicity and therefore their relatively low therapeutic selectivity. By synthetic modification, new naphthindazole-4,9-quinones (structure 44, Fig. 3) have been tested, showing high antileishmanial activity combined with reduced toxicity in vivo . 5-chlor-naphthindazole-4,9-quinone is active in vitro at IC50=0.21 µg ml–1. In vivo, 5-chlor-naphthindazole-4,9-quinone reduces the liver parasite load by >95% when given i.v. at 15 mg kg–1 day–1 for 4 days consecutively (Kayser et al. 2001c).