New medicines from nature's armamentarium

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2 Scholte, E.J. et al. (2005) An entomopathogenic fungus for control of adult African malaria mosquitoes. Science 308, 1641–1642 3 Imler, J.L. and Bulet, P. (2005) Antimicrobial peptides in Drosophila: structures, activities and gene regulation. Chem Immunol Allergy 86, 1–21 4 Levashina, E.A. (2004) Immune responses in Anopheles gambiae. Insect Biochem. Mol. Biol. 34, 673–678 5 Lamberty, M. et al. (2001) Insect immunity. Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect. J. Biol. Chem. 276, 4085–4092 6 Scholte, E.J. et al. (2004) Entomopathogenic fungi for mosquito control: a review. J. Insect Sci. 4, 19 7 Martinez-Torres, D. et al. (1998) Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s. Insect Mol. Biol. 7, 179–184

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8 Costantini, C. et al. (1999) Mosquito behavioural aspects of vector– human interactions in the Anopheles gambiae complex. Parassitologia 41, 209–217 9 Genthner, F.J. et al. (1998) Toxicity and pathogenicity testing of the insect pest control fungus Metarhizium anisopliae. Arch. Environ. Contam. Toxicol. 35, 317–324 10 Clark, T.B. et al. (1968) Field and laboratory studies on the pathogenicity of the fungus Beauveria bassiana to three genera of mosquitoes. J. Invertebr. Pathol. 11, 1–7 11 Scholte, E.J. et al. (2003) Infection of malaria (Anopheles gambiae s.s.) and filariasis (Culex quinquefasciatus) vectors with the entomopathogenic fungus Metarhizium anisopliae. Malar. J. 2, 29 12 Khetan, S.K. (2000) Microbial Pest Control, 1st edn, Marcel Dekker 1471-4922/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2005.12.008

New medicines from nature’s armamentarium Andy Crump 2-7-11-1707 Shibaura, Minato-Ku, Tokyo 108-0023, Japan

Nature frequently unleashes a barrage of new and frightening diseases against humans – such as HIV, severe acquired respiratory syndrome, Ebola virus and avian flu recently – in addition to the seemingly everpresent scourges such as malaria and tuberculosis. Fortunately, nature also provides the wherewithal to help conquer the diseases that it sets loose. All that is needed is the human ingenuity to discover, develop and apply the solutions in an optimal fashion. Participants at the 9th Max Tishler Memorial Symposium (Tokyo, July 2005) were told about several new advances in the search for new anti-infective drugs derived from natural sources and were able to learn how one of the most effective drugs ever, ivermectin, made its way through what was, at the time, uncharted territory and how precedents were set at nearly every stage to form a model for all subsequent public–private partnerships.

The promise of natural remedies Traditional medicine has long focused on the power of natural products to treat and cure diseases. Such products are now being actively sought to combat global diseases such as HIV–AIDS and malaria. Most of the powerful new antimalarials, for example, are based on artemisinin, which is derived from the Chinese herb Artemisia annua. Indeed, w140 new drugs originated directly or indirectly from Chinese medicinal plants through the application of modern scientific methods [1]. Many potential anti-HIV compounds that originate from naturally occurring organisms are also being discovered, especially when sophisticated screening techniques are used and molecular modification is brought to bear in developing new leads Corresponding author: Crump, A. ([email protected]).

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for drug candidates. Haruo Tanaka and colleagues at Kitasato University and the Kitasato Institute (http:// www.kitasato.or.jp/rcb/eng/intro.html) developed a novel syncytium-formation screening system and used it to discover the potent anti-HIV protein actinohivin [2], which is produced by the recently discovered actinomycete Longispora albida novel genus, novel species [3]. Actinohivin, a chain of 114 amino acids with internal-sequence triplication (segments 1–3), blocks the infection of susceptible cells by various strains of T-cell (T)-tropic and macrophage (M)-tropic HIV-1 and HIV-2 by binding to the high-mannose-type sugar chain of HIV gp120 (Figure 1). The envelope glycoprotein gp120–gp41 of HIV-1 normally binds to cellular receptors and mediates fusion between viral and cellular membranes [4]. The potent anti-HIV function of actinohivin [5] is brought about by linking to three high-mannose sugar chains of one gp120 molecule, thus binding to gp120 specifically – much like an antibody. The compound will be developed in collaboration with the World Health Organization [WHO (http://www.who.int/en/)] as a topical agent to prevent HIV transmission, with previous work having discovered that actinohivin has no detrimental effect on vaginal cells or sperm (H. Tanaka et al., unpublished). It is thought that more-potent derivatives could be developed as injectable anti-HIV–AIDS drugs, although no industrial partner has yet been identified. In 1994, Kuo-Hsiung Lee and colleagues isolated the pyranocoumarin suksdorfin from Lomatium suksdorfii by bioactivity-directed fractionation and anti-HIV-1 assays in H9 lymphocytes [6]. The original compound has now been modified into 3 0 R,4 0 R-di-O-camphanoyl-(C)-cis-khellactone (DCK). Mechanism-of-action studies indicate that DCK and its analogues have a novel effect, preventing the production of double-stranded viral DNA from the

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Tyr23 Tyr32

Asp15 Leu25

Gln33

Asn28

Figure 1. Docking model between actinohivin segment 1 and a(1–2) mannobiose. Essential amino acids of actinohivin segment 1 that are needed for binding to highmannose sugar chains of gp120 are shown. The affinities of actinohivin for a(1–2) mannobiose, mannotriose, MAN8 and MAN9 are weak. However, when the three segments of actinohivin bind to three high-mannose-type sugar chains of gp120, a strong and specific affinity occurs by the so-called ‘cluster effect’ of lectin, causing potent and specific anti-HIV activity [5]. Image created by S. Hirono using the FAMS modelling software developed by H. Umeyama [15].

single-stranded intermediate; this is different from traditional reverse transcriptase inhibitors, which block the generation of single-stranded DNA from the RNA template. Another anti-HIV lead compound – triterpene betulinic acid, which is derived from Syzigium claviflorum – has been modified into a more potent analogue, 3-O(3 0 dimethylsuccinyl)-betulinic acid (DSB). DSB strongly inhibits primary HIV isolates (with low nanomolar EC50 values in vitro) and, more importantly, remains effective against HIV strains that are resistant to currently available drugs. Furthermore, DSB functions synergistically when delivered with existing approved drugs and, moreover, its mode of action is distinct from other inhibitors, making it a ‘first-in-class maturation inhibitor’ that targets the CA-SP1 region of Gag [7]. DSB is licensed to Panacos Pharmaceuticals (http://www.panacos.com/) and is in Phase II trials in the USA under the name PA-457.

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Vietnam, was in preclinical development with the WHO Special Programme for Research and Training in Tropical Diseases (TDR) and Tibotec (http://www.tibotec.com/), but toxicity and mixture problems proved to be insurmountable. Nevertheless, licochalcone A, which is extracted from the root of Chinese liquorice, is active against Leishmania donovani and Leishmania major and is now in development with URL Pharmaceuticals (http://www. licapharma.com/frame.cfm?sprogZ1&grpZ1&menuZ1) [8]. In addition, the propylquinolones – notably chimanines B and D, which are derived from the Bolivian plant Galipea longiflora – have activity against Leishmania amazonensis, decreasing the weight of skin lesions by 70%. Chimanine D and 2-n-propylquinolone are also extremely effective against L. donovani and reduce the parasite burden in the liver by 85–99% [9]. At the 9th Max Tishler Memorial Symposium (Tokyo, July 2005), Alan Fairlamb had relatively little to report about antitrypanosomal drugs from natural sources, despite being buoyed by the publication of the genomes of three key trypanosomatids (Trypanosoma brucei, Trypanosoma cruzi and Leishmania major) [10]. Having determined that trypanothione reductase (TryR) is unique to trypanosomatids and is an essential component in the mechanism of hydrogen peroxide detoxification, Fairlamb’s research group has identified TryR as a target for drug discovery to combat African sleeping sickness, leishmaniasis and Chagas disease [11]. Thus, the postgenomic era offers enormous potential for the treatment and control of these diseases. Studies using molecular modelling have identified two natural products, cadabacine and lunarine, as being selective TryR inhibitors. Lunarine, which is extracted from the honesty plant (Lunaria biennis), inhibits TryR and, although this effect is time- and concentration-dependent, research is proceeding [12]. The model product: ivermectin The Kitasato Institute has long been a world leader in the search for useful bioactive substances from naturally occurring microorganisms, providing a wide variety of compounds, including ivermectin [13] (Table 1). The organism from which ivermectin is produced, Streptomyces avermectinius, was isolated from soil in Japan and sent to the Merck, Sharp and Dohme (http://www.merck. com/mrl/) research laboratories in the early 1970s. Table 1. Antiparasitic compounds originating from the Kitasato Institutea

Natural products for neglected diseases Simon Croft of the Drugs for Neglected Diseases Initiative [DNDi (http://www.dndi.org/)] indicates that screening and investigation of natural products would be a strong component of DNDi activities, and a collaborative research agreement now exists between the DNDi and the Kitasato Institute. Using leishmaniasis as an example, DNDi work is beginning to gather momentum in the search for natural lead compounds, despite the disappointment caused by the failure of the antileishmanial compound PX-6518. This triterpene saponiside, derived from the leaves of Maesa spp. harvested in www.sciencedirect.com

Year 1979 1981 1987 1993 1999 2000 2000 2001 2001 2002 2002

Compound Avermectin (partner: Merck, Sharp and Dohme) Hitachimycin Jietacin Hynapenes Viridomycin F Argadin Argifin Nafuredin Oligomycin G Antimycin A9 Miyakamide

a For a complete list of bioactive microbial products originating from the Kitasato Institute, see http://www.kitasato.or.jp.

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A collaborative research programme then led to the discovery of avermectin. Merck scientists drove this through the development process and produced the more potent ivermectin, which was one of the most effective broad-spectrum antiparasitic compounds ever developed. Introduced into the global animal health market in 1981, ivermectin quickly became a blockbuster drug, earning more than US$1 billion in sales annually. In 1988 – following work by Merck, WHO, TDR and the Onchocerciasis Control Programme in West Africa (OCP), in what could justifiably be called the forerunner of public–private partnerships – ivermectin was found to be highly effective at curing onchocerciasis (river blindness), an ancient disease that has devastated Africa and parts of Latin America. In what was the first and largest drug-donation scheme, ivermectin (under the brand name Mectizanw) was provided free of charge by Merck for the treatment of onchocerciasis for as long as it was needed. Onchocerciasis is due to be eliminated from the Americas as a public health problem by the end of 2005. In Africa, where the disease situation is far worse, the African Programme for Onchocerciasis Control (APOC) plans to use principally the annual distribution of ivermectin to rid the continent of the disease. Ivermectin is so safe that even illiterate villagers can distribute it using a unique system of community-directed treatment in which communities assume all responsibilities, financial and logistical, for collecting and distributing the drug [14]. However, severe adverse neurological reactions, including several deaths, have been reported in people with a high intensity of Loa loa infection. This has curtailed ivermectin treatment programmes in areas that are coendemic for L. loa (i.e. most of Central Africa) and there is a pressing need to identify communities in which this parasite is endemic (http://www.who.int/tdr/diseases/ oncho/direction.htm). By 2007, sustainable community-driven systems will be in place to ensure that 90 million Africans receive ivermectin annually. Ivermectin is also effective against lymphatic filariasis, and extension of the Mectizan Donation Programme by Merck to cover onchocerciasis and lymphatic filariasis where they coexist – in addition to donation of albendazole by GlaxoSmithKline (http://www. gsk.com/index.htm) to treat lymphatic filariasis, and financial support from bilateral and public sector sources – has driven the Global Programme to Eliminate Lymphatic Filariasis to the point at which O130 million people in 38 nations are receiving annual drug treatments (see http://www.filariasis.org). Ivermectin – already used extensively in animal health and in eliminating both onchocerciasis and lymphatic filariasis, two of the most disfiguring and deleterious human diseases – is now being used commercially for the treatment of strongyloidiasis, mites and scabies. The foundation of the model The advent of ivermectin was partly due to the fact that ¯ mura (now president of the Kitasato Institute) Satoshi O was invited in the early 1970s to take up a post as visiting professor in the department of Chemistry at Wesleyan University (http://www.wesleyan.edu/); the department www.sciencedirect.com

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was run by Max Tishler, who had retired from a successful stint running Merck to take up the new university post. The liaison between the two men, the application of human innovation and resourcefulness in the spirit of friendship, and the subsequent liaison with Merck and its extensive, highly skilled staff and resources accounted for the rest. The expertise of the various research and administrative teams at Merck and the Kitasato Institute, the drive and vision of global leaders in WHO, TDR and OCP, the forceful action of a large cadre of committed nongovernment development organizations who distributed the drug initially, and the skills, ingenuity and unwavering commitment of affected communities wishing to take their health and wellbeing into their own hands brought about a trail-blazing, standard-setting example for all other private-sector–public-sector and multidisciplinary public health partnerships to emulate. Through the efforts of multisectoral collaborations and the input of committed and talented individuals, it has been possible to exploit natural products to solve some of the major disease problems of the world. Smallpox has been eradicated by concerted human action, and several other diseases – including leprosy, onchocerciasis, polio and dracunculiasis – are being eliminated, unless nature intervenes once more. Supplementary data Supplementary data associated with this article can be found at doi:10.1016/j.pt.2005.12.009

References 1 Bale, H.E. (2004) Globalising innovation in healthcare technology. In Global Forum Update on Research for Health, pp. 155–157, Global Forum for Health Research and Pro-Book Publishing 2 Chiba, H. et al. (2001) A simple screening system for anti-HIV drugs: syncytium formation assay using T-cell line tropic and macrophage tropic HIV env expressing cell lines – establishment and validation. J. Antibiot. (Tokyo) 54, 818–826 3 Matsumoto, A. et al. (2003) Longispora albida gen. nov., sp. nov., a novel genus of the family Micromonosporaceae. Int. J. Syst. Evol. Microbiol. 53, 1553–1559 4 Chiba, H. et al. (2004) Actinohivin, a novel anti-HIV protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chain of gp120. Biochem. Biophys. Res. Commun. 316, 203–210 5 Takahashi, A. et al. (2005) Essential regions for antiviral activities of actinohivin, a sugar-binding anti-HIV protein from an actinomycete. Arch. Biochem. Biophys. 47, 233–240 6 Lee, T.T. et al. (1994) Suksdorfin: an anti-HIV principle from Lomatium suksdorfii, its structure–activity correlation with related coumarins, and synergistic effects with anti-AIDS nucleosides. Bioorg. Med. Chem. 2, 1051–1056 7 Li, F. et al. (2003) PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing. Proc. Natl. Acad. Sci. U. S. A. 100, 13555–13560 8 Zhai, L. et al. (1999) The antileishmanial activity of novel oxygenated chalcones and their mechanism of action. J. Antimicrob. Chemother. 43, 793–803 9 Chan-Bacab, M.J. and Pen˜a-Rodrı´guez, L.M. (2001) Plant natural products with leishmanicidal activity. Nat. Prod. Rep. 18, 674–688 10 Ash, C. and Jasny, B.R. (2005) Trypanosomatid genomes. Introduction. Science 309, 399 11 Fairlamb, A.H. and Cerami, A. (1992) Metabolism and functions of trypanothione in the Kinetoplastida. Annu. Rev. Microbiol. 46, 695–729

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12 Hunter, W.N. et al. (2003) Targeting metabolic pathways in microbial pathogens: oxidative stress and anti-folate drug resistance in trypanosomatids. Biochem. Soc. Trans. 31, 607–610 ¯ mura: in pursuit of 13 Crump, A. and Otoguro, K. (2005) Satoshi O nature’s bounty. Trends Parasitol. 21, 126–132 ¯ mura, S. and Crump, A. (2004) Life and times of ivermectin: a 14 O success story. Nat. Rev. Microbiol. 2, 984–989

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15 Ogata, K. and Umeyama, H. (2000) An automatic homology modeling method consisting of database searches and simulated annealing. J. Mol. Graph. Model. 18, 258–272, 305–306

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Letters

The re-emergence of trichinellosis in China? Jing Cui1, Zhong Quan Wang1 and Malcolm W. Kennedy2 1

Department of Parasitology, Medical College, Zhengzhou University, Zhengzhou 450052, China Environmental and Evolutionary Biology, Institute of Biomedical and Life Sciences, Graham Kerr Building, University of Glasgow, Glasgow, UK, G12 8QQ 2

Between 1964 and 1999, there were 548 outbreaks of human trichinellosis in China (23 004 cases and 236 deaths), 525 of which arose from the consumption of pork [1]. In China, Trichinella infection in swine is thought to derive mainly from food waste and is endemic in southwestern, central and northeastern areas of the country [2] (Figure 1). There is evidence that trichinellosis is re-emerging in areas of China that were relatively free from this infection, such as Qinghai Province, which is located in the west of the country and has a population of approximately five million, including several minority ethnic groups (Tibetan, Muslim and Mongolian) that account for 46% of the total population of the area. Beef and mutton are the main meats consumed by the minority ethnic groups, with pork consumption limited by religious customs. Routine surveillance confirmed the absence of domestic trichinellosis from Qinghai before 1989, with microscopic examination of O35 000 pigs killed in the capital (Xining) revealing no infections [3]. However, the Western Region Development strategy of the 1990s elicited the migration and settlement of large numbers of people from central to western areas, resulting in the increased importation of pork products to the latter areas, either commercially or privately. The areas of central China from which potentially infected meat derives have pig prevalences of 6.76% (Hubei) and 4.27% (Henan), as estimated from abattoir inspections and surveys in markets [4]. In the new western communities, pigs are reared under relatively primitive conditions in which they consume raw waste materials, can scavenge animal carcasses from both wild and domestic sources, and come into contact with rodents. Therefore, there is potential for a substantial increase in the incidence of human trichinellosis in western areas of China, and this is supported by recent surveys. In 1990, the incidence of infected pork samples from markets in the city of Xining was only w0.1% [3] but recent abattoir surveys in Qinghai reveal increases to 15.9% in Huangyuan County in 1997 and to 23.0% in the city of Delingha Corresponding author: Wang, Z.Q. ([email protected]). Available online 27 December 2005 www.sciencedirect.com

in 2004. More alarmingly, the prevalence was as high as 33.3% in one migrant community of Delingha in which all pigs were reared in the open [5] and in which most were slaughtered at private premises without inspection. From 2000 to 2003, sampling from markets indicated an increase in the prevalence of infected pork to 3.2% in Qinghai [6]. No cases of human trichinellosis have yet been confirmed but the province represents an area of growing risk. These changes also pose a potential danger to the rapidly developing tourist industry. The prevalence of infection in pigs slaughtered in the Liaoning Province was 0.34% in 1980 [4] and 0.02% in 2000 [7] but, in a survey carried out in the city of Shengyang (Liaoning Province) in 1999, the prevalence had increased to 52.94%, and in the Dongling tourist area prevalence was 100.00% [8]. In the tourist area of Nanyang (Henan Province), the overall prevalence of trichinellosis in pigs has risen from 0.1% to 22.8% in piggeries

Liaoning Beijing Qinghai

Henan Hubei

Hainan?

Taiwan? Guangxi

Figure 1. The distribution of animal and human trichinellosis in China. Large black dots denote regions in which both human trichinellosis and animal trichinellosis have been recorded, covering 18 provinces, autonomous regions and municipalities. Small black dots denote regions in which only animal Trichinella infections have been reported, covering 12 provinces, autonomous regions and municipalities. Question marks denote regions in which no human or animal trichinellosis has been reported.