In tropical regions, protozoan parasites can cause severe diseases with malaria, leishmaniasis, sleeping sickness, and Chagas disease standing in the forefront. Many of the drugs currently being used to treat these diseases have been developed more than 50 years ago and can cause severe adverse effects. Above all, resistance to existing drugs is widespread and has become a serious problem threatening the success of control measures. In order to identify new antiprotozoal agents, more than 600 commercial agrochemicals have been tested on the pathogens causing the above mentioned diseases. For all of the pathogens, compounds were identified with similar or even higher activities than the currently used drugs in applied in vitro assays. Furthermore, in vivo activity was observed for the fungicide/oomyceticide azoxystrobin, and the insecticide hydramethylnon in the Plasmodium berghei mouse model, and for the oomyceticide zoxamide in the Trypanosoma brucei rhodesiense STIB900 mouse model, respectively.
The Protozoan parasites of the genera Plasmodium spp. , Leishmania spp. , Trypanosoma brucei spp. and Trypanosoma cruzi, are the disease causative agents threatening entire populations in mainly resource poor countries around the world. Malaria, due to infection with Plasmodium spp. , is one of the most devastating diseases in developing countries, with 216 million cases in 2010, causing an estimated 655,000 deaths per year [1]. Other recent estimates assume up to 1. 2 million deaths per year [2]. For the treatment of malaria several highly active drugs are available, like chloroquine, quinine, mefloquine, atovaquone, artesunate, and their analogs. Thus, malaria is often not included in the list of the neglected tropical diseases. Unfortunately, significant resistance to almost all of these drugs has developed; even to the “last resort” artemisinin-derivatives, first cases of delayed clinical efficacy have been reported [3]. Recently, large libraries from pharma companies have been screened against protozoan parasites and some interesting hits [4], [5], [6], [7] have been found, especially against malaria with the spiroindolones currently undergoing clinical evaluation [8], [9]. Most of the promising compounds in the development pipeline are in a rather early clinical stage, so that a high failure rate is expected [10]. Considering the rapid development of resistance, and the challenges seen with the development of malaria vaccines [11], a continuous refilling of research pipelines with compounds in preclinical/clinical evaluation will be necessary, for the long term perspective. Therefore new compounds for resistance management would be highly desirable, even if they might not show the same remarkably high activity levels as the recently promoted peroxide candidates like OZ439 [12]. In addition, the global malaria agenda has shifted from the mere control of clinical cases to malaria elimination and eventually eradication urgently requiring transmission blocking agents [13]. Human African trypanosomiasis (HAT), also known as sleeping sickness, is caused by infections of T. b. rhodesiense and T. b. gambiense. Populations living in remote rural areas of sub-Saharan Africa are at risk of acquiring HAT. The disease burden in 2000 was estimated at 1. 3 Mio DALYs (Disability-Adjusted Life Years) and the estimated number of cases up to 70,000 in 2006 [14]. In recent years the public health situation has improved due to increased monitoring and chemotherapy, resulting in the decrease of reported HAT cases to approximately 10,000 [15]. Only 4 drugs are currently registered as HAT treatment. Pentamidine and suramin are used to treat the hemolymphatic stage (stage 1) of the disease, while melarsoprol and eflornithine (DFMO) are used in stage 2 of the disease when the parasites have invaded the central nervous system (CNS) and which is lethal if untreated. The available drugs are unsatisfactory due to cost, toxicity, poor oral bioavailability, long treatment and lack of efficacy. Melarsoprol is highly toxic, and up to 5% of the second stage patients treated with melarsoprol die of a reactive encephalopathy. Eflornithine treatment is expensive and logistically difficult; it requires four daily intravenous infusions over fourteen days. Recently the eflornithine-nifurtimox combination therapy (NECT) was introduced [16]. The requirement of intravenous administration although reduced to a quarter of injections as compared to monotherapy is still a limitation, with a need for new and more easily administrable drugs. Trypanosoma cruzi infection elicts Chagas disease and is an important public health problem causing approximately 14,000 deaths and 0. 7 Mio DALY annually [17]. Treatment options are limited due to toxicity of available drugs, parasite resistance, and poor drug activity during the chronic phase of the disease. Currently there are two medications being used to treat Chagas disease, nifurtimox and benznidazole [18]. Severe toxicity and long treatment requirements are associated with both drugs [19]. Therefore new medications are badly needed for treating this disease especially in its chronic phase. Leishmaniasis causes approximately 50,000 deaths and 2. 1 Mio DALY annually [20]. It threatens about 350 million people around the world and 12 million people are believed to be infected, with 1–2 million estimated new cases every year [21]. Widely used medications are still based on i. v. application of antimony compounds like stilbogluconate, resulting in severe side effects. More modern, but also more expensive medications are liposomal amphotericin B, miltefosine, and paromomycin [22]. Thus new affordable and effective therapies are urgently needed to combat these disastrous diseases. Registration requirements for agrochemicals are in some aspects even more stringent than for pharmaceuticals, as side effects that are tolerated for drugs against many life threatening diseases, are not acceptable for agrochemicals that potentially could enter the food chain [23], [24], [25]. As a consequence, all commercialized agrochemicals must go through broad toxicological profiles including e. g. chronic and reprotoxicological studies in different mammalian species, covering at least part of the preclinical studies required for drug development. Furthermore, agrochemicals are highly optimized on agrochemical pest targets with often good selectivities in mammals and excellent temperature and storage stability. Another interesting feature of commercial agrochemicals is the very low production cost of only a few cent/g, as the compounds are produced in highly optimized processes on the multi-ton scale. Surprisingly, these aspects have not led to a systematic evaluation of agrochemicals for pharmacological use so far [26]. Here we present data of over 600 commercial agrochemicals which have been systematically tested for the first time for their antiparasitic activity.
A library of over 600 compounds (for a list of CAS-numbers and common names of the tested agrochemicals see Supporting Information S1), that are or have been active ingredients in commercial agrochemical products, has been compiled from the BASF compound depository and was dissolved in DMSO stock solutions in a concentration of 10 mg/ml. These samples were then further diluted according to the requirements of the assays. The structural integrity of the dissolved samples has been confirmed subsequently by LCMS-analysis. All work was conducted in accordance to relevant national and international guidelines. The in vivo efficacy studies were approved by the veterinary authorities of the Canton Basel-Stadt. The in vivo studies were carried out under license No. 1731 and license No. 739 of the Kantonales Veterinäramt, CH-4025 Basel, Switzerland adhering to the Tierschutzverordnung from 23. 04. 2008 (based on the Tierschutzgesetz from 26. 12. 2005).
38 agrochemicals with sub-µM activity on T. cruzi were identified, many of which being azoles with P450-inhibiting activity (Figure 2). P450-monoxygenases have been discussed before as targets against T. cruzi, especially the sterol 14α-demethylase [49]. The standard drug benznidazole (LD50 rat p. o. not available) [50], [51] has an IC50 of 1871 nM in this assay. Ipconazole (LD50 rat p. o. 888 mg/kg), has an IC50 of 3. 0 nM, the most active agrochemical against T. cruzi. It is a fungicide used predominantly in seed dressing. The tested material is, like the commercial material, racemic and a mixture of diastereomers, therefore an enantiopure isomer could potentially have even higher activity. Difenoconazole (LD50 rat p. o. 1453 mg/kg), a broad spectrum and systemic fungicide, showed an IC50 value of 7. 4 nM. This commercial agrochemical is again a racemic diasteromeric mixture and could therefore also have intrinsically higher activity as a pure isomer. Clotrimazole (14 nM), and viniconazole [52] (26 nM), are two azole drugs used against fungal skin infections, that have also been discussed as agro fungicides and therefore have been tested in this screen. As they have a complete pharmacological dossier they might also be interesting drug candidates. Zoxamide (LD50 rat p. o. >5000 mg/kg), a broadspectrum oomyceticide used in fruits and vegetables, showed 27 nM activity. It is sold and was tested as a racemate. Its mode of action against oomycetes is the inhibition of microtubule formation. Pyridaben (30 nM), and tolfenpyrad (55 nM), are insecticides/acaricides inhibiting the complex 1 in the mitochondrial electron transport chain. A number of further azole fungicides showed activities below 100 nM including metconazole 31 nM, tebuconazole 36 nM, bitertanol 35 nM, climbazole 55 nM, prochloraz 69 nM, hexaconazole 73 nM, and fenapanil 99 nM. Further agrochemicals with high activity in this assay were penconazole (130 nM), epoxyconazole (136 nM), imazalil (148 nM), propiconazole (160 nM), fenarimol (193 nM), fluquinconazole (199 nM), picoxystrobin (248 nM), cyproconazole (257 nM), myclobutanil (374 nM), tetraconazole (478 nM), and pyrifenox (491 nM). In spite of the excellent in vitro activity initial experiments in a T. cruzi mouse model did so far not show in vivo efficacy for selected hits (personal communication Nazaré Soiro). Against L. donovani only two agrochemicals showed sub-µM activity (Figure 3). The standard miltefosine (LD50 rat p. o. 246 mg/kg) showed in this assay an IC50 value of 250 nM. Zoxamide (LD50 rat p. o. >5000 mg/kg) showed an IC50 of 250 nM. The oomyceticidal compound has been discussed in the T. cruzi section. Tolylfluanid (LD50 rat p. o. >5000 mg/kg) resulted in an IC50 value of 861 nM. It is a protective fungicide and oomyceticide with presumed thiol conjugating activity. Other agrochemicals with moderate activity against L. donovani were flocumafen (2451 nM), dimoxystrobin (3248 nM), bromofenoxin (3839 nM), cyhexatin (4517 nM), and cyazofamid (4988 nM). Due to the split of most life science companies into their agro- and pharma branches in the 1990s, the companies active in agrochemistry have not been involved in the recent screening activities to identify new drugs against infectious tropical diseases, even though agrochemicals might have a high potential to yield interesting hits for these applications. In this cooperation between industrial and public partners, it was shown for several commercial agrochemicals that they are highly active against some of the most important pathogens of infectious tropical diseases. Interestingly as anticipated, several of the oomyceticides (strobilurins against P. falciparum, zoxamide against T. b. rhodesiense and L. donovani) were active against these protozoans, but also other agrochemicals (e. g. hydramethylnon against P. falciparum; azoles like iproconazole against T. cruzi) showed very interesting activities. Exemplified by one of the major commercial agrochemicals, the fungicide azoxystrobin, as well as for the insecticide hydramethylnone, the reduction of parasitemia, and significant life extension for P. berghei infected mice was achieved. For zoxamide, an effect against T. brucei in the mouse model was also demonstrated. This successful in vitro– in vivo transfer without galenic optimization could not be taken for granted, as these agrochemicals have not been optimized for mammalian pharmacokinetics. There is still a high probability that the identified hits in the end might not be suitable for human use, as there are still several hurdles to overcome. However, the results of this highly focussed and relatively low input approach are more promising than could have been hoped for. It is especially noteworthy, that the screen of less than 700 agrochemical resulted in e. g. 24 new sub-µM hits against P. falciparum, compared to 4 new sub-µM hit in over 2687 recently tested commercial drugs (excluding known antimicrobial and anti-cancer a. i.) [54], [55]. This clearly demonstrates that agrochemistry can be a very interesting and so far untapped source of new leads, and maybe even drug candidates, against protozoal diseases. It would also be very interesting to screen commercial agrochemicals against the pathogens of other neglected diseases, like schistosomes, nematodes, food borne trematodes, diarrhoeal amoebas and also tropical bacterial pathogens, for which good antibiotic cures are missing. These studies are still to be done.
Even though agrochemistry and infectious disease control have the same principle goal – the suppression of harmful organisms without harming human health and the environment – there have been only very limited activities to exploit this overlap for the development of new antiinfectious drugs so far. In this study and for the first time, over 600 commercial agrochemicals were systematically screened against the infectious pathogens causing malaria, sleeping sickness, Chagas disease and leishmaniasis. Many highly active compounds with known low mammalian toxicity were identified in cell based assays, and the activity of some of them could even be confirmed in first animal model studies. Further expansion of this concept to other pathogens and the examination of analogues of the identified hits, potentially available from agrochemical companies, would allow for a very efficient source of novel drug candidates.
plasmodium falciparum
medicine
biotechnology
agrochemicals
infectious diseases
chagas disease
tropical diseases (non-neglected)
african trypanosomiasis
leishmaniasis
neglected tropical diseases
malaria
agriculture
Agrochemicals against Malaria, Sleeping Sickness, Leishmaniasis and Chagas Disease