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Three series of monoquinolines consisting of a 1,4-bis(3-aminopropyl)piperazine linker and a large variety of terminal groups were synthesized. Our aim was to prove that in related bisquinoline, it is the second quinoline moiety that is responsible for cytotoxicity and that it is not an absolute requirement for overcoming resistance to chloroquine (CQ). Eleven compounds displayed a higher selectivity index (ratio CC50/IC50 activity) than CQ, and one of them cured mice infected by Plasmodium berghei.

Eight antioxidants from five different polyphenolic classes (cinnamic acids, benzoic acids, flavonoids, proanthocyanidins and stilbenes), and the water-soluble vitamin E derivative trolox were examined for their antioxidant activity in-vitro. In addition, the compounds were tested for their cytotoxicity on growing fibroblasts and their inhibition of the classical pathway of the complement system. Procyanidin C1 was shown to be a good scavenger of both DPPH* and HO*, and a strong inhibitor of lipid peroxidation and the classical pathway of the complement system. Consequently, procyanidin C1 was classified as the most promising antioxidant in-vitro of all compounds tested. In contrast, genistein exhibited a very low antioxidant activity in both the lipid peroxidation and the DPPH* scavenging assay, a high cytotoxicity and a low complement-inhibiting activity.

These guidelines have been prepared to assist in the planning, operation and interpretation of studies designed to assess the effectiveness of drugs against helminth parasites of chickens and turkeys. They are the first to be compiled under the auspices of the World Association for the Advancement of Veterinary Parasitology (WAAVP) for these parasites. The advantages and disadvantages of the widely used critical and controlled tests are discussed. Information is provided on the selection of animals for experiments, animal housing, feed, dose determination studies, confirmatory and field trials, record keeping and necropsy procedures. This document should help investigators and those involved in product approval and registration in conducting and evaluating studies concerned with determining the effectiveness and safety of anthelmintic drugs.

A series of 5-nitro-2-furancarbohydrazides were synthesized. In vitro antitrypanosomal activities of these compounds were determined against the closely related protozoa Trypanosoma cruzi Trypanosoma brucei and discussed in relation to potential targets.

On the basis of the original lead neocryptolepine or 5-methyl-5H-indolo[2,3-b]quinoline, an alkaloid from Cryptolepis sanguinolenta, derivatives were prepared using a biradical cyclization methodology. Starting from easily accessible educts, this approach allowed the synthesis of hitherto unknown compounds with a varied substitution pattern. As a result of steric hindrance, preferential formation of the 3-substituted isomers over the 1-substituted isomers was observed when cyclizing N-(3-substituted-phenyl)-N'-[2-(2-trimethylsilylethynyl)phenyl]carbodiimides. All compounds were evaluated for their activity against chloroquine-sensitive as well as chloroquine-resistant Plasmodium falciparum strains, for their activity against Trypanosoma brucei and T. cruzi, and for their cytotoxicity on human MRC-5 cells. Mechanisms of action were investigated by testing heme complexation using ESI-MS, inhibition of beta-hematin formation, DNA interactions (DNA-methyl green assay and linear dichroism), and inhibition of human topoisomerase II. Neocryptolepine derivatives with a higher antiplasmodial activity and a lower cytotoxicity than the original lead have been obtained. This selective antiplasmodial activity was associated with inhibition of beta-hematin formation. 2-Bromoneocryptolepine was the most selective compound with an IC(50) value against chloroquine-resistant P. falciparum of 4.0 microM in the absence of cytotoxicity (IC(50)32 microM). Although cryptolepine, a known lead for antimalarials also originally isolated from Cryptolepis sanguinolenta, was more active (IC(50) = 2.0 microM), 2-bromoneocryptolepine showed a low affinity for DNA and no inhibition of human topoisomerase II, in contrast to cryptolepine. Although some neocryptolepine derivatives showed a higher antiplasmodial activity than 2-bromocryptolepine, these compounds also showed a higher affinity for DNA and/or a more pronounced cytotoxicity. Therefore, 2-bromoneocryptolepine is considered as the most promising lead from the present work for new antimalarial agents. In addition, 2-bromo-, 2-nitro-, and 2-methoxy-9-cyanoneocryptolepine exhibited antitrypanosomal activity in the micromolar range in the absence of obvious cytotoxicity.

Glutathione (GSH), which is known to guard Plasmodium falciparum from oxidative damage, may have an additional protective role by promoting heme catabolism. An elevation of GSH content in parasites leads to increased resistance to chloroquine (CQ), while GSH depletion in resistant P. falciparum strains is expected to restore the sensitivity to CQ. High intracellular GSH levels depend inter alia on the efficient reduction of GSSG by glutathione reductase (GR). On the basis of this hypothesis, we have developed a new strategy for overcoming glutathione-dependent 4-aminoquinoline resistance. To direct both a 4-aminoquinoline and a GR inhibitor to the parasite, double-drugs were designed and synthesized. Quinoline-based alcohols (with known antimalarial activity) were combined with a GR inhibitor via a metabolically labile ester bond to give double-headed prodrugs. The biochemically most active double-drug 7 of this series was then evaluated as a growth inhibitor against six Plasmodium falciparum strains that differed in their degree of resistance to CQ; the ED(50) values for CQ ranged from 14 to 183 nM. While the inhibitory activity of the original 4-aminoquinoline-based alcohol followed that of CQ in these tests, the double-drug exhibited similar efficiency against all strains, the ED(50) being as low as 28 nM. For the ester 7, a dose-dependent decrease in glutathione content and GR activity and an increase in glutathione-S-transferase activity were determined in treated parasites. The drug was subsequently tested for its antimalarial action in vivo using murine malaria models infected with P. berghei. A 178% excess mean survival time was determined for the animals treated with 40 mg/kg 7 for 4 days. No cytotoxicity due to this compound was observed. Work is in progress to extend and validate the strategy outlined here.

Amodiaquine (AQ) is an antimalarial which is effective against chloroquino-resistant strains of Plasmodium falciparum but whose clinical use is severely restricted because of associated hepatotoxicity and agranulocytosis. "One-pot" synthesis of formamidines likely to be transformed into AQ derivatives is reported. Compared with AQ, the new compounds were devoid of in vitro cytotoxicity upon human embryonic lung cells and mouse peritoneal macrophages. One showed a potent in vivo activity in mice infected with P berghei. Transformation of this compound by reductive amination led to a new type of AQ derivatives that displayed an in vitro activity similar to that of AQ but did not lead to toxic quinone-imines.

A series of symmetrically substituted 1,4-bis(3-aminopropyl)piperazines was synthesized and tested towards trypanothione reductase and for its in vitro trypanocidal potency. The most trypanocidal amongst them was found to be totally inactive towards the enzyme and thus constitutes a lead structure for the identification of new potential Trypanosoma cruzi target(s).

A molecular modeling study meant to detect pharmacophore-like patterns in the active site of trypanothione reductase (TR) offered hints about the opportunity of synthesizing and testing diphenylsulfide derivatives with prolonged or branched polyamino side chains as putative TR inhibitors. The inhibition results within the synthesized series confirmed the main working hypothesis inspired by the molecular modeling study. The different compounds were tested in vitro on the enzyme and on Trypanosoma cruzi and Trypanosoma brucei trypomastigotes as well as in vivo in infected mice.

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis (VL). Antimonials (SSG) have long been the first-line treatment against VL, but have now been replaced by miltefosine (MIL) in the Indian subcontinent due to the emergence of SSG-resistance. Our previous study hypothesised that SSG-resistant L. donovani might have increased in vivo survival skills which could affect the efficacy of other treatments such as MIL. The present study attempts to validate these hypotheses. Fourteen strains derived from Nepalese clinical isolates with documented SSG-susceptibility were infected in BALB/c mice to study their survival capacity in drug free conditions (non-treated mice) and in MIL-treated mice. SSG-resistant parasites caused a significant higher in vivo parasite load compared to SSG-sensitive parasites. However, this did not seem to affect the strains' response to MIL-treatment since parasites from both phenotypes responded equally well to in vivo MIL exposure. We conclude that there is a positive association between SSG-resistance and in vivo survival skills in our sample of L. donovani strains which could suggest a higher virulence of SSG-R strains compared to SSG-S strains. These greater in vivo survival skills of SSG-R parasites do not seem to directly affect their susceptibility to MIL. However, it cannot be excluded that repeated MIL exposure will elicit different adaptations in these SSG-R parasites with superior survival skills compared to the SSG-S parasites. Our results therefore highlight the need to closely monitor drug efficacy in the field, especially in the context of the Kala-azar elimination programme ongoing in the Indian subcontinent.

Bioguided fractionation of Syzygium cumini (Myrtaceae) bark decoction for antiplasmodial activity was performed, leading to the isolation of three known ellagic acid derivatives (ellagic acid, ellagic acid 4-0-a-L-2"-acetylrhamnopyranoside, 3-0-methylellagic acid 3'-O-a-L-rhamnopyranoside), as well as the new derivative 3-0-methylellagic acid 3'-O-f)-D­glucopyranoside. Activity investigation was based on the reduction of P. Jalciparum (PjKl) parasitaemia in vitro and the inhibition of f)-hematin formation, a known mechanism of action of some antimalarial drugs. Among the investigated ellagic acid derivatives, only ellagic acid was able to reduce P. Jalciparum parasitaemia in vitro and inhibit f)-hematin formation, suggesting that free hydroxyl groups are necessary for activity within this class ofcompounds. Keywords: Syzygium cumini, Myrtaceae, ellagic acid derivatives, antiplasmodial, f)-hematin.

The current drug R&D pipeline for most neglected diseases remains weak, and unlikely to support registration of novel drug classes that meet desired target product profiles in the short term. This calls for sustained investment as well as greater emphasis in the risky upstream drug discovery. Access to technologies, resources, and strong management as well as clear compound progression criteria are factors in the successful implementation of any collaborative drug discovery effort. We discuss how some of these factors have impacted drug discovery for tropical diseases within the past four decades, and highlight new opportunities and challenges through the virtual NorthSouth drug discovery network as well as the rationale for greater participation of institutions in developing countries in product innovation. A set of criteria designed to facilitate compound progression from screening hits to drug candidate selection is presented to guide ongoing efforts.

The effect of diclazuril, fed to chickens at 1 ppm in the feed, was studied to determine whether the drug interfered with the development of immunity to Eimeria tenella. Group A was not treated, Groups B and C received diclazuril from Day 1 until Day 15, after which time the medicated feed was replaced by blank feed for the remainder of the experiment from Day 16 until Day 42. Immunization was performed in Groups A and B by artificial trickle infections with 2,000 sporulated oocysts per bird on Days 4, 6, 8, 11, and 13. On Day 29, a challenge infection was given using 200,000 oocysts per bird. The unmedicated birds (Group A) developed subclinical coccidiosis after the trickle infections with excretion of oocysts and a slightly decreased growth performance. At challenge, a good protective unimmunity was present, reflected by a good growth performance and a low oocyst excretion. The unimmunized birds (Group C) developed a severe clinical disease after challenge with nigh oocyst output, increased mortality, and poor growth performance. The diclazuril-medicated, trickle-infected birds (Group B) were well protected, both against the immunizing trickle infections and the challenge infection. After challenge, no clinical disease developed, although some lesions and oocyst excretion were present. It is concluded that, under the conditions of the trial, diclazuril did not significantly interfere with protective immunity formation against E. tenella.

The relative importance of peak level- and residual level-related flukicidal activity of closantel against immature and mature Fasciola hepatica was evaluated in a comparative efficacy trial using two animal species with a different plasma elimination pattern, that is, the rat and the sheep with an elimination half-life of less than one week and of two to three weeks, respectively. The rats were dosed orally with closantel at 20 mg kg−1 at two, four, six, eight and 10 weeks; the sheep at 10 mg kg−1 at eight, 10 and 12 weeks after artificial infection. Necropsy was performed either one week after treatment or 12 weeks after infection. Efficacy rates and the length of the recovered flukes were evaluated. It was demonstrated that the flukicidal effect of closantel is directly related to its peak plasma levels and less to its residual plasma concentrations. In the rat, a high efficacy (P

Racing pigeons were artificially infected with a mixed inoculum of Eimeria labbeana (85 per cent) and E columbarum (15 per cent) and treated orally with 2·5 mg clazuril either on day 1, 2, 3, 4, 5, 6 or 7 after infection. The impact of the treatment on the different developmental stages was evaluated by oocyst output and by histological examination of the duodenum and jejunum. The life cycle always became completely interrupted, but maximal effects were noted when treatment was given on day 4, 5 or 6 after infection. Treatment during patency completely interrupted oocyst excretion within three days after dosing. Degenerative changes in schizonts and gametocytes were always observed. The histology revealed a reduced number and abnormal structure of developing merozoites; a ballooned aspect and presence of numerous small vacuoles in the microgametocytes; the absence of typical wall-forming bodies in macrogametocytes and a complete absence of oocysts. It is concluded that clazuril has a coccidiocidal effect on the asexual and sexual developmental stages of both Eimeria species, resulting in a complete interruption of the life cycle.

To evaluate in vitro the antiprotozoal and cytotoxic activies of extracts obtained from leaves,stem bark and root bark of Vismia guineensis. For each plant party, three types of extracts (chloroform, ethyl acetate and ethanol 70%) were prepared by maceration in accordance with the increasing polarity of these solvents. Extracts denoted as Vgl1, Vgl2, Vgl3 for leaves, Vgb1 Vgb2, Vgb3 for stem bark, Vgr1,Vgr2, Vgr3 for root bark. These plant extracts were tested in vitro against Trypanosoma brucei brucei, Trypanosoma cruzi, Leishamania infantum, chloroquine-sensitive Ghanaian strain of Plasmodium falciparum , and MRC-5 cell lines. The most pronounced antiprotozoal activities and cytotoxicity were observed of apolar extracts from leaves, stem bark and root bark. Five extracts: Vgl2, Vgb1, Vgb2, Vgr1, Vgr2 exhibited pronounced inhibitory activity (IC5 against at least one selected protozoa. Ethyl acetate extracts from leaves (Vgb2) and stem bark (Vgb2) were found to exhibit pronounced and good antiprotozoal activity against Trypanosoma sp with IC50 values Vgl2 : 6.80 (T. brucei􀀂􀀝􀀂􀀵􀀫􀀂􀀔􀀐􀀒􀀛􀀋􀀎􀀂􀀔􀀐􀀒􀀗􀀂􀀊T.cruzi􀀂􀀝􀀂 SI 6,97), Vgt2: 6.75 (T. brucei􀀂􀀝􀀂􀀵􀀫􀀂􀀓􀀐􀀓􀀗􀀋􀀎􀀂􀀔􀀐􀀓􀀖􀀂􀀊T. cruzi 􀀝􀀂􀀵􀀫􀀂􀀖􀀐􀀒􀀓􀀋􀀐 Extract from stem bark (Vgt1) showed ytotoxicity Plasmodium falciparum IC50 Phytochemical investigations led to the isolation anthraquinone type compounds from these, 3-geranyloxy-6- methyl 1,8 dihydroxyanthraquinone, 3-O-(E-3-hydroxymethylbut-2 enyl)-emodin, 3-O-(3-hydroxymethyl-4- hydroxybut-2-enyl)-emodin. These results can partly support the traditional use of Vismia guineensis for the treatment of parasitic diseases.

A series of twelve analogs carrying fluoro, chloro, bromo and iodo halogens on the ortho, meta and para positions of a benzoyloxytropane skeleton were synthesized by a simple acylation of 8-methyl-8-aza-bicyclo[3.2.1]octan- 3α-ol by halogenobenzoyl chlorides. The compounds were evaluated in vitro against Plasmodium falciparum (P. f.), Trypanosoma brucei brucei (T. b. b.), Trypanosoma cruzi (T. c.) and Leishmania infantum (L. i.). This study shows that the presence of a halogenated atom and its position on the aromatic ring are important for in vitro activity. Compounds 4 (IC50 = 3.6 µM), 8 (IC50 = 6.7 µM), 5 (IC50 = 8.1 µM) and 7 (IC50 = 9.5 µM) were found the most active against P. f., whereas compounds 12 (IC50 = 5.1 µM), 11 (IC50 = 5.6 µM) and 9 (IC50 = 5.8 µM) exhibited the most pronounced activity against T. b. b. This series of compounds can be considered as non-toxic to the human cell line MRC-5.

Background Worldwide particularly in developing countries, a large proportion of the population is at risk for tropical parasitic diseases. Several medicinal plants are still used traditionally against protozoal infections in Yemen and Saudi Arabia. Thus the present study investigated the in vitro antiprotozoal activity of twenty-five plants collected from the Arabian Peninsula. Methods Plant materials were extracted with methanol and screened in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxic activity was determined against MRC-5 cells to assess selectivity. The criterion for activity was an IC50 T. brucei) and selectivity index of >4. Results Antiplasmodial activity was found in the extracts of Chrozophora oblongifolia, Ficus ingens, Lavandula dentata and Plectranthus barbatus. Amastigotes of T. cruzi were affected by Grewia erythraea, L. dentata, Tagetes minuta and Vernonia leopoldii. Activity against T. brucei was obtained in G. erythraea, L. dentata, P. barbatus and T. minuta. No relevant activity was found against L. infantum. High levels of cytotoxicity (MRC-5 IC50 Cupressus sempervirens, Kanahia laniflora and Kniphofia sumarae. Conclusion The results endorse that medicinal plants can be promising sources of natural products with antiprotozoal activity potential. The results support to some extent the traditional uses of some plants for the treatment of parasitic protozoal diseases.

Two new hygroline and tropane alkaloids, 4-hydroxybenzenepropanoylhygroline (1) and 3 alpha,4 beta-dihydroxy-6 beta-angeloyoxytropane (2) have been isolated from the aerial parts of Schizanthus hookeri and S. tricolor, respectively, two plants indigenous from Chile. Their structures were elucidated by spectroscopic methods and high resolution mass spectrometry. Their antiparasitic activity and cytotoxicity were measured.

Five methanol extracts obtained from five plant species selected according ethnobotanical data were screened for their antiprotozoal activity against Plasmodium falciparum, Leishmania infantum, Trypanosoma cruzi and Trypanosoma brucei. Cytotoxicity was evaluated on MRC-5 cells. Withania somnifera methanol extract showed low activity against P. falciparum (IC50 17.7 μg ml-1), but with non-selective antimalarial activity. Most of the extracts tested against L. infantum exhibited low activity with IC50 ranging from 20 to 32 μg ml-1, except W. somnifera which showed a remarkable but non-selective leishmanicidal activity (IC50 of 2.0 μg ml-1). W. somnifera methanol extract also exhibited a marked activity against T cruzi (IC50 0.6 μg ml-1, SI=3.5).

SUMMARYLeishmaniaparasites are able to survive in the macrophage, one of the most hostile environments of the vertebrate host. The present study investigated howLeishmaniainfection influences these host cell defence mechanisms. Macrophages were infected with antimony-susceptible and -resistantLeishmaniastrains. Free radical production inLeishmania-infected macrophages was measured by electron paramagnetic resonance. Apoptosis was detected with fluorescence microscopy using Annexin-V FITC labelling and with Western blotting to detect caspase-3 cleavage. Independent of their drug susceptibility profile or species background, all studiedLeishmaniastrains induced a similar increase in free radical production in macrophages. O2●−production was significantly elevated during phagocytosis of the stationary phase promastigotes. Conversely, NO levels increased later in the infection and none of the strains induced capsase-3 cleavage.Leishmania donovaniinfection led to phosphatidylserine externalization only in RAW 264.7 cells. After an initial burst of O2●−during phagocytosis of promastigotes, amastigotes protect themselves by decreasing the O2●−production to the basal level. An increased NO production was observed 6 h after infection. Finally, induction of cell death is probably not essential in the survival of the parasite within the macrophage.

Diclazuril, a new benzeneacetonitrile anticoccidial, has potent activity against various stages of Eimeria tenella. A single treatment of experimentally infected chickens during the prepatent phase (up to day 5) results in a complete interruption of the life cycle and oocyst shedding. The first- and second-generation schizonts show extensive degenerative changes that finally result in a complete loss of the parasitic stage. The degeneration is characterized by loss of internal structure, the appearance of many intracytoplasmic vacuoles, and incomplete merogony. The merozoites themselves show similar degenerative changes, including the presence of numerous small vacuoles in the cytoplasm. Diclazuril is also effective against both the micro- and macrogametocytes that have a ballooned appearance and loose their internal structure completely. In the macrogametocytes, wall-forming bodies either do not develop or disappear rapidly. Development of typical caecal lesions is prevented when treatment with diclazuril is initiated before large numbers of second-generation schizonts appear, i.e., day 3. It is concluded that diclazuril is lethal against both the asexual and the sexual stages of E. tenella. At the proposed use level of 1 ppm in the feed, the life cycle is interrupted at a very early stage and lesion development and oocyst shedding are completely prevented.