Parasite prolyl oligopeptidases and the challenge of designing chemotherapeuticals for Chagas disease, leishmaniasis and African trypanosomiasis.

The trypanosomatids Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp. cause Chagas disease, leishmaniasis and human African trypanosomiasis, respectively. It is estimated that over 10 million people worldwide suffer from these neglected diseases, posing enormous social and economic problems in endemic areas. There are no vaccines to prevent these infections and chemotherapies are not adequate. This picture indicates that new chemotherapeutic agents must be developed to treat these illnesses. For this purpose, understanding the biology of the pathogenic trypanosomatid- host cell interface is fundamental for molecular and functional characterization of virulence factors that may be used as targets for the development of inhibitors to be used for effective chemotherapy. In this context, it is well known that proteases have crucial functions for both metabolism and infectivity of pathogens and are thus potential drug targets. In this regard, prolyl oligopeptidase and oligopeptidase B, both members of the S9 serine protease family, have been shown to play important roles in the interactions of pathogenic protozoa with their mammalian hosts and may thus be considered targets for drug design. This review aims to discuss structural and functional properties of these intriguing enzymes and their potential as targets for the development of drugs against Chagas disease, leishmaniasis and African trypanosomiasis.

[Effect of serum on the outcome of in vitro testing of the chemosensitivity of Plasmodium falciparum]. / Influence de la nature du sérum sur la réalisation de test de chimiosensibilité in vitro de Plasmodium falciparum.

Plasmodium falciparum, the parasite responsible for potentially fatal malaria, is a major cause of morbidity and of mortality in tropical countries. In vitro culture of Plasmodium remains indispensable for identification of phenotype and surveillance of the efficacy of antimalarial drugs. Culture of Plasmodium falciparum requires the use of RPMI 1640 medium, the efficacy of which depends on addition of human serum. Culture with medium containing either reference human serum of (RS) or serum containing Plasmodium antibodies (SND) provides a plasmodial maturation rate exceeding 20% (lower threshold of validity according to WHO). Comparison of these two culture supplements for in vitro testing of the chemosensitivity of Plasmodium strains to pyrimethamine showed no difference in IC50: < 2000 nM for sensitive strains (FCB1 and PFB) and > 2000 nM for resistant strains (K1).

Synthesis and antimalarial activity of new amino analogues of amodiaquine.

Amodiaquine remains one of the most prescribed antimalarial 4-aminoquinoline. To assess the importance of the 4'-hydroxyl group and subsequent hydrogen bond in the antimalarial activity of amodiaquine (AQ), a series of new analogues in which this functionality was replaced by various amino groups was synthesized. The incorporation of a 3'-pyrrolidinamino group instead of the 3'-diethylamino function of AQ allowed the development of a parallel series of amopyroquine derivatives. The compounds were screened against both chloroquine (CQ)-sensitive and -resistant strains of Plasmodium falciparum and their cytotoxicity evaluated upon the MRC5 cell line. Antimalarial activity in a low nanomolar range was recorded showing that the 4'-hydroxy function can be successfully replaced by various amino substituents in terms of activity without any influence of the level of CQ-resistance of the strains. Furthermore the ability of the compounds to inhibit beta-hematin formation was measured in order to discuss the mechanism of action of these new compounds. Compounds 7d and 8d exhibit a high selectivity index and may be considered as promising leads for further development.

Discovery of new targets for antimalarial chemotherapy.

The understanding of the biology and the biochemistry of malaria parasites has considerably increased over the past two decades with the discovery of many potential targets for new antimalarial drugs. The decrypted genomes of several Plasmodium species and the new post-genomic tools further enriched our "reservoir" of targets and increased our ability to validate potential drug targets or to study the entire parasite metabolism. This review discusses targets involved in calcium metabolism, protein prenylation and apicoplast functions that have emerged by different approaches.

In vitro antiplasmodial activity of Brazilian Cerrado plants used as traditional remedies.

Twenty-seven species of native Brazilian Cerrado plants commonly used by traditional healers to treat malaria and other diseases were collected and 204 hexanic and ethanolic extracts were obtained by maceration. The antiplasmodial activity of the extracts was tested in vitro against a chloroquine resistant strain (FcB1) of Plasmodium falciparum, and cytotoxicity against the cell lines L-6 of rats and MRC-5 of human was evaluated. Thirty-two extracts showed significant inhibition rates of Plasmodium falciparum growth and of these six showed cytotoxicity against the cell lines. The strongest antiplasmodial activity was found for the hexanic extracts of Xylopia aromatica root wood (IC(50)=4.7 microg/ml), Xylopia emarginata root bark (IC(50)=4.9 microg/ml), Casearia sylvestris var. lingua leaves, stem wood and stem bark, and root wood and root bark (IC(50) values from 0.9 to 2.3 microg/ml), and Cupania vernalis leaves (IC(50)=0.9 microg/ml); and for the ethanolic extract of Aspidosperma macrocarpon root bark (IC(50)=4.9 microg/ml). However, the best selectivity towards Plasmodium falciparum was observed for the hexanic root bark extract of Matayba guianensis (IC(50) on Plasmodium falciparum=6.1 microg/ml, SI=16.4 for MRC-5) and the ethanolic root bark extract of Aspidosperma macrocarpon (IC(50) on Plasmodium falciparum=4.9 micro/ml, SI=16.2 for MRC-5).

In vitro and in vivo antiplasmodial activity of Momordica balsamina alone or in a traditional mixture.

BACKGROUND: Because of the dramatic situation of malaria in Africa, there is an urgent need to find new and cheap drugs, such as herbal medicines. Here we report the study of the in vitro and in vivo antimalarial activity of Momordica balsamina alone or in a traditional mixture used in Niger. METHODS: Extracts were obtained with different solvents and tested in vitro on Plasmodium falciparum and in vivo on Plasmodium vinckei. RESULTS: The best extracts are methanolic and present promising results in vivo by intraperitoneal and oral administration. CONCLUSION: The antimalarial activity of M. balsamina, traditionally used in Niger, is confirmed in vitro and in vivo without any toxicity in healthy mice.

[Stabwound of the cervical spinal cord. Two case reports]. / Plaie médullaire cervicale par arme blanche. Présentation de deux cas.

Two cases of Brown-Sequard syndrome following a stab wound of the cervical spinal cord are reported. Spinal cord hemisection was confirmed by magnetic resonance imaging and surgical exploration. Both patients presented leakage of the cerebrospinal fluid and underwent surgical repair. In the first case, the pia-mater was sutured to close the wound and decrease the risk of post-traumatic syringomyelia. Outcome at ten and two years follow up was good in both patients who were able to walk. One of them returned to work. The contribution of surgical repair of spinal cord stab wounds and mechanisms of recovery are discussed.

Design, synthesis and antimalarial activity of a glyoxylylhydrazone library.

Synthesis of a new family of quinolylhydrazone derivatives and evaluation of their activity against a chloroquine-resistant strain of Plasmodium falciparum are described. The best compound displayed an activity 6-fold higher than chloroquine. None of the active compounds were found to inhibit beta-hematin formation in vitro in the same range as chloroquine and five among them displayed lower calculated vacuolar accumulation ratios, suggesting the implication of a different mechanism of action.

[Neoadjuvant chemotherapy for symptomatic non operable grade II fibrillary astrocytoma in adults]. / Chimiothérapie néoadjuvante dans les astrocytomes fibrillaires de grade II symptomatiques non opérables de l'adulte.

We collected 6 case-reports of symptomatric non removable low grade fibrillary astrocytoma of adults treated with a procarbazine-CCNU-vincristine chemotherapy regimen. All patients had drug-resistant epilepsy but brain imaging was stable. Total gross resection was rejected because of Volume or tumor location. After 4 to 7 cycles of chemotherapy, 2 patients had partial response and one minor response on brain MRI. All of them were seizure-free. Progression free survival was not reached at 5 Years. Up-front chemotherapy for low-grade astrocytomas may be useful and has to be prospectively evaluated.

Antiparasitic activities of medicinal plants used in Ivory Coast.

During an ethnopharmacological survey of antiparasitic medicinal plants used in Ivory Coast, 17 plants were identified and collected. Polar, non-polar and alkaloidic extracts of various parts of these species were evaluated in vitro in an antiparasitic drug screening. Antimalarial, leishmanicidal, trypanocidal, antihelminthiasis and antiscabies activities were determined. Among the selected plants, Anogeissus leiocarpus and Terminalia glaucescens were strongly active against Plasmodium falciparum. Lawsonia inermis, selectively prescribed against trypanosomiasis shows interesting trypanocidal activities as did other 15 plants. Anthelmintic activities were found for 10 active species and 2 species (Uvaria afzelli and Monodora myristica) were actives against mites.

Parallel synthesis and anti-malarial activity of a sulfonamide library.

Solution-phase synthesis and evaluation of a library of 31 sulfonamides as inhibitors of a chloroquine-resistant strain of Plasmodium falciparum are described. The most potent compound displayed an activity 100-fold better than chloroquine. Experiments using a fluorescent sulfonamide derivative suggest that their site of action inside the parasite is different to that of chloroquine.

The IGF system in neuroblastoma xenografts: focus on IGF-binding protein-6.

With a view to investigating the implication of IGF-binding protein-6 (IGFBP-6) in the growth of neuroblastomas, nude mice were injected with IGFBP-6-expressing or control IGR-N-91 human neuroblastoma cells and the resulting xenografts examined. Expression of IGFBP-3, IGFBP-4 and type 1 and type 2 IGF receptor messengers was similar in control tumours and equal-sized IGFBP-6-expressing tumours that had developed. IGF-II was more strongly expressed in control tumours, and IGFBP-6-expressing tumours contained less IGFBP-2 than controls. In both populations, there was a significant positive correlation between IGF-II and IGFBP-2 expression. In small IGFBP-6-expressing xenografts where tumour development had apparently been arrested, haematoxylin--eosin and TUNEL staining revealed numerous apoptotic cells. In situ hybridization indicated homogeneous distribution of the IGFBP-6 signal in test tumours. In cell culture, IGFBP-6-expressing cells expressed similar amounts of IGFBP-2, IGF-II and N-myc mRNAs as control cells; but media conditioned by IGFBP-6-expressing cells contained less intact IGFBP-2 protein, with no increase in its proteolytic fragment. In media treated with plasminogen, in which IGFBP-2 was proteolysed, IGFBP-6 was increased. With its especially strong affinity for IGF-II and its resistance to proteolysis, IGFBP-6 would act by sequestering IGF-II, hence inhibiting its mitogenic and anti-apoptotic effects. In excess, IGFBP-6 would displace IGF-II from IGFBP-2 whose potentiation of IGF-II action would cease and whose susceptibility to degradation would be increased. This study therefore shows that IGFBP-6 plays a role in neuroblastoma cell growth in vivo and in vitro and that stable overexpression of IGFBP-6 leads to alteration of the initial balance between the IGFBPs.

A prodrug form of a Plasmodium falciparum glutathione reductase inhibitor conjugated with a 4-anilinoquinoline.

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.

Trypanosoma cruzi prolyl oligopeptidase Tc80 is involved in nonphagocytic mammalian cell invasion by trypomastigotes.

Trypanosoma cruzi is an intracellular protozoan parasite able to invade a wide variety of mammalian cells. To have access to the target organs/cells, the parasite must cross the basal laminae and the extracellular matrix (ECM). We previously characterized an 80-kDa proteinase (Tc80) secreted by the infective trypomastigotes that hydrolyzes native collagens and might be involved in infection by degrading ECM components. Here, we present evidence indicating a role for Tc80 in the invasion of nonphagocytic cells. Tc80 was classified as a member of the prolyl oligopeptidase (POP) family of serine proteases and was also found to hydrolyze fibronectin. Selective inhibitors for POP Tc80 were synthesized that blocked parasite entry into cells. Blockage occurred when trypomastigotes were preincubated with irreversible inhibitors but not after host cell preincubation, and the blockage correlated with inhibition of POP Tc80 activity in treated parasites. These data and the enzyme location inside a vesicular compartment close to the flagellar pocket, a specialized domain in endocytosis/exocytosis, strongly suggest a role for POP Tc80 in the maturation of parasite protein(s) and/or, after secretion, in a local action on parasite or host cell/ECM components required for invasion.

Antiplasmodial activity of nitroaromatic and quinoidal compounds: redox potential vs. inhibition of erythrocyte glutathione reductase.

Prooxidant nitroaromatic and quinoidal compounds possess antimalarial activity, which might be attributed either to their formation of reactive oxygen species or to their inhibition of antioxidant enzyme glutathione reductase (GR, EC 1.6.4.2). We have examined the activity in vitro against Plasmodium falciparum of 24 prooxidant compounds of different structure (nitrobenzenes, nitrofurans, quinones, 1,1'-dibenzyl-4,4'-bipyridinium, and methylene blue), which possess a broad range of single-electron reduction potentials (E(1)(7)) and erythrocyte glutathione reductase inhibition constants (K(i(GR))). For a series of homologous derivatives of 2-(5'-nitrofurylvinyl)quinoline-4-carbonic acid, the relationship between compound K(i(GR)) and concentration causing 50% parasite growth inhibition (IC(50)) was absent. For all the compounds examined in this study, the dependence of IC(50) on their K(i(GR)) was insignificant. In contrast, IC(50) decreased with an increase in E(1)(7) and positive electrostatic charge of aromatic part of molecule (Z): log IC(50) (microM) = -(0.9846 +/- 0.3525) - (7.2850 +/- 1.2340) E(1)(7) (V) - (1.1034 +/- 0.1832) Z (r(2) = 0.8015). The redox cycling activity of nitroaromatic and quinoidal compounds in ferredoxin:NADP(+) reductase-catalyzed reaction and the rate of oxyhemoglobin oxidation in lysed erythrocytes increased with an increase in their E(1)(7) value. Our findings imply that the antiplasmodial activity of nitroaromatic and quinoidal compounds is mainly influenced by their ability to form reactive oxygen species, and much less significantly by the GR inhibition.

Synthesis and in vitro and in vivo antimalarial activity of new 4-anilinoquinolines.

A new series of 4-anilinoquinolines with two proton-accepting side chains has been synthesized. Antimalarial activity and levels of cytotoxicity upon both MRC-5 cells and macrophages were found to be highly dependent upon the features of these side chains. Several compounds were found to be active in the low nanomolar range, against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum in vitro. From among them, a morpholino derivative cured mice infected by Plasmodium berghei and displayed a lower toxicity than amodiaquine upon mouse macrophages.

Antimalarial in-vivo activity of bis(9-amino-6-chloro-2-methoxyacridines).

In the fight against malaria, chemotherapy using bisacridines may represent an alternative method to overcoming chloroquine-resistance. Eight bis(9-amino-6-chloro-2-methoxyacridines), in which acridine moieties were linked by polyamines substituted with a side chain, were tested for their in-vivo activity upon mice infected by Plasmodium berghei. Three of the compounds revealed antimalarial activity but no relationship could be deduced from a comparison of in-vitro and in-vivo activities. N-alkylation of the central amino group generated toxicity and, therefore, only compounds N-acylated in this position can be selected as leads.

"One-pot" synthesis and antimalarial activity of formamidine derivatives of 4-anilinoquinoline.

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.

[Factors influencing recurrence and role of radiotherapy in filum terminale ependymomas. 14 cases and review of the literature]. / Analyse des facteurs de récidive et rôle de la radiothérapie dans les épendymomes du filum terminale. A propos d'une série de 14 cas et revue de la littérature.

PURPOSE: We reviewed a series of 14 cases of filum terminale ependymoma and 264 cases in the literature, to study the characteristics of these tumors and specifically to determine factors influencing recurrence. MATERIAL AND METHODS: This series analyzed data between 1984 and 1998. The mean follow-up period was 5.5 years and the median age 40 years. Pain was the first symptom, except in one case, with progressive sensitive-motor deficit. Four patients had a myelography and a CT scan, and ten a MRI. All patients were operated with a total resection in 12 cases. All tumors, except one, were a myxopapillary ependymoma. RESULTS: Clinical results were excellent in 2 cases, good in 8, stable in 2 and worse in 2. Two patients had a recurrence after an initial sub-total resection, both were operated on again, followed by post-operative radiotherapy. Among the 278 filum terminale ependymoma, removal was total in 200 (72%). A recurrence occurred in 15% of them after total removal, and in 43% after partial removal (p<0.001). Among patients with partial removal, recurrence was observed in 33% of them if they had post-operative radiotherapy, and in 55% of them if they did not have post-operative radiotherapy (p<0.05). CONCLUSION: The extent of tumor removal has a statistically significant effect upon recurrence. This review is in favor of post-operative radiotherapy in case of partial removal, but this systematic attitude can be discussed after a critical analysis of this study. Long term follow-up is mandatory due to the possibility of late recurrence.

IGFBPs are involved in xenograft development in nude mice.

BACKGROUND: The insulin-like growth factors (IGFs) are involved in the growth and differentiation of neuroblastoma cells. In all biological fluids, they are non-covalently bound to high-affinity binding proteins (IGFBP-1 to -6) which modulate their bioavailability. We previously showed that IGFBP-6 expression is linked to the arrest of growth in neuroblastoma cells, whereas IGFBP-2 is associated with proliferation. PROCEDURE: To study the role of IGFBP-6 in cell growth, we stably IGR-N-91 neuroblastoma cells with a plasmid containing sequences coding for IGFBP-6 under the control of the cytomegalovirus (CMV) promoter. RESULTS: The incidence and size of tumors generated by injecting IGFBP-6-expressing cells into nude mice were reduced by factors of 2 and 5, respectively, as compared with those generated by injection by control cells. Northern blot analyses if xenografts revealed weaker expression of IGF-II, type 2 IGF receptor and IGFBP-2 mRNAs in IGFBP-6-expressing cthan in control xenografts. IGFBP-6 may therefore reduce the expression of IGF-II (which induces tumour development) at a transcriptional level. Conversely, containing IGFBP-2 cDNA under the control of CMV promoter grew three to four times as fast as normal control xenografts. Northern blot analyses revealed weaker expression of intact IGFBP-3 and IGFBP-1 in IGFBP-2-expressing than in control xenografts. CONCLUSIONS: IGFBP-1 and intact IGFBP-3 expression both enhance IGF bioavailability which promotes tumour growth. Although the mechanisms of action of IGFBP-2 and IGFBP-6 remain to be elucidated, an inverse relationship appears to exist between the two binding proteins, IGFBP-2 being involved in proliferation and IGFBP-6 in its arrest.