Evaluating drug resistance in visceral leishmaniasis: the challenges.

For decades antimonials were the drugs of choice for the treatment of visceral leishmaniasis (VL), but the recent emergence of resistance has made them redundant as first-line therapy in the endemic VL region in the Indian subcontinent. The application of other drugs has been limited due to adverse effects, perceived high cost, need for parenteral administration and increasing rate of treatment failures. Liposomal amphotericin B (AmB) and miltefosine (MIL) have been positioned as the effective first-line treatments; however, the number of monotherapy MIL-failures has increased after a decade of use. Since no validated molecular resistance markers are yet available, monitoring and surveillance of changes in drug sensitivity and resistance still depends on standard phenotypic in vitro promastigote or amastigote susceptibility assays. Clinical isolates displaying defined MIL- or AmB-resistance are still fairly scarce and fundamental and applied research on resistance mechanisms and dynamics remains largely dependent on laboratory-generated drug resistant strains. This review addresses the various challenges associated with drug susceptibility and -resistance monitoring in VL, with particular emphasis on the choice of strains, susceptibility model selection and standardization of procedures with specific read-out parameters and well-defined threshold criteria. The latter are essential to support surveillance systems and safeguard the limited number of currently available antileishmanial drugs.

Preclinical safety and tolerability of a repeatedly administered human leishmaniasis DNA vaccine.

The leishmaniases are a complex of vector-borne diseases caused by protozoan parasites of the genus Leishmania. LEISHDNAVAX is a multi-antigen, T-cell epitope-enriched DNA vaccine candidate against human leishmaniasis. The vaccine candidate has been proven immunogenic and showed prophylactic efficacy in preclinical studies. Here, we describe the safety testing of LEISHDNAVAX in naive mice and rats, complemented by the demonstration of tolerability in Leishmania-infected mice. Biodistribution and persistence were examined following single and repeated intradermal (i.d.) administration to rats. DNA vectors were distributed systemically but did not accumulate upon repeated injections. Although vector DNA was cleared from most other tissues within 60 days after the last injection, it persisted in skin at the site of injection and in draining lymph nodes. Evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after i.d. administration to naive, non-infected mice did not reveal any safety concerns. LEISHDNAVAX was also well tolerated in Leishmania-infected mice. Taken together, our results substantiate a favorable safety profile of LEISHDNAVAX in both naive and infected animals and thus, support the initiation of clinical trials for both preventive and therapeutic applications of the vaccine.

Kinetoplastida: new therapeutic strategies.

New formulations and therapeutic switching of the established drugs, amphotericin B and paromomycin, together with the discovery of miltefosine, have significantly improved the opportunities for treatment of visceral leishmaniasis (VL) chemotherapy. However, for human African trypanosomiasis (HAT), Chagas disease and cutaneous leishmaniases there has been limited progress. For HAT, a novel diamidine, parfuramidine, is in phase III clinical trial for early-stage disease, but for the treatment of late-stage disease there are no new drugs and combinations of eflornithine with melarsoprol or nifurtimox have been the focus of clinical studies. For Chagas disease, different classes of compounds that have validated biochemical targets, sterol biosynthesis methylases and cysteine proteases, are in various stages of development. The genome sequences that are now available for the pathogens that cause the leishmaniases and trypanosomiases, and new methods for rapid validation of targets, are part of the solution to discover new drugs. The integration of medicinal chemistry, pharmacokinetics, project planning and interaction with the pharma/biotech sector are essential if progress is to be made. Although there are financial constraints, the appearance of new funding sources and not-for-profit product development partnerships offers hope for drug development.

Transmission and scanning EM-immunogold labeling of Leishmania major lipophosphoglycan in the sandfly Phlebotomus papatasi.

Previous studies using immunostaining and light microscopy demonstrated expression of Leishmania major lipophosphoglycan (LPG) on parasites developing in the sandfly gut from 2 days post infection. By days 4 to 7 post infection, there appeared to be large amounts of parasite-free LPG deposited on/in the microvilli and epithelial cells lining the thoracic midgut, while forward migration of parasites and the morphological changes which accompany metacyclogenesis were associated with developmental modification of the LPG molecules. Studies presented here examine this process with much greater precision using electron microscopy and immunogold labeling techniques to study the different developmental forms (nectomonads, haptomonads, paramastigotes, and metacyclics) of promastigotes in the sandfly gut. Results obtained using LPG-specific monoclonal antibodies (WIC79.3, 45D3 and the metacyclic-specific 3F12) show (1) gold labeling over the cell surface, within the flagellar pocket, and extending along the entire length of the flagellum of electron-dense nectomonads observed in the abdominal and thoracic midgut regions on days 4 and 7 post infection, and of electron-lucid haptomonads in the foregut, (2) dense labeling around the flagellar tips, by which nectomonad forms bind to the midgut microvilli, but not on the microvilli themselves or within the epithelial cells lining the midgut, (3) significant metacyclic-specific (3F12) labeling on nectomonad forms in the lumen of the midgut and attached to the microvilli, and (4) dense labeling on the cell surface of electron-lucid paramastigotes in the esophagus and in the filamentous matrix surrounding paramastigote and metacyclic forms in the esophagus and pharynx. These results are discussed in the light of the proposed roles for LPG in parasite attachment to, and survival in, the sandfly gut.

The farnesyltransferase inhibitor manumycin A is a novel trypanocide with a complex mode of action including major effects on mitochondria.

Eukaryotes modify numerous proteins, including small GTPases of the ras superfamily, with isoprenes as a mechanism for membrane attachment. Inhibition of farnesylation of ras has been successfully exploited to control cell growth, with promise in the clinic for treatment of human tumours. Using an in vitro screen of mammalian farnesyltransferase inhibitors, we have identified manumycin A as potently active against growth of both bloodstream and procyclic forms of Trypanosoma brucei. Other structural classes of farnesyltransferase inhibitors were far less effective. Exposure of T. brucei for brief periods to lethal concentrations of manumycin A resulted in subsequent cell death whilst the concentration required to achieve killing was dependent on serum concentration, suggesting partitioning of manumycin A into hydrophobic cellular sites. Manumycin A did not affect trypanosomal protein and DNA synthesis or cell cycle progression but altered incorporation of prenyl groups into several polypeptides indicating a specific effect on the prenylation without effect on other mevalonate pathway products, most importantly prenyl pyrophosphate levels. Morphological analysis indicated that manumycin A caused significant mitochondrial damage suggesting an additional site of action. Structural analogues of manumycin A containing a quinone were also highly trypanocidal and altered mitochondrial morphology, suggesting interference with electron/proton transport systems. Furthermore, manumycin A also elicited mitochondrial alterations in mammalian cells indicating that the effect is not confined to lower eukaryotes. Manumycin A is well tolerated in vivo but failed to cure experimental trypanosomiasis in mice.

Proton NMR lipid profile of Leishmania donovani promastigotes.

The proton nuclear magnetic resonance (NMR) lipid profile of Leishmania donovani was obtained in the one-dimensional and two-dimensional modes. Partial assignments of lipid classes and individual lipids were obtained purely from the proton NMR spectrum of the mixture. A more complete assignment and quantitative analysis was achieved by prior separation of the lipids by high pressure liquid chromatography (HPLC) followed by proton NMR analysis of the fractions. This work showed that proton NMR spectroscopy could facilitate lipid analysis and classification of various parasitic protozoa and serve as a basis for rapid studies of comparative lipid metabolism in parasites.

QSAR study on the contribution of log P and E(s) to the in vitro antiprotozoal activity of glutathione derivatives.

A series of N-S-blocked glutathione monoester and diester derivatives based on N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)glutathione were evaluated for activity against the pathogenic parasites Trypanosoma brucei brucei, Trypanosoma cruzi, and Leishmania donovani in vitro.Only monoesters 7-9 with a log P value of >2.7 were active inhibitors of T.b. brucei bloodstream form trypomastigotes. Diester compounds 10-15 and 17-27 in most cases were better inhibitors of T.b. brucei than monoester compounds, and some displayed high activity against T. cruzi 14 and L. donovani 17, 19, 29. Compounds 14, 24, and 25 were the most active compounds identified against T.b. brucei having ED(50) values of <0.4 microM. Analysis of the inhibition data (ED(50)) vs calculated log P and E(s) values provided evidence to support membrane penetration and steric factors as the key component in the activity of these compounds. The optimum values for log P and E(s) determined were 5.8 and -0.70, respectively. A QSAR equation relating log(1/ED(50)) vs log P and E(s) was determined and interpreted within the proposed mechanism of activity for these compounds.

Use of an additional hydrophobic binding site, the Z site, in the rational drug design of a new class of stronger trypanothione reductase inhibitor, quaternary alkylammonium phenothiazines.

Improved rationally designed lead drug structures against African trypanosomiasis, Chagas disease, and leishmaniasis were obtained against trypanothione reductase from Trypanosoma cruzi. Substituted-benzyl [3-(2-chloro-4a, 10a-dihydrophenothiazin-10-yl)propyl]dimethylammonium salts, synthesized by Menschutkin quaternization of the tertiary alkylamine omega-nitrogen atom of chlorpromazine, were linear, competitive inhibitors of recombinant trypanothione reductase from T. cruzi, with either trypanothione disulfide or N-benzyloxycarbonyl-L-cysteinylglycyl 3-dimethylaminopropylamide disulfide as substrate. The permanent positive charge on the distal nitrogen atom of the tricyclic side chain contribution to binding was estimated as >/=5.6 kcal.mol(-1) by comparison with the analogue with the cationic nitrogen atom of the quaternary replaced by an ether oxygen atom. A further major contribution to improving K(i) values and inhibition strength was the hydrophobic natures and structures of the N-benzyl substituents. The strongest inhibitor, the [3-(2-chloro-4a,10a-dihydrophenothiazin-10-yl)propyl](3, 4-dichlorobenzyl)dimethylammonium derivative (K(i) 0.12 microM), was approximately 2 orders of magnitude more inhibitory than the parent chlorpromazine. Several of these quaternary phenothiazines completely inhibited T. brucei parasite growth in vitro at <1 microM. Antiparasite activity was not solely determined by inhibition strength against trypanothione reductase, there being a strong contribution from hydrophobicity (for example, benzhydryl-quaternized chlorpromazime had ED(50) < 1 microM). Although active against Leishmania donovani, none of the analogues showed major improvement in this activity relative to chlorpromazine or other nonquaternized phenothiazines. The p-tert-butylbenzyl-quaternized analogue very strongly inhibited (ED(50) < 1 microM) growth of the amastigote stage of T. cruzi.

Cytotoxicity of (2,2':6',2''-terpyridine)platinum(II) complexes to Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei.

A range of (2,2':6',2''-terpyridine)platinum(II) complexes are shown to possess antiprotozoal activity in vitro against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei,the causative organisms of tropical diseases leishmaniasis and trypanosomiasis. The best compounds caused 100% and 78% inhibition of growth of the intracellular amastigote forms of L. donovani and T. cruzi, respectively, at a concentration of 1 microM and 100% inhibition of growth of the bloodstream trypomastigote forms of T. brucei at a concentration of 0.03 microM. The results obtained with complexes in which the fourth ligand to platinum(II) is capable of being substituted with a substitution inert hydroxyethanethiolate complex are compared. The ammine complexes show high antiprotozoal activity suggesting that the trans influence of the 2,2':6',2''-terpyridine ligand has a profound effect on the ease of displacement of the fourth ligand in (2,2':6',2'' -terpyridine)platinum(II) complexes, although nonbonded interaction between the ammine ligand and the 6 and 6' ' hydrogens probably also weakens the ligation to Pt(II).

Structure-activity relationships for the antileishmanial and antitrypanosomal activities of 1'-substituted 9-anilinoacridines.

Members of the class of 9-anilinoacridine topoisomerase II inhibitors bearing lipophilic electron-donating 1'-anilino substituents are active against both the promastigote and amastigote forms of the parasite Leishmania major. A series of analogues of the known 1'-NHhexyl lead compound were prepared and evaluated against L. major in macrophage culture to further develop structure-activity relationships (SAR). Toxicity toward mammalian cells was measured in a human leukemia cell line, and the ratio of the two IC50 values (IC50(J)/IC50(L)) was used as a measure of the in vitro therapeutic index (IVTI). A 3,6-diNMe2 substitution pattern on the acridine greatly increased toxicity to L. major without altering mammalian toxicity, increasing IVTIs over that of the lead compound. The 2-OMe, 6-Cl acridine substitution pattern used in the antimalarial drug mepacrine also resulted in potent antileishmanial activity and high IVTIs. Earlier suggestions of the utility of 2'-OR groups in lowering mammalian cytotoxicity were not borne out in this wider study. A series of very lipophilic 1'-NRR (symmetric dialkylamino)-substituted analogues showed relatively high antileishmanial potency, but no clear trend was apparent across the series, and none were superior to the 1'-NH(CH2)5Me subclass. Subsets of the most active 1'-N(R)(CH2)5Me- and 1'-N(alkyl)2-substituted compounds against L. major were also evaluated against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei, but no consistent SAR could be discerned in these physiologically diverse test systems. The present study has confirmed earlier conclusions that lipophilic electron-donating groups at the 1'-position of 9-anilinoacridines provide high activity against L. major, but the SAR patterns observed do not carry over to the other parasites studied.

Phenothiazine inhibitors of trypanothione reductase as potential antitrypanosomal and antileishmanial drugs.

Given the role of trypanothione in the redox defenses of pathogenic trypanosomal and leishmanial parasites, in contrast to glutathione for their mammalian hosts, selective inhibitors of trypanothione reductase are potential drug leads against trypanosomiasis and leishmaniasis. In the present study, the rational drug design approach was used to discover tricyclic neuroleptic molecular frameworks as lead structures for the development of inhibitors, selective for trypanothione reductase over host glutathione reductase. From a homology-modeled structure for trypanothione reductase, replaced in the later stages of the study by the X-ray coordinates for the enzyme from Crithidia fasciculata, a series of inhibitors based on phenothiazine was designed. These were shown to be reversible inhibitors of trypanothione reductase from Trypanosoma cruzi, linearly competitive with trypanothione as substrate and noncompetitive with NADPH, consistent with ping-pong bi bi kinetics. Analogues, synthesized to define structure-activity relationships for the active site, included N-acylpromazines, 2-substituted phenothiazines, and trisubstituted promazines. Analysis of Ki and I50 data, on the basis of calculated log P and molar refractivity values, provided evidence of a specially favored fit of small 2-substituents (especially 2-chloro and 2-trifluoromethyl), with a remote hydrophobic patch on the enzyme accessible for larger, hydrophobic 2-substituents. There was also evidence of an additional hydrophobic enzymic region available to suitable N-substituents of the promazine nucleus. Ki data also indicated that the phenothiazine nucleus can adopt more than one inhibitory orientation in its binding site. Selected compounds were tested for in vitro activity against Trypanosoma brucei, T. cruzi, and Leishmania donovani, with selective activities in the micromolar range being determined for a number of them.

Design, synthesis, and evaluation of inhibitors of trypanosomal and leishmanial dihydrofolate reductase.

This paper concerns the design, synthesis, and evaluation of inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Initially study was made of the structures of the leishmanial and human enzyme active sites to see if there were significant differences which could be exploited for selective drug design. Then a series of compounds were synthesized based on 5-benzyl-2, 4-diaminopyrimidines. These compounds were assayed against the protozoan and human enzymes and showed selectivity for the protozoan enzymes. The structural data was then used to rationalize the enzyme assay data. Compounds were also tested against the clinically relevant forms of the intact parasite. Activity was seen against the trypanosomes for a number of compounds. The compounds were in general less active against Leishmania. This latter result may be due to uptake problems. Two of the compounds also showed some in vivo activity in a model of African trypanosomiasis.

2- and 3-substituted 1,4-naphthoquinone derivatives as subversive substrates of trypanothione reductase and lipoamide dehydrogenase from Trypanosoma cruzi: synthesis and correlation between redox cycling activities and in vitro cytotoxicity.

Trypanothione reductase (TR) is both a valid and an attractive target for the design of new trypanocidal drugs. Starting from menadione, plumbagin, and juglone, three distinct series of 1,4-naphthoquinones (NQ) were synthesized as potential inhibitors of TR from Trypanosoma cruzi (TcTR). The three parent molecules were functionalized at carbons 2 and/or 3 by various polyamine chains. Optimization of TcTR inhibition and TcTR specificity versus human disulfide reductases was achieved with the 3,3'-[polyaminobis(carbonylalkyl)]bis(1,4-NQ) series 19-20, in which an optimum chain length was determined for inhibition of the trypanothione disulfide reduction. The most active derivatives against trypanosomes in cultures were also studied as subversive substrates of TcTR and lipoamide dehydrogenase (TcLipDH). The activities were measured by following NAD(P)H oxidation as well as coupling the reactions to the reduction of cytochrome c which permits the detection of one-electron transfer. For TcTR, 20(4-c) proved to be a potent subversive substrate and an effective uncompetitive inhibitor versus trypanothione disulfide and NADPH. Molecular modeling studies based on the known X-ray structures of TcTR and hGR were conducted in order to compare the structural features, dimensions, and accessibility of the cavity at the dimer interface of TcTR with that of hGR, as one of the putative NQ binding sites. TcLipDH reduced the plumbagin derivatives by an order of magnitude faster than the corresponding menadione derivatives. Such differences were not observed with the pig heart enzyme. The most efficient and specific subversive substrates of TcTR and TcLipDH exhibited potent antitrypanosomal activity in in vitro T. brucei and T. cruzi cultures. The results obtained here confirm that reduction of NQs by parasitic flavoenzymes is a promising strategy for the development of new trypanocidal drugs.

Synthesis and evaluation of cryptolepine analogues for their potential as new antimalarial agents.

The indoloquinoline alkaloid cryptolepine 1 has potent in vitro antiplasmodial activity, but it is also a DNA intercalator with cytotoxic properties. We have shown that the antiplasmodial mechanism of 1 is likely to be due, at least in part, to a chloroquine-like action that does not depend on intercalation into DNA. A number of substituted analogues of 1 have been prepared that have potent activities against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and also have in common with chloroquine the inhibition of beta-hematin formation in a cell-free system. Several compounds also displayed activity against Plasmodium berghei in mice, the most potent being 2,7-dibromocryptolepine 8, which suppressed parasitemia by 89% as compared to untreated infected controls at a dose of 12.5 mg kg(-1) day(-1) ip. No correlation was observed between in vitro cytotoxicity and the effect of compounds on the melting point of DNA (DeltaT(m) value) or toxicity in the mouse-malaria model.

Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy.

We have investigated the effects in vitro of a series of bisphosphonates on the proliferation of Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum. The results show that nitrogen-containing bisphosphonates of the type used in bone resorption therapy have significant activity against parasites, with the aromatic species having in some cases nanomolar or low-micromolar IC(50) activity values against parasite replication (e.g. o-risedronate, IC(50) = 220 nM for T. brucei rhodesiense; risedronate, IC(50) = 490 nM for T. gondii). In T. cruzi, the nitrogen-containing bisphosphonate risedronate is shown to inhibit sterol biosynthesis at a pre-squalene level, most likely by inhibiting farnesylpyrophosphate synthase. Bisphosphonates therefore appear to have potential in treating parasitic protozoan diseases.

Antileishmanial actions of tricyclic neuroleptics appear to lack structural specificity.

There is continuing interest in the antimicrobial effects of tricyclic neuroleptics and antidepressants, particularly against trypanosomatid protozoa, but few studies have attempted to dissociate antiparasitic actions from effects on the central nervous system. In this report, we have measured the antileishmanial potency of a range of structural analogues in this drug group, and conclude that no one structural determinant is critical for activity. Instead, potency may be predicted from an overall determination of the lipophilic and steric components of the structure by a simple combination of two descriptive parameters, valence connectivity and an interactive term.

The activity of alkyl phosphorylcholines and related derivatives against Leishmania donovani.

Several alkyl phosphorylcholines and related derivatives were tested against Leishmania donovani amastigotes in mouse peritoneal macrophages in vitro and ED50 values were determined in the range of 1-12 microM. The three alkyl phosphorylcholines tested against L. donovani in BALB/c mice were active, an ED50 of 12.8 mg/kg/day X 5 was ascertained for one compound, but an alkyl phosphorylethanolamine was inactive.

Nitrofuran drugs as common subversive substrates of Trypanosoma cruzi lipoamide dehydrogenase and trypanothione reductase.

Lipoamide dehydrogenase (LipDH), trypanothione reductase (TR), and glutathione reductase (GR) catalyze the NAD(P)H-dependent reduction of disulfide substrates. TR occurs exclusively in trypanosomatids which lack a GR. Besides their physiological reactions, the flavoenzymes catalyze the single-electron reduction of nitrofurans with the concomitant generation of superoxide anions. Here, we report on the interaction of clinically used antimicrobial nitrofurans with LipDH and TR from Trypanosoma cruzi, the causative agent of Chagas' disease (South American trypanosomiasis), in comparison to mammalian LipDH and GR. The compounds were studied as inhibitors and as subversive substrates of the enzymes. None of the nitrofurans inhibited LipDH, although they did interfere with the disulfide reduction of TR and GR. When the compounds were studied as substrates, T. cruzi LipDH showed a high rate of nitrofuran reduction and was even more efficient than its mammalian counterpart. Several derivatives were also effective subversive substrates of TR, but the respective reaction with human GR was negligible. Nifuroxazide, nifuroxime, and nifurprazine proved to be the most promising derivatives since they were redox-cycled by both T. cruzi LipDH and TR and had pronounced antiparasitic effects in cultures of T. cruzi and Trypanosoma brucei. The results suggest that those nitrofuran derivatives which interact with both parasite flavoenzymes should be revisited as trypanocidal drugs.

In vitro and in vivo activity of amphotericin B-lipid formulations against experimental Trypanosoma cruzi infections.

The activities of four amphotericin B formulations, Fungizone, AmBisome, Amphocil, and Abelcet, were compared in vitro and in vivo against Trypanosoma cruzi infections. In vitro, Fungizone and Amphocil were highly active against T. cruzi Y strain amastigotes in macrophages with 50% effective dose (ED50) values of 0.027-0.028 microg/ml, which were 7-fold and 42-fold more active than AmBisome and Abelcet, respectively. In vitro activities of all formulations against T. cruzi amastigotes in Vero cells were similar, with ED50 values in the range of 2.0-4.2 microg/ml. Acute infections of the T. cruzi Y strain in BALB/c mice were suppressed in all animals by a single 25 mg/kg dose of the liposomal formulation AmBisome. At the same dose, the two other lipid formulations, Amphocil and Abelcet, increased the survival rate but did not suppress infection in all animals.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers for targeted delivery of 8-aminoquinoline antileishmanial drugs.

A challenge to successful chemotherapy of visceral leishmaniasis is the dose-limiting toxicity of antileishmanial agents. One approach to increase the efficacy and reduce the toxicity of these agents is to direct the drug to the phagolysosomes of the reticuloendothelial system (RES) where the leishmanial parasites reside. In this work a series of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-antileishmanial drug conjugates containing lysosomally degradable side chains and with or without sugar targeting moieties were synthesized, characterized and investigated for their in vivo efficacy in mice infected with Leishmania. An 8-aminoquinoline analog, namely 8-[(4-amino-1-methylbutyl)amino]-5-[3,4-dichlorophenoxy]-6-methoxy-4-methylquinoline (NPC1161) was used as a model antileishmanial agent. At 5 mg/kg body weight drug equivalent dose, all HPMA copolymer-drug conjugates which contained lysosomally degradable side chains showed significant in vivo antileishmanial activity (>99% inhibition), comparable to the activity of the free drug. At 2 mg dose, the same conjugates were significantly more effective (84-90% inhibition) than the free drug (67% inhibition). These results indicate the potential of lysosomotropic HPMA copolymers for the targeted delivery of antileishmanial compounds in the treatment of visceral leishmaniasis.