Development of new antileishmanial drugs – current knowledge and future prospects

Leishmaniasis is a protozoan vector borne disease prevalent throughout the world and present in at least 88 countries. The parasite is transmitted by infected phlebotomine sandfly bites. While conventional therapies i.e. pentavalent antimonials, amphotericin B and pentamidine continue to play a major role, it is evident that new drugs or strategies must circumvent the limitations, such as a long-term parenteral administration, toxicity, the high cost in endemic countries and the emergence of resistance, that prevail. One of the most promising drugs is miltefosine, a new oral, approved alkylphospholipid for visceral leishmaniasis with only slight adverse effects. Although we have now this recent and encouraging advance, there is still a need to develop safe, efficient and affordable new treatments for the different clinical forms that exist. This review summarises conventional therapy and the current efforts in the discovery of drugs to treat leishmaniasis with the emphasis on drug combinations to enhance efficiency and prevent the emergence of resistance, the investigation of natural products with the objective of offering new bioactive chemical structures and the development of novel antileishmanial targets.     

Practical progress and new drugs for changing patterns of leishmaniasis

The problems surrounding leishmaniasis are changing. An increase in travel, the Indian and Sudanese epidemics of visceral leishmaniasis, parasite resistance to antimony and the emergence of AIDS-related leishmaniasis have all increased the urgency for new drugs, and led to reappraisals of the old ones, as discussed here by Piero Olliaro and Anthony Bryceson.     

Antimony transport mechanisms in resistant leishmania parasites

Antimonial compounds have been used for more than a century in the treatment of the parasitic disease leishmaniasis. Although pentavalent antimonials are still first-line drugs in several developing countries, this class of drugs is no longer recommended in the Indian sub-continent because of the emergence of drug resistance. The precise mechanisms involved in the resistance of leishmania parasites to antimony are still subject to debate. It is now well documented that drug resistance in leishmania parasites is a multifactorial phenomenon involving multiple genes whose expression pattern synergistically leads to the resistance phenotype. The reduction of intracellular antimony accumulation is a frequent change observed in resistant leishmania cells; however, no comprehensive transport model has been presented so far to explain this change and its contribution to Leishmania resistance. The present review firstly covers the actual knowledge on the metabolism of antimonial drugs, the mechanisms of their transmembrane transport and intracellular processing in Leishmania. It further describes both the functional and molecular changes associated with Sb resistance in this organism. Possible transport models based on the actual knowledge are then presented, as well as their functional implications. Biophysical and pharmacological strategies are finally proposed for the precise identification of the transport pathways.     

Prokaryotic expression, purification, and polyclonal antibody production against a novel drug resistance gene of Leishmania donovani clinical isolate

Diseases produced by protozoan parasites are one of the main causes of morbidity and mortality around the world, affecting millions of people. Among these, leishmaniasis has become the second most common cause of death and the problem is further complicated by the expansion of parasite resistance to the conventional drugs. The high rate of therapeutic failure thus calls for new rational approaches to develop alternative drugs. Understanding resistance mechanisms may help identify new targets for drug development. So we present here the cloning, expression, purification, and antibody production of a gene implicated in imparting resistance to pentavalent antimony (SbV) in clinical isolates of kala azar with a view to gain insight into the novel mechanism of its drug resistance.     

New drugs for the treatment of human parasitic protozoa

Whereas parasitic diseases are always a heavy burden for humanity, few are the new antiparasitic molecules marketed during the last 25 years. Thus on the 1393 new molecules marketed between 1975 and 1999, only 7 have antiprotozoan properties. This talk will detail the progress made in the treatment of the intestinal protozoa, malaria, visceral leishmaniasis and toxoplasmosis, problems with which are especially confronted the European parasitologists. The treatment of Giardia and intestinal amoebas is based on 5-nitro-imidazoles derivatives. Single-dose treatments can be used with tinidazole or secnidazole. Resistance to these compounds of Giardia were described and in these cases, treatment by quinacrine or nitazoxanide are possible alternatives. Nitazoxanide is marketed in the United States and in Australia. It seems to be a well tolerated antiparasitic agent with a broad spectrum because it is active on a lot of intestinal protozoa and helminths. It acts on the same metabolic way as the 5-nitro-imidazoles (inhibition of the ferredoxine reductase) but without synthesis of free radicals and DNA deterioration of the target cell. It is thus neither teratogenic nor mutagenic. Artemisinin derivatives allowed considerable progress in the treatment of malaria. They have short half-lifes, allowing a fast parasitic clearance and these derivatives do no provoke resistance. They are first line drugs for the treatment of malaria in areas of drug resistance. The arthemeter-lumefantrine association (Riamet, Coartem) ensures a rapid disappearance of the circulating parasites and is well tolerated. Atovaquone-proguanil (Malarone) is usable in the treatment of acute malaria but also in disease prevention with the advantage of continuing drug intake for only 7 days after having left the infected area. The treatment of leishmaniasis is always delicate and is characterized by the worrying development of antimony resistances, probably related in the European zones to the treatment of dogs. Liposomal amphotricin is an alternative of choice but remains very expensive. The heating of amphotericin to 70 degrees C during 20 minutes gives it experimental properties and efficacies comparable with that of liposomal amphotericin, but at a less cost. Miltefosine, an alkyl-phospholipide antimetabolite, is very active on visceral leishmaniasis resistant to antimonial treatment. However, its long half-life could induce the emergence of resistances. Miltefosine induces much less side effects than conventional amphotericin B. The commonly used anti-toxoplasmic drugs (sulphadiazine and pyrimethamine) were marketed some 50 years ago and are only active on the rapid forms in multiplication. No drug is really efficient on the cysts although preliminary tests with atovaquone are encouraging to treat ophthalmologic forms in immunocompetent patients. To conclude, it is important to continue to search for new antiprotozoan molecules because, for some parasites, drug resistance is an important problem. Moreover, the treatment of the pregnant women, particularly during the first trimester, is often impossible and there is a lack of galenic forms easily usable in children. A better knowledge of the metabolic pathways of protozoa (particularly the apicoplast of Apicomplexa parasites) would certainly open the posssibility to identify new drugs. To reduce and delay the appearance of resistances, mass-treatments of mass should be avoided and targeted treatments prefered as well as the use of associations of molecules having different modes of action.     

Present status of antileishmanial vaccines

The term leishmaniasis refers collectively to various clinical syndromes that are caused by obligate intracellular protozoa of the genus Leishmania. Approximately 350 million people in 8 countries are estimated to be threatened by the disease [1]. The World Health Organization estimated that there are 12 million cases of all forms of leishmaniasis worldwide, with over 500,000 new cases of visceral disease occurring each year [1]. Most of the drugs commonly used to treat different forms of leishmaniasis are toxic and have unacceptable side effects. Moreover, cases of drug resistant leishmaniasis are on the rise. Due to non-existence of effective vaccine to date, improved immunoprophylactic approaches still remain desirable to combat leishmaniasis. Antileishmanial vaccines developed around the globe are discussed.     

The application of gene expression microarray technology to kinetoplastid research

Protozoan parasites in the order Kinetoplastida cause severe disease primarily in tropical and subtropical areas. Vaccines to control these diseases have shown some promise, but none are in active clinical use. Drug treatments are available for all of the acute infections, but the emergence of resistance and an unresponsive chronic phase are current problems. Rapid advances in genomic technology open the possibility of discovering new genes that can contribute to vaccine initiatives or serve as targets for development of new drugs. The DNA microarray is a genomic technology, which is being applied to new gene discovery in kinetoplastid parasites. Both cDNA and genomic microarrays for Leishmania major have identified a number of new genes that are expressed in a stage-specific fashion and preliminary results from a L. donovani genomic microarray also demonstrated new gene discovery. A microarray of Trypanosoma brucei genomic fragments identified new genes whose expression differs between the insect borne stage and the human infectious stage of the parasite. The next few years, building on this foundational work, should witness the most exciting stage as microarrays are applied to questions such as the basis of drug resistance, post kala azar dermal leishmaniasis, the regulation of differentiation to infectious stages, linking coordinately regulated pathways of genes and development of genetically defined parasites that may have potential as live attenuated vaccines.     

Research and development of antibacterial agents

Several new antibacterial agents are currently being developed in response to the emergence of bacterial resistance to existing drugs. The new agents include compounds that inhibit macromolecular synthesis or interfere with bacterial membrane function. Apart from the oxazolidinones and cationic peptides, the remainder of these new compounds are analogues of earlier antibiotic classes; therefore, it is probable that existing resistance mechanisms will adapt to accommodate the new derivatives. To minimise the potential for emergence of resistance to new agents, research strategies should be chosen that not only enhance the discovery of structurally novel drugs, but also direct these to new molecular targets that may themselves have decreased potential to give rise to drug-resistant variants.     

Extensively drug-resistant tuberculosis: current challenges and threats

Extensively drug-resistant tuberculosis (XDR-TB) is defined as tuberculosis caused by a Mycobacterium tuberculosis strain that is resistant to at least rifampicin and isoniazid among the first-line antitubercular drugs (multidrug-resistant tuberculosis; MDR-TB) in addition to resistance to any fluroquinolones and at least one of three injectable second-line drugs, namely amikacin, kanamycin and/or capreomycin. Recent studies have described XDR-TB strains from all continents. Worldwide prevalence of XDR-TB is estimated to be c. 6.6% in all the studied countries among multidrug-resistant M. tuberculosis strains. The emergence of XDR-TB strains is a reflection of poor tuberculosis management, and controlling its emergence constitutes an urgent global health reality and a challenge to tuberculosis control activities in all parts of the world, especially in developing countries and those lacking resources and as well as in countries with increasing prevalence of HIV/AIDS.     

Inhibitors of dihydrofolate reductase in Leishmania and trypanosomes

The protozoan diseases leishmaniasis, Chagas' disease and African trypanosomiasis are major health problems in many countries, particularly developing countries, and there are few drugs available to treat these diseases. Dihydrofolate reductase (DHFR) inhibitors have been used successfully in the treatment of a number of other diseases such as cancer, malaria and bacterial infections; however they have not been used for the treatment of these diseases. This article summarises studies on leishmanial and trypanosomal DHFR inhibitor development and evaluation. Possible mechanisms of resistance to DHFR inhibitors are also discussed.     

Timing the Emergence of Resistance to Anti-HIV Drugs with Large Genetic Barriers

New antiretroviral drugs that offer large genetic barriers to resistance, such as the recently approved inhibitors of HIV-1 protease, tipranavir and darunavir, present promising weapons to avert the failure of current therapies for HIV infection. Optimal treatment strategies with the new drugs, however, are yet to be established. A key limitation is the poor understanding of the process by which HIV surmounts large genetic barriers to resistance. Extant models of HIV dynamics are predicated on the predominance of deterministic forces underlying the emergence of resistant genomes. In contrast, stochastic forces may dominate, especially when the genetic barrier is large, and delay the emergence of resistant genomes. We develop a mathematical model of HIV dynamics under the influence of an antiretroviral drug to predict the waiting time for the emergence of genomes that carry the requisite mutations to overcome the genetic barrier of the drug. We apply our model to describe the development of resistance to tipranavir in in vitro serial passage experiments. Model predictions of the times of emergence of different mutant genomes with increasing resistance to tipranavir are in quantitative agreement with experiments, indicating that our model captures the dynamics of the development of resistance to antiretroviral drugs accurately. Further, model predictions provide insights into the influence of underlying evolutionary processes such as recombination on the development of resistance, and suggest guidelines for drug design: drugs that offer large genetic barriers to resistance with resistance sites tightly localized on the viral genome and exhibiting positive epistatic interactions maximally inhibit the emergence of resistant genomes.     

Leishmania spp.: development of pentostam-resistant clones in vitro by discontinuous drug exposure

Antimony unresponsiveness in mucocutaneous and visceral leishmaniasis is a serious clinical problem. Information on the mechanisms and characteristics of drug resistance in parasites that suggest chemotherapeutic strategies to overcome resistance is of practical importance. We developed nine lines of Leishmania resistant to drugs, the major emphasis being on pentavalent antimony (Sb) complexed to carbohydrate in the form of sodium stibogluconate (Pentostam), one of the only two antileishmanial agents with a clearly favorable therapeutic index. Resistance to Pentostam (33- to 212-fold increase) was obtained in promastigotes of Leishmania in vitro by exposure to gradually increasing concentrations of drug over several passages. Resistance to Sb was found to be either stable or unstable. Stable resistance to Sb required (greater than 3) exposures of the initial sensitive clones to Pentostam and tended to stabilize with increased time under pressure. In general, resistance obtained in a clone after only a few (less than or equal to 3) step treatments was low and unstable. Differences in the susceptibility to Pentostam were found between strains isolated from patients with American cutaneous leishmaniasis. In addition, natural isolates of Leishmania from patients represented a heterogeneous population of parasites as demonstrated by a biphasic concentration response to Sb (typical of mixed population dynamics) and by marked differences in susceptibility to Pentostam among clones prepared from single isolates. These results suggest that the emergence of parasite resistance to antimonial treatment is a potential risk of inadequate dose therapy.     

Potent antiprotozoal activity of a novel semi-synthetic berberine derivative

Treatment of diseases such as African sleeping sickness and leishmaniasis often depends on relatively expensive or toxic drugs, and resistance to current chemotherapeutics is an issue in treating these diseases and malaria. In this study, a new semi-synthetic berberine analogue, 5,6-didehydro-8,8-diethyl-13-oxodihydroberberine chloride (1), showed nanomolar level potency against in vitro models of leishmaniasis, malaria, and trypanosomiasis as well as activity in an in vivo visceral leishmaniasis model. Since the synthetic starting material, berberine hemisulfate, is inexpensive, 8,8-dialkyl-substituted analogues of berberine may lead to a new class of affordable antiprotozoal compounds.     

Glibenclamide modulates glucantime activity and disposition in Leishmania major

A source of chemotherapeutic failure in anti-infective therapies is the active movement of drugs across membranes, through ATP-binding cassette (ABC) transporters. In fact, simultaneous administration of therapeutic drugs with ABC transporter blockers has been invoked to be the way to actively prevent the emergence of drug resistance. Herein, we demonstrate that glucantime’s efficacy in decreasing the infection rate of Leishmania-infected macrophages is strongly enhanced when used in combination with glibenclamide, a specific blocker of ABC transporters. Intracellular ABC transporters mediate glucantime sequestration in intracellular organelles. Their selective inhibition may effectively increase the cytoplasmic concentration of glucantime and its leishmanicidal activity. Our results reveal for the first time that glibenclamide targets in Leishmania major a compartment associated with a multivesicular system that is simultaneously labeled by the acidic marker LysoTracker-red and may represent the organelle where antimonials are sequestered. These results constitute a proof of concept that conclusively demonstrates the potential value that combination therapy with an ABC transporter blocker may have for leishmaniasis therapy.     

Mechanisms of drug resistance in Leishmania

The emergence of drug resistance in protozoan parasites is a major obstacle to their control. Since vaccines are not yet in sight for several of these parasites, there is on urgent need to develop new and better drugs. These antimicrobial agents will possibly be more expensive, and will therefore impose on additional burden in health-care costs and in the planning of public health policies of the developing countries. A better understanding of drug resistance, to try to circumvent or overcome it, and the search for new specific cellular targets of parasites are warranted. The development, in vitro, of drug-resistant parasite cell lines has been instrumental in our understanding of the mechanisms of drug resistance in parasitic protozoans. Marc Ouellette and Barbara Popodopoulou here present on overview of the recent progress on the elucidation of mechanisms of drug resistance in the protozoan parasite Leishmania, selected under laboratory conditions.     

Developing imidazole analogues as potential inhibitor for Leishmania donovani trypanothione reductase: virtual screening,molecular docking,dynamics and ADMET approach

Visceral leishmaniasis (VL) affects Indian subcontinent, African and South American continent, and it covers 70 countries worldwide. Visceral form of leishmaniasis is caused by Leishmania donovani in Indian subcontinent which is lethal if left untreated. Extensive resistance to antileishmanial drugs such as sodium stibogluconate, pentamidine and miltefosine and their decreased efficacy has been reported in the endemic region. Amphotericin B drug has shown good antileishmanial activity with significant toxicity, but its cost of treatment has limited the outreach of this treatment to affected people living in endemic zone. So, there is an urgent need to identify new antileishmanial drugs with excellent activity and minimal toxicity issues. Trypanothione reductase, a component of antioxidant system, is necessary for parasite growth and survival to raise infection. To develop potential inhibitor, we docked nine hundred and eighty-four 5-nitroimidazole analogues along with clomipramine which is a well-known inhibitor for TR. Total one hundred and forty-seven 5-nitroimidazole analogues with better docking score than clomipramine were chosen for ADMET and QikProp studies. Among these imidazole analogues, total twenty-four imidazole analogues and clomipramine were chosen on the basis of their ADMET, QikProp, and prime MM-GBSA study. Later on, two analogues with best MM-GBSA dG bind were undergone molecular dynamic simulation to ensure protein–ligand interactions. Using above approach, we confirm that ethyl 2-acetyl-5-[4-butyl-2-(3-hydroxypentyl)-5-nitro-1H-imidazol-1-yl]pent-2-enoate can be a drug candidate against L. donovani for the treatment of VL in the Indian subcontinent.     

Anti-leishmanial activity of alkaloidal extract from Aspidosperma ramiflorum

Infections due to protozoa of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials (SbV), which present renal and cardiac toxicity. Besides, the precise chemical structure and mechanism of action of these drugs are unknown up to date. In order to find new drugs against leishmaniasis, we have been studying extracts of Brazilian trees. In the present study, we have evaluated the effectiveness of an alkaloid extract of Aspidosperma ramiflorum Muell. Arg. (Apocynaceae), against the extracellular forms promastigotes of L. (L.) amazonensis and L. (V.) braziliensis. The alkaloid extract of A. ramiflorum was much more effective against L. (L.) amazonensis (LD50 < 47 microg/ml) than L. (V.) braziliensis. Based on these in vitro results against L. (L.) amazonensis new studies should be made to find the compounds with anti-leishmanial activity.     

In vitro activity of nicotinamide/antileishmanial drug combinations

To improve the management of leishmaniasis, new drugs and/or alternative therapeutic strategies are required. Combination therapy of antileishmanial drugs is currently considered as one of the most rational approaches to lower treatment failure rate and limit drug resistance spreading. Nicotinamide (NAm), also known as vitamin B3 that is already is used in human therapy, exerts in vitro antileishmanial activity. Drug combination studies, performed on L. infantum axenic amastigotes, revealed that NAm significantly improves the antileishmanial activity of trivalent antimony in a synergistic manner while it shows additive activity with amphotericin B and slightly antagonizes pentamidine activity. NAm also significantly increases the toxicity of pentavalent antimony against the intracellular forms of L. infantum, L. amazonensis and L. braziliensis. The potential of NAm to be used as adjuvant during leishmaniasis chemotherapy is further discussed.     

Management of insecticides for use in disease vector control: Lessons from six countries in Asia and the Middle East

Interventions to control the vectors of human diseases, notably malaria, leishmaniasis and dengue, have relied mainly on the action of chemical insecticides. However, concerns have been raised regarding the management of insecticides in vector-borne disease-endemic countries. Our study aimed to analyze how vector control insecticides are managed in selected countries to extract lessons learned.A qualitative analysis of the situation of vector control insecticides management was conducted in six countries. Multi-stakeholder meetings and key informer interviews were conducted on aspects covering the pesticide lifecycle. Findings were compared and synthesized to extract lessons learned. Centrally executed guidelines and standards on the management of insecticides offered direction and control in most malaria programs, but were largely lacking from decentralized dengue programs, where practices of procurement, application, safety, storage, and disposal were variable between districts. Decentralized programs were better at facilitating participation of stakeholders and local communities and securing financing from local budgets. However, little coordination existed between malaria, visceral leishmaniasis and dengue programs within countries. Entomological capacity was concentrated in malaria programs at central level, while dengue and visceral leishmaniasis programs were missing out on expertise. Monitoring systems for insecticide resistance in malaria vectors were rarely used for dengue or visceral leishmaniasis vectors. Strategies for insecticide resistance management, where present, did not extend across programs or sectors in most countries. Dengue programs in most countries continued to rely on space spraying which, considering the realities on the ground, call for revision of international guidelines.Vector control programs in the selected countries were confronted with critical shortcomings in the procurement, application, safety measures, storage, and disposal of vector control insecticides, with implications for the efficiency, effectiveness, and safety of vector control. Further international support is needed to assist countries in situation analysis, action planning and development of national guidelines on vector control insecticide management.     

Sitamaquine as a putative antileishmanial drug candidate: from the mechanism of action to the risk of drug resistance

Sitamaquine is a 8-aminoquinoline in development for the treatment of visceral leishmaniasis by oral route, no activity being observed on the experimental cutaneous leishmaniasis experimental models. Recent data explain how sitamaquine accumulate in Leishmania parasites, however its molecular targets remain to be identified. An advantage of sitamaquine is its short elimination half-life, preventing a rapid resistance emergence. The antileishmanial action of its metabolites is not known. The selection of a sitamaquine-resistant clone of L. donovani in laboratory and the phase II clinical trials pointing out some adverse effects such as methemoglobinemia and nephrotoxicity are considered for a further development decision.