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Publicly Available Published by De Gruyter June 11, 2019

Current and promising novel drug candidates against visceral leishmaniasis

  • Rosa M. Reguera , Yolanda Pérez-Pertejo , Camino Gutiérrez-Corbo , Bárbara Domínguez-Asenjo , César Ordóñez , Carlos García-Estrada , María Martínez-Valladares and Rafael Balaña-Fouce EMAIL logo


Leishmaniasis is a group of zoonotic diseases caused by a trypanosomatid parasite mostly in impoverished populations of low-income countries. In their different forms, leishmaniasis is prevalent in more than 98 countries all over the world and approximately 360-million people are at risk. Since no vaccine is currently available to prevent any form of the disease, the control strategy of leishmaniasis mainly relies on early case detection followed by adequate pharmacological treatment that may improve the prognosis and can reduce transmission. A handful of compounds and formulations are available for the treatment of leishmaniasis in humans, but only few of them are currently in use since most of these agents are associated with toxicity problems such as nephrotoxicity and cardiotoxicity in addition to resistance problems. In recent decades, very few novel drugs, new formulations of standard drugs or combinations of them have been approved against leishmaniasis. This review highlights the current drugs and combinations that are used medical practice and recent advances in new treatments against leishmaniasis that were pointed out in the recent 2nd Conference, Global Challenges in Neglected Tropical Diseases, held in San Juan, Puerto Rico in June 2018, emphasizing the plethora of new families of molecules that are bridging the gap between preclinical and first-in-man trials in next future.

Leishmaniasis in the context of neglected tropical diseases

Neglected tropical diseases (NTDs) are a serious challenge within the global health issue, especially in underdeveloped and developing countries, where their prevalence is high [1]. NTDs affect nearly one billion people mainly in tropical and subtropical regions, with an estimated cost for developing economies of billions of dollars every year [2]. World Health Organization (WHO) has included, up to now, 20 NTDs mostly caused by helminths, protozoa parasites and viruses. Despite this etiological diversity, NTDs have in common their little morbidity and relatively low mortality, their over-representation within the most vulnerable layers of population of poor or developing countries, and the responsibility of causing stigma and discrimination, especially in children and women [3]. Although these diseases are rather infrequent in countries of the developed world, several factors like global warming, the increased tourism to NTDs-bearing regions, and migration fluxes from endemic areas, have intensified the incidence of these diseases in formerly non-endemic countries. As an example, American trypanosomiasis (Chagas disease), which is endemic from South American regions where the transmission vector lives, can be found nowadays in North America [4] and several European countries [5] as an emerging disease.

Kinetoplastids are a group of protozoa responsible for some of the most neglected and deadly diseases in humankind: Human African trypanosomiasis (HAT or sleeping sickness) and American trypanosomiasis, and leishmaniasis. This group of microorganisms cause a spectrum of acute and sometimes chronic systemic and often disfiguring diseases, which entail a high socioeconomic impact on patients, their families and the whole community in endemic regions. Leishmaniasis is considered a zoonotic NTD transmitted by the bite of female phlebotomine sandflies, although some species can have anthroponotic transmission (for a recent review see: [6]. According to WHO, over 20 different species of Leishmania spp. are responsible for more than one billion people being at risk of infection in 98 endemic tropical and subtropical countries. Incidence of 0.7–1 million new cases of leishmaniasis and 310 million people vulnerable to infection occurs annually. Similar to other NTDs, these figures are pushed up by environmental, demographic and behavioral factors of human communities, which contribute to the changing epidemiology, the cyclic recurrence in endemic regions, as well as to its recent spread to non-endemic countries [6], [7].

Different presentations of the disease occur depending on the geographic region, the infecting species, and host immunity to infection, and are characterized by several clinical manifestations. Disfiguring cutaneous leishmaniasis (CL), the most prevalent form with an incidence of 0.7–1.2 million new cases per year in the Old World, is characterized by a variety of lesions in exposed skin areas. Mucocutaneous leishmaniasis (MCL) is a highly disabling variety of CL that destroys the mucosal tissues of the oral cavity, nose and throat of population sectors living in South America. However, the most serious form of the disease is the visceral form (VL), which can be fatal if left untreated. This presentation is endemic in many regions of the world, but more than 90% of the global burden is confined to seven countries: India, Somalia, Kenya, Sudan, South Sudan, Ethiopia and Brazil, where most of deceases are accounted. However, a successful eradication campaign in Asia has left the Eastern Africa region with the highest burden of VL [8]. Although any of the presentations of leishmaniasis is considered a public health challenge to Europe and other developed areas, Southern European countries are not free of this disease. An example is represented by the outbreak occurred in Spain in 2009 in a wealthy area near Madrid, where an unusual incidence of more than 20 cases per 10 000 inhabitants was declared in 2011 [9].

Host’s immunocompetence is crucial for leishmaniasis development. In this context, Leishmania–HIV coinfections – firstly reported in European countries and now practically disappeared – is considered a serious complication in 35 countries of the Indian subcontinent, East Africa and Brazil. In these countries, figures are rising in the last years, which represents a major issue in the management of both infections [10], [11], [12]. A second problem associated to VL is post-kala-azar dermal leishmaniasis (PKDL), a disfiguring cutaneous sequela of VL characterized by macular, papular or nodular skin lesions that are developed by 50% of patients with VL in Sudan and 1–3% of patients with VL in the Indian subcontinent [13].

WHO has established an elimination target for VL of <1 case/10 000 population [14]. In order to achieve this goal, a combination of health policy strategies focused on preventing the transmission from the sandfly vector to the human host, and to control animal reservoir host, are recommended by WHO. Unfortunately, despite the initial success of leishmanization in the Middle East against CL [15], the multiple efforts done during the last decades, and the fact that the majority of people who recover from infection become immune, nowadays there is no reliable preventive human vaccine in clinical use against leishmaniasis [16]. In this regard, the management strategy for leishmaniasis falls on the control of vectors and reservoirs, and the early diagnosis of cases, followed by an adequate treatment that can improve the prognosis and can reduce transmission [17]. Eventually, when the disease is established, medical management is based on a limited and – in many cases controversial – number of antileishmanial drugs that with exceptions, are the same as those used 70 years ago.

There are several problems with the current drugs used to treat leishmaniasis. Most of them are associated with adverse side effects and/or with treatment failures and relapses due to drug resistance. Furthermore, this bunch of antileishmanial medicines has several pitfalls that prevent their use in certain population cohorts or under determined circumstances: e.g. some of them are unsuitable for child-bearing women and newborns due to teratogenic issues. A second concern is the poor oral bioavailability of most of these drugs. This implies the parenteral administration of most of them, sometimes by slow intravenous infusions, which involves patient hospitalization throughout the treatment. The latter implies high costs that further weaken the health systems of these countries. Parenteral administration is particularly concerning, since 38% of the leishmaniasis cases worldwide occur in children below the age of 15 years [18]. Finally, some of these medicines require an intact cold chain in order to keep them chemically unchanged under the environmental warm conditions of the target regions where they have to be delivered.

These and other issues related to the development of new drugs, the understanding of mechanisms of action and resistance of the medicines in clinical use, were some of the hot topics presented in the last 2th Global Challenges in Neglected Tropical Diseases Conference held in San Juan (Puerto Rico), hosted by Prof. Nestor Carballeira (University of Puerto Rico, Rio Piedras PR, USA) and Colegio de Químicos de Puerto Rico.

Current treatment of leishmaniasis

Treatment of VL varies from one endemic region to another. WHO recommended regimes for major-VL-endemic foci, as it is summarized in Table 1. In general, the treatment options are inadequate and new drugs are urgently needed. Most of the drugs used in the treatment of leishmaniasis are included within the 19th edition of WHO Model List of Essential Medicines (April 2015), namely pentavalent antimonials (SbV), miltefosine, amphotericin B deoxycolate or formulated in liposomal formulations, paromomycin and pentamidine (Fig. 1).

Table 1:

Current pharmacological treatment against VL.

SbV-based drugsParenteral (im) 20 mg/kg/day for 28–30 daysLow costDrug resistance in Bihar (India), PKDLPain in the injection site, cardiotoxicity, pancreatitis[6], [27]
Amphotericine B deoxycholateSlow iv infusion 1 mg/kg/day for 30 daysEffective against SbV resistant strainsRequire hospitalizationNephrotoxicity[37], [40]
AmBisomeSlow iv infusion 10 mg/kg single doseEffective at single doseCostly, chemically unstableFever during infusion, back pain, nephrotoxicity[9], [20], [38]
MiltefosineOral 50–100 mg/kg/day for 28 daysNo hospitalizationNeed allometric administration in childrenGastrointestinal complications, teratogenic[53], [54], [56], [132]
ParomomycinParenteral (im) 15 mg/kg/day for 21 daysLow costPoor results against African VL as monotherapyPain in the injection site, hepatotoxicity[58], [61]
PentamidineSlow iv infusion 4 mg/kg monthly for 12 monthsUse in HIV positive co-infectionsMultiple adverse effectsInsuline-dependent diabetes, myocarditis, nephrotoxicity[63]
SbV-paromomycin combinationParenteral (im) SbV 20 mg/kg/day+paromomycin for 17 daysReduce the number of im injectionsRequire hospitalizationProblems regarding SbV administration[64], [65], [66], [67]
Fig. 1: Chemical structure of antileishmanial drugs in current clinical use.
Fig. 1:

Chemical structure of antileishmanial drugs in current clinical use.

Pentavalent antimonials

Organometallic complexes derived from pentavalent antimony (SbV) have been used as drugs of choice in the treatment of VL and CL for more than 70 years. However, despite their proven leishmanicidal efficacy, SbV-based drugs are not free of serious issues, including their high death rate in certain population subsets (age below 2 or over 45, or in cases of advanced disease), severe side-effects and parenteral route of administration, which justify their urgent replacement by other safer medicines [19].

Two drugs based on organic complexes with SbV fall under this denomination: meglumine antimoniate (1) (Glucantime®) (85 mg SbV/100 mL) and sodium stibogluconate (2) (Pentostam®) (100 mg SbV/100 mL). Both formulations have been used extensively in the Indian subcontinent until their replacement with by single dose of liposomal amphotericin B [20]. However, despite the report of a growing incidence of failures and relapses are being reported, the use of Pentostam® and other generic forms of SbV are still in use in Eastern African as first choice antileishmanial medicines [21]. They can be used alone – as monotherapy – or in combination with other drugs such as amphotericin B, paromomycin and miltefosine to enhance their antileishmanial effect and to prevent the occurrence relapses.

Both compounds are prodrugs, since metabolic transformation is required for their activation within the parasite. In fact, SbV must be reduced to the more toxic form SbIII by the reductive metabolism of Leishmania amastigotes [22], which is favored by trypanothione, the specific free-radical scavenger of trypanosomatids [23]. The reduced SbIII form not only causes enzyme inhibition of energy metabolism and macromolecules biosynthesis but also alters the thiol redox balance, which is responsible for preventing the toxic effects of reactive oxygen species in the parasite [24].

SbV-based drugs have poor gastrointestinal absorption, and patients have to be hospitalized for 3–4 weeks for parenteral (intravenous or intramuscular) injections. Pentostam® was administered in India at 20 mg/kg/day for 28−30 days, which represents a clear increase of tolerance to SbV since the early administrations of the drug. This regime is currently recommended in Eastern Africa to fight VL produced by L. donovani, but it has been superseded in Southern European countries by AmBisome® as first-line drug to fight human – but not canine VL in order to avoid the risk of emergence of resistant strains [25]. After rapid absorption from the injection site, SbV are distributed by means of a two- compartment model with a first-order absorption rate [26]. Since the leishmanicidal effect of SbV depends on the concentration reached in target organs (spleen, liver and bone marrow), a high bioavailability and distribution volume are desired.

The chemical nature of SbV formulations causes severe pain at the injection site as well as phlebotoxicity, muscular fibrosis and abscesses. Cardiotoxicity is a common side-effect of SbV formulations Arthralgia and myalgia, elevated hepatic enzymes and pancreatitis are other common adverse events [27].

A serious concern about the use of SbV-based medicines is an increasing incidence of resistance to these compounds. This has been reported in some districts of the Bihar state in North-Eastern of India, where ~90% of VL cases occurs. In this particular region, the effectiveness of Pentostam® gradually declined, and more than 60% of VL cases are now refractory [28]. Resistance may be a consequence of large-scale misuse and may be triggered either by host/parasite or by environmental factors. Host’s factors may involve modifications of pharmacokinetic parameters or alterations in the immunological response to the parasite. Parasite’s factors include structural modifications of target proteins or mechanisms to evade the host immunological system [29], [30]. However, there are serious concerns that the emergence of resistant strains is due to an increase in the tolerance due to long-term exposure of general population – including that infected with Leishmania – to high trivalent arsenic concentrations in drinking water [31].

Amphotericin B

Broad-spectrum macrocyclic polyene antibiotic with strong antifungal activity isolated from the actinomycete Streptomyces nodosus that early showed high antileishmania effect. Amphotericin B deoxycholate (Fungizone®) is one of the currently available formulations. However, it is poorly absorbed from the gastrointestinal tract and must be intravenously administered, which may lead to hemolytic anemia [32], [33]. This undesirable side-effect can be avoided by using lipid-based formulations of amphotericin B, such as AmBisome®, Amphocyl or Abelcet, which represent a further step in the treatment of VL [34]. The mechanism of action is exerted by binding to the ergosterol present in the Leishmania plasma membrane, thus forming aqueous pores in cell membranes. This causes metabolic disturbance, leakage of intracellular small molecules and cell death [35]. Lipid formulations of amphotericin B have better tissue penetration (in liver and spleen) with sustained levels and stability in blood, macrophages, and tissues. These are the main features that increase efficacy and minimize toxicity of liposomal amphotericin B [36].

One of the most serious concerns in the use of any formulation of amphotericin B is the need of repeated intravenous injections. Several posologies have been used in different countries with very good outcomes. In India, a dose regime of 1 mg/kg amphotericin B deoxycholate on alternate days for up to 30 days (15 mg/kg total dose) is near 100% efficient, but toxicity can be unbearable for many patients [37]. However, a single injection of 10 mg/kg of AmBisome® produced a cure rate of 96.3%, and was less expensive than the repeated injections of amphotericin B deoxycholate [20]. This promising outcome prompted the WHO to recommend single dose AmBisome® as a first option treatment for VL, with combinations with paromomycin and miltefosine as a second option in Indian Subcontinent. However, the efficacy of AmBisome® (15–49 mg/kg over 6 doses) is barely 55% in East Africa and even worse in HIV-positive groups [38]. In Southern Europe, liposomal amphotericin B is considered first-line treatment for VL. AmBisome® dosed at 3−5 mg/kg per day, up to a total of 20 mg/kg was effective in the Spanish outbreak of 2009 with up to 99−100% effectiveness [9].

Despite the high efficacy of amphotericin B formulations, several issues have reduced its use in field conditions. First of all, the low oral bioavailability of any amphotericin B formulation is considered a serious concern [39], since it involves intravenous administration by slow infusion and mandatory hospitalization of patients, which jeopardizes health care systems in low-income settings. Moreover, the deoxycholate form of the drug is responsible for nephrotoxic outputs that can worsen the effects of the disease [40]. Other sever-side effects are hypokalemia and myocarditis, which require close monitoring and hospitalization. In addition, any formulation of the drug is fairly unstable at the target point of care, mainly tropical regions where the high temperatures can degrade the drug, and therefore, a costly cold chain is necessary [41]. Finally, drug pressure with new AmBisome® protocols is producing the emergence of first cases of resistances in Bihar (India) [42].


Miltefosine (4) (hexadecylphosphocholine, an alkyl phospholipid-like compound similar to lecithins) was originally conceived for the topical treatment of cutaneous metastases in human breast cancer [43]. However, it was rapidly observed that the compound had strong anti-leishmanial activity against human VL [44]. The most notable advantage of miltefosine is a consequence of its zwitterionic nature, which makes it readily dissolvable in water. Due to this property, the bioavailability of miltefosine after oral administration is good enough to allow self-administration by the patients, thus avoiding the serious disturbances of hospitalization [45].

The mechanism of action of miltefosine is not clearly defined, and a unique target of interaction seems to be ruled out. Glycosylphosphatidylinositol anchor biosynthesis, perturbation of ether−lipid metabolism, and signal transduction are possible targets of miltefosine. In Leishmania, in vitro studies showed that miltefosine interferes with fatty acid and sterol metabolism in amastigotes, this mechanism being responsible for alterations in the composition and fluidity of parasite plasmatic membrane [46], [47]. Furthermore, miltefosine induces the apoptosis-like mechanism of cell death described in kinetoplastids [48], and mitochondrial dysfunction [49]. Recently, Pinto-Martinez and coworkers have shown that miltefosine is able to disrupt Ca2+ homeostasis in L. donovani, hence inducing the rapid alkalization of acidocalcisomes [50]. Apart from being toxic for Leishmania spp. cells, experimental data suggest that miltefosine induces IFN-γ, TNF-α, and IL-12 production from macrophages [51], which stimulates the innate immune response of the host [52].

The currently recommended dose of miltefosine as monotherapy for VL in Indian Subcontinent is 50–100 mg/day for a total of 28 days with a cure output of 94% [53]. Similar results were found in East Africa using a posology of 100 mg/day for a total of 28 days [54]. Miltefosine pharmacokinetics has an impact on the clinical practice in humans: (i) miltefosine shows a slow absorption process after oral administration and a high bioavailability (estimated in 94%); (ii) the elimination half-life in dogs has been estimated in 1 week, which represents a low plasma clearance. Consequently, the rate of accumulation in blood is 5- to 6-fold higher after repeated administration at steady-state conditions; (iii) miltefosine is not a substrate of enzymes of oxidative metabolism. However, miltefosine can be metabolized by phospholipases C and D in intact hepatocytes to choline, which may be used for the biosynthesis of acetylcholine or lecithin in the host [55].

The adverse effects of miltefosine include disturbance of the gastrointestinal tract, intestinal pain, vomits, diarrhea, anorexia and occasional hepato- and nephrotoxicity. A serious issue of miltefosine administration is its teratogenic effect and therefore, it is contraindicated during pregnancy. In this regard, women of child-bearing age have to take contraceptives during the treatment and for an additional period of 3 months afterward, due to the long half-life of miltefosine [56].


Paromomycin (5) is an off-patent aminoglycoside antibiotic isolated from the actinomycete Streptomyces krestomuceticus, which has been revealed as an effective and safe treatment for human VL in combination with miltefosine [57]. Other advantage of paromomycin is its affordability: the cost of the treatment is only ~$10 per patient [58]. Its pharmacological activity is based on protein synthesis inhibition by targeting several ribosomal proteins, respiratory dysfunction and mitochondrial membrane depolarization in the parasite [59].

Like other antileishmanial drugs, paromomycin is poorly absorbed by the oral route. Therefore, the intramuscular or intravenous routes are used with repeated administrations. Pharmacokinetic studies of paromomycin showed that it is quickly absorbed through intramuscular injection, with peak plasma levels being reached within 1 h [60]. Paromomycin sulfate at a regimen of 15 mg/kg via i.m. for 21 days, showed similar efficacy to amphotericin B (1 mg/kg i.v. on alternate days for 30 days) in a phase III study in India, with final cure rates of 94.6% [58]. However, the outcomes obtained in East Africa were not very promising. Paromomycin as monotherapy, provided only an 80% average cure rate at 15 mg/kg per day for 21 days. This value was much lower than the 94.1% cure rates obtained with the standard treatment, which consisted of daily doses of sodium stibogluconate 20 mg/kg for 30 days [61]. Common adverse effects include pain at the injection-site, and with lesser prevalence, ototoxicity, and an increase in hepatic transaminases and nephrotoxicity [62].


Pentamidine (6) is a cationic diamidine used in the 1980s for the treatment of pneumonia caused by Pneumocystis carinii associated to HIV patients. Pentamidine has antileishmanial activity, but its use was discontinued due to serious toxicity issues, such as insulin-dependent diabetes mellitus and declining efficacy. Nevertheless, infusions of 4 mg/kg pentamidine-isethionate, administered once in a month for 12 months are being used in HIV–VL coinfection in Ethiopia, and has been found to have a probability of relapse-free survival of 79% [63].

Combination therapies

Over the last decades, very few novel drugs or new formulations of standard drugs have been approved against VL worldwide. On the contrary, combinations of drugs have been screened for clinical use, and are currently recommended by DNDi and WHO. Drug combinations are replacing monotherapy, enhancing efficacy and reducing the emergence of resistant strains. It is remarkable that specific combinations of drugs differ according to the geographical region and depend on the type of transmission (anthroponotic or zoonotic), on the presence of confirmed cases of resistance, and on the cost and chemical stability of the drugs at the point of care.

Combinations of AmBisome®+paromomycin and Ambisome®+miltefosine, were tested in Asia, where DNDi conducted safety and effectiveness studies, including a pilot project in the Bihar State of India (2012–2015). Here, combination therapies at the primary healthcare level, and single-dose AmBisome® at the hospital level, were implemented [64]. These regimens were considered safe and effective and, based on the results obtained in the study, the Indian National Roadmap for Kala-Azar Elimination recommended use of single dose AmBisome® in August 2014 as a first option treatment for VL [65].

The combination of Pentostam® and paromomycin was introduced in East Africa in 2010 by the Leishmaniasis East Africa Platform (LEAP) and DNDi. The treatment consisted of daily intramuscular administration of Pentostam® (20 mg/kg) given concurrently with paromomycin (11 mg/kg base dose) for 17 days. This combination was as efficacious and safe as Pentostam® alone [66], with two important advantages: (i) reduced time of treatment (17 days instead of the initial 30 days), which improves the adherence of patients, and (ii) a more affordable cost treatment [67]. A phase III clinical trial is in progress in East Africa in order to compare the efficacy and safety of two combination regimens of miltefosine and paromomycin with the aforementioned combination in use. The goal is to replace the painful intramuscular repeated Pentostam® and paromomycin injections.

Leishmaniasis drug discovery

The introduction of a new medicine from de novo drug discovery stage to agencies approval is estimated to take an average of 15 years, with an approximate cost of $2 billion [68], [69]. However, since new medicines for NTDs are addressed to impoverished population targets, the interest by the pharma-industry has been traditionally poor due to the low economic return, which results in low private investment. Therefore, much of early drug discovery and hit-to-lead optimization for NTDs have been done in academic laboratories, which lack, in the majority of cases, the economic muscle required to complete the extremely high-cost and complex discovery process, and translate the basic research into further stages.

However, the efforts in drug discovery and development for NTDs have expanded in the last 10–15 years, fueled by significant investments done by governmental, non-governmental and non-profit organizations. Some Big Pharma companies have contributed to these efforts by sharing their libraries of compounds and technical facilities with academic groups for the testing of new molecules or innovative models of the disease. This can accelerate the translation process from the bench to the first-in-man clinical trials of new or repurposed molecules [70]. Any consortium established between different stakeholders, either public or private, to provide new medicines against NTDs, must overcome the inherent shortcomings of the academic world with the translational expertise and business vision and drug development know-how of the pharmaceutical industry.

In this context, and before starting a drug discovery campaign to eradicate VL, a defined target product profile (TPP) common to all lead candidates, is required. The TPP defines the characteristics of the most outstanding molecules, not only in terms of effectiveness to kill Leishmania, but also in terms of security, with the absence of side effects in the treated patients [71]. However, and due to the specificities of the population to be addressed, this TPP should include oral administration and if possible, short dosing schedule in order to avoid the hospitalization of the patient. In addition, any new candidate molecule should be easy to manufacture and scale-up, have an affordable price, and long-term stability in storage and transport, which avoids the need for a cold chain in the point of care. Finally, and in addition to the medical characteristics and wherever feasible, the future drug candidate against VL should be free from intellectual property and licensing constraints that could restrain the availability of the medicine to the target population. DNDi has gathered all the requirements for a drug against VL in the TPP of Table 2.

Table 2:

Ideal and acceptable TPP recommended by DNDi for any drug candidate to fight VL.

CharacteristicIdeal target profileAcceptable target profile
Target label|VL and PKDLVL
Leishmania spp.All speciesL. donovani
DistributionAll areasIndia and East Africa
Target populationBoth immunocompromised and immunocompetentImmunocompetent
Clinical efficacy>95%>90%
ResistanceActive against resistant strainsActive against resistant strains
Safety and TolerabilityNo adverse effects requiring monitoringOne monitoring visit at mid-end point of treatment
InteractionsNone. Compatible for combination therapyNone for malaria, HIV and tuberculosis therapies
FormulationOral/im depotOral/im depot
TreatmentOrally 1 per day×10 days 3 shots over 10 daysBid for <10 days po; or <3 shots over 10 days
Stability3 years in zone 4Stable under conditions that are reasonable considered in target region (over 2 years)

Once the objectives have been defined, the next step is the selection of the screening platform. Reymond and co-workers estimated the potential chemical space in 166.4 billion molecules of up to 17 atoms (GDB-17 database) [72]. However, after discarding unstable or non-synthesizable molecules, this number was reduced by at least 10 orders of magnitude. The goal is to identify those molecule candidates that fulfill the TPP principles shown in Table 2 against VL. In this regard, a recent work by Parker and co-workers showed that only 17% of the human proteome have known ligands [73]. For trypanosomatids, the scenario can be even worse, given the large number of genes encoding hypothetical and putative proteins with unknown function that are present in their genome databases [74].

Some methodological obstacles prevent proper therapeutic development. One of them is the lack of consensus regarding appropriate in vitro and in vivo screening protocols. The initial steps in drug discovery include the identification of active compounds using the hit-to-lead strategy in an iterative process until reaching the clinical candidate. For VL, the potency is the first, but not unique, selection parameter obtained from screening assays. The selection of hit compounds is done according to target-based (TDD) or phenotype-based drug discovery (PDD) criteria, which must be established at the beginning of any antileishmanial drug discovery campaign [75]. Although based on opposed paradigms, these criteria can complement each other, thus increasing the odds of discovering and developing new drugs [76]. TDD hypothesizes that a defined molecular target (protein) has a crucial role in a disease. On the contrary, PDD evaluates phenotypic changes introduced by an external agent on a cell, tissue or entire organism, independently of a particular mechanism of action. According to Nagle and coworkers, the PDD strategy is being more relevant in introducing novel drugs against VL than target-based efforts, although a successful drug discovery campaign will only progress if the mechanism of action is deconvoluted [77].

However, hit selection based only in the potency to kill parasites, may be misleading. Prioritization of hits should also include selectivity criteria based on the comparative toxicity for the parasite over mammalian cell lines, as well as predictive pharmacology/toxicology profiles (ADMET) based on their physicochemical properties.

Leads that move forward to preclinical stages of drug discovery, will be subjected to tests that will assess the efficacy on in vivo disease models, which will recreate the human disease in relation to its etiology, pathophysiology, symptomatology and response to therapeutic agents. However, and according to recent clinical trials, it seems that the current methodology for evaluating efficacy in animal models is less accurate than initially thought. This has led to a reconsideration of the screening sequence in the drug discovery process and to assess new technologies and methods for in vivo testing. Experimental models using rodents, such as BALB/c mice and Syrian golden hamsters, are preferred for initial preclinical stages. This is because they facilitate management at the acute and chronic stages of the disease, although none of them accurately reproduces the visceral human disease [78], [79].

Combined with existing techniques, novel non-invasive bioimaging techniques have demonstrated a tremendous breakthrough in that field, due to their high sensitivity, rapidity, and provision of quantitative results to assess parasite dissemination into tissues, drug penetration and semi-quantitative evaluation of efficacy through time [80]. This approach uses transgenic parasites transfected with foreign genes encoding fluorescent or bioluminescent reporters, which allow the precise detection of parasite burden in a non-invasive animal model using sensitive Charge-Coupled Cameras [81], [82], [83], [84]. Real-time in vivo imaging might also help understand the physical and temporal dynamics of the parasite at tissue/organ level in the mammalian host [85], [86].

Preclinical studies will show good pharmacokinetic/pharmacodynamic properties of the drug candidate after oral administration in in vivo studies, which must be performed under GLP standards prior scale-up synthesis. This requires GMPs before submission of an Investigational Medicinal Product Dossier that allows the first-in-man phase I study [87], [88].

New molecules entering in clinical trials against VL

Drug repurposing is the preferred strategy to accelerate drug discovery in a field with scarce funds and urgent need to obtain a cost-effective final product. This strategy that consist on the finding of novel uses for marketed therapeutics or highly characterized investigational compounds, has become increasingly common for NTDs. Repurposing avoids the risk associated with the costs of drug discovery and development, up to (and often including) preclinical assays, and reduces the time required to bring the drug to market. It is worth bearing in mind that the drug repurposing strategy will accelerate the overall process and reduce the cost of drug discovery as long as the chemical libraries are well annotated and the pharmacokinetic and safety data of the compounds are available to researchers. The main challenge would reside in establishing the connection between the old drug and the new target. A handful of new candidates against VL are entering phase I trials as a result of repositioning processes for compounds used for other pathologies [89].

Nitroaromatic compounds

Nitroaromatic, including nitroimidazole, nitroimidazo-oxazole and nitroimidazo-oxazine derivatives are a well-known class of pharmacologically active compounds. In the search for more effective drugs for VL and other NTDs, researchers have repositioned the therapeutic value of some nitroaromatic compounds from anti-tubercular drug discovery programs (Fig. 2). Despite the prevention of medicinal chemists to metabolic activation of the nitro group associated to potential mutagenicity and carcinogenicity issues [90], nitroaromatic drugs are nowadays an important part of DNDi portfolio for human African trypanosomiasis (HAT). Metronidazole (7) (Flagyl) is an antibiotic effective against microaerophilic and anaerobic bacteria and other infections produced by parasites in low oxygen environment, such as trichomoniasis, giardiasis and amoebiasis [91]. By its part, benznidazole (8) is one of the first-line medicines against acute Chagas disease produced by T. cruzi, and has the active nitroimidazole scaffold responsible for good oral bioavailability and killing effects on the pathogen after enzymatic nitro activation [92]. However, despite the phylogenetical proximity of these species with Leishmania, none of these compounds has shown in vivo significative effectiveness against this parasite, although they have inspired the synthesis of new drugs, which are nowadays near of clinical trials.

Fig. 2: Chemical structure of nitroimidazole compounds.
Fig. 2:

Chemical structure of nitroimidazole compounds.

Fexinidazole (9) is an oral nitroimidazole derivative with proven efficacy against HAT [93]. Fexinidazole is entering in phase 3b trials to test the feasibility of home-based treatments and new drug application should be expected shortly. Therapy consists of one daily dose of pills for 10 days (days 1–4: 1800 mg, days 5–10: 1200 mg), this protocol being similar for both (haemolymphatic and neurological) stages of the disease (NCT01685827) [94]. This oral administration is addressed to substitute current parenteral administration of the Nifurtimox/Eflornithine Combination Therapy (NECT) in Africa, which requires hospitalization and a minimum of four nurses to give the infusions to the patient [95]. Fexinidazole was proven in a mouse model of visceral leishmaniasis infected at a dose of 200 mg/kg for 5 days, which led to 98.4% suppression of parasite load, equivalent to that found with the standard treatment with miltefosine [96]. Based on this efficacy, a phase II proof-of-concept study was initiated in 2013 with the aim to assess the safety and efficacy of fexinidazole for the treatment of primary VL in adult patients in Sudan (NCT01980199). Despite all patients showed clinical improvement during treatment, the study was interrupted in 2014, since it failed to prove conclusive efficacy in the majority of patients [97]. As an ultimate goal is being developed a combination of fexinidazole and miltefosine. In this regard, a drug-drug interaction study in normal healthy volunteers is being supported by DNDi in order to assess the pharmacokinetics and safety of this combination against VL.

Several antitubercular drugs with nitroimidazole structure have become leads in antileishmanial therapy but just one candidate and two backup compounds are entering in first-to-man studies at present. The bicyclic nitroimidazopyran, (PA-824) – pretomanid (10) – exhibited potent bactericidal activity against multidrug-resistant strains of M. tuberculosis and promising oral activity in animal infection models [98]. Pretomanid exists as two R and S enantiomers where the R form is six-fold more effective than the S form. [R]-PA-824 shows potent cidal effect against promastigotes and intracellular amastigotes of L. donovani with EC50 values of 160 nM and 930 nM, respectively. Furthermore, PA-824 is not activated by leishmanial nitroreductase unlike other nitroimidazoles, such as nifurtimox and fexinidazole, which indicates that this enzyme is not the primary target of these compounds [99]. In a murine model of VL, (R)-PA-824 exhibited >99% suppression of parasite burden at a dose of 100 mg/kg b.i.d. when administered orally for 5 days. However, despite (R)-PA-824 offered good ADMET conditions for further lead optimization for VL [100], this compound has not entered pre-clinical development. Nevertheless, it has served of inspiration for other compounds entering phase I, such as DNDI-0690 (see below).

Extensive medicinal chemistry work in developing bicyclic nitroimidazofurans derivatives yielded 6-nitro-2,3-dihydroimidazo[2,1-b]-oxazoles with antitubercular activity, including delamanid ((R)-OPC-67683; Deltyba) (11) [101]. It is an oral nitroimidazo-oxazine compound that introduces lipophilic phenoxy groups to the 4-position of the piperidine ring to circumvent active multidrug-resistant tuberculosis. Both [R] and [S] enantiomers of delamanid are active in killing L. donovani parasites, the R-enantiomer being one order of magnitude more potent against promastigotes, axenic amastigotes and intracellular amastigotes, with EC50 values of 15.5, 5.4 and 86.5 nM, respectively [102]. In addition, bacterial nitroreductase does not play a role in the activation of delamanid either in L. donovani promastigotes or amastigotes, which points to a mechanism of activation different from that of fexinidazole [103]. The efficacy of delamanid administered orally to L. donovani-infected mice at 30 and 50 mg kg−1, b.i.d. for 5–10 days correlates well with that of miltefosine (98.8–99.8% suppression), which points to potential repurposing for VL [104].

An in-depth medicinal chemistry effort yielded 72 nitroimidazoles belonging to four chemical subclasses and identified the R-enantiomer of 6-nitroimidazo-oxazole derivative (VL-2098) (12), as potent antileishmanial drug. It showed in vitro antileishmanial activity and was curative in a model of VL in hamster at a minimum dose of 6.25 mg/kg for 5 days. It was also effective with once-daily oral dose regimen in L. donovani infected Balb/c mice with a marked potential to boost host immunity via activation of host-protective Th1-dominated immune responses. However, the development of VL-2098 by DNDI was interrupted due to adverse effects observed during toxicological studies [105].

The antileishmanial effect of the family of reversed C-6 linker analogues of 7-substituted nitroimidazooxazine compounds, identified DNDI-0690 (13), which was effective against intramacrophage forms of both L. donovani and L. infantum (EC50=60 nM and 93 nM, respectively) and practically non-toxic (SI>1000) for mammalian cells. The compound administered orally at 25 and 50 mg/kg bw once daily for 5 consecutive days reduced the parasite burden in the target organs by more than 98% in mouse and hamster models of disease, showing high oral bioavailability. In addition, no mutagenic issues have been described. These optimal characteristics of DNDI-0690 pointed to this compound as a preclinical candidate in September 2015, with the first-in-man studies being carried out by the end 2018 [105]. An extensive structure−activity relationship investigation of 6-substituted nitroimidazooxazine compounds yielded DNDI-8219 (14), which is a trifluoromethoxy 3 phenoxy derivative with optimal solubility and excellent oral bioavailability. No mutagenic issues have been described for this compound. The efficacy in the mouse model of L. donovani VL is similar to the that of the early curative L. infantum hamster model, showing >97% parasite clearance in target organs at 25 mg/kg administered twice daily [106]. These authors provided enough information to earmark DNDI-8219 as the favorite backup development candidate for phase I studies.


Benzoxaboroles are cyclic boron-containing drugs developed against several infectious diseases under the initiative of the biotech company Scynexis and Anacor Pharmaceuticals (Fig. 3). Two benzoxaborole derivatives are approved by the US Food and Drug Administration as topic ointments for the treatment of toenail onychomycosis (AN-2690: tavaborole) (15) and atopic dermatitis (AN-2728: crisaborole) (16) [107]. In the first instance, a series of 6-carboxiamide derivatives of the benzoxaborole scaffold yielded the fluorinated AN-4169 (17), which showed prominent in vitro killing effects on HAT (T. brucei, both T.b. gambiensis and T.b. rhodesiensis) at submicromolar concentrations [108]. This compound was curative when administered orally at 2.5 mg/kg b.i.d. for four consecutive days in an acute model of T. brucei trypanosomiasis. However, this dose had to be increased to 50 mg/kg b.i.d. for fourteen consecutive days in CNS models of murine trypanosomiasis [108].

Fig. 3: Chemical structure of benzoxaborole compounds.
Fig. 3:

Chemical structure of benzoxaborole compounds.

The modest results obtained with AN-4169 in the in vivo CNS model, encouraged further extensive efforts of lead optimization. With regard to, the substitution of two methyl groups in 3 position of the oxaborole scaffold of AN-4169, yielded SCYX-7158 (AN5568) – acoziborole (18) – a compound with better pharmacokinetic profile despite its lower in vitro trypanocidal effect [109]. In fact, the bioavailability of SCYX-7158 in brain is six to eight times higher than that of other benzoxaboroles tested. Due to these notable pharmacokinetic parameters and the absence of toxicity, acoziborole was able to cure mice infected with trypanosomes in the CNS following once-daily oral administration of 25 mg/kg for 7 days [110]. After succeed in phase I/II and waiting for results of phase III, submission of a regulatory dossier to the European Medicines Agency is planned in a near future. Remarkably, the 3-methyl substituted benzoxaborole AN7973 (19) has shown to be efficacious after single 10 mg/kg intramuscular injection in cattle infected with T. congolense, thus pointing to a future veterinary use of this compound in Africa [111]. Further development of a library of these compounds has yielded AN-11736, which is also effective on T. vivax [112]. These molecules will likely be key to the World Health Organization’s target of disease control, and will replace NECT against human sleeping sickness and berenil against cattle trypanosomiasis as first election medicines by 2030.

Several mechanisms of action have been reported to explain the cidal action of these molecules. The most plausible one was early elucidated in fungi, showing that the boron atom of the oxaborole ring binds to the 2- and 3-oxygen atoms of the tRNA’s 3-terminal adenosine, thus trapping Leucyl-tRNA synthetase bound to tRNALeu and arresting protein synthesis, which ultimately kills the pathogens [113], [114]. Genomics, proteomics and metabolomic approaches, using analogs of acoziborole in trypanosomas, yielded a list of candidate genes implicated in the antitrypanosomal mode of action. They included the Cleavage and Polyadenylation Specificity Factor 3 [115], as well as enzymes involved in S-adenosyl-L-methionine metabolism [116].

Since no CNS accumulation is required for the development of leishmaniasis, those compounds showing effect lacked the dimethyl substituents at C3 of oxaborole scaffold. In this regard SCYX-6759 (AN 4169) – a demethylated form of acoziborole – and recently, a 5-carboxamide derivative called LSH003, showed curative outcomes when administered orally to Balb/c mice suffering VL and L. major CL, respectively. In 2016, the 6-carboxamide benzoxaborole DNDI-6148 (20) concluded preclinical VL studies. This compound resulted as curative as miltefosine in mice at a minimum dose of 25 mg/kg/day b.i.d. after a 10-day treatment. This remarkable effectiveness has proposed this compound as lead candidate for first-in-man studies of safety, tolerability, and pharmacokinetics after single oral ascending dose in 2018. In addition, two other oxaboroles with better profile, dubbed DNDI-5421 and DNDI-5610, could replace DNDI-6148 if the latter does not successfully complete the preclinical studies [117].

Compounds from PDD screening

New technologies based on bioimaging have transformed the field of drug discovery during the last decade. High-content screening (HCS) technology aided by robotic automation, have permitted the assessment of millions of small molecules under PDD conditions [118]. This has provided multiparametric information from intramacrofage amastigote infections, which up to now, is one of the most physiological relevant assays to find high quality hits under HTS conditions [119], [120]. Unfortunately, the mechanism of action of many of these compounds has not yet being elucidated, and deconvolution studies are in process in order to optimize the basic hit scaffolds with antileishmanial activity (Fig. 4).

Fig. 4: Chemical structure of aminopyrazole (left), pyrazolopyrimidine (top right) and the proteasome inhibitor GNF6702 (right bottom).
Fig. 4:

Chemical structure of aminopyrazole (left), pyrazolopyrimidine (top right) and the proteasome inhibitor GNF6702 (right bottom).


The finding of 5-aminopyrazole and 2-aminothiazole ureas with potent antibacterial activity in vitro vs. Staphylococcus aureus, was described by Kane and co-workers in 2003 [121]. In addition, Pevarello and co-workers tested a series of 3-phenylacetamidoaminopyrazoles targeting a platform based on cyclin-dependent kinases (CDK2/A) with interesting antitumour effects both in vitro and in vivo when administered orally [122]. According to Mowbray, as a result of the DNDi-Pfizer’s agreement to test for a subset of 26 500 small molecules from a 95 000 compounds included in Pfizer’s library, molecules with pyrazole ring were initially identified to be effective against L. donovani amastigotes in THP-1 cells using high-content screening assay [120], [123].

Consequently, a lead optimization campaign to discover additional compounds was designed in order to establish whether 3-aminopyrazole represented a genuinely potent scaffold to be exploited. A series of arylpiperdine/piperazine substituted aminopyrazole ureas yielded the early lead compound, which displayed good potency against both L. infantum and L. donovani intramacrophage amastigotes (IC50=2.37 and 1.31 μM, respectively) and excellent oral bioavaliability and metabolic stability against liver microsomes in vitro. In addition, when hamster infected with L. infantum were treated orally with 50 mg/kg b.i.d. for 5 days, parasite burden in liver and spleen was reduced in 92.7% and 95%, respectively, without any obvious signs of toxicity [123]. Despite the good balance between effectivity and pharmacokinetic parameters of 26, several rounds of lead optimization yielded the arylpyrrolidine derivatives DNDI-1044 (21) and DNDI-8012 (22), with 10-fold higher antileishmanial activity on both L. infantum and L. donovani, and on a list of field resistant clinical strains. DNDI-1044 reduced the parasite burden – in liver and spleen – in more than 95% in the early curative L. infantum model of hamster at a minimum of 12.5 mg/kg b.i.d. for 5 days. DNDI-8012 followed the same pattern, but at a dose of 50 mg/kg bw [124].

To this end, DNDi with support from Takeda Pharmaceutical Company Ltd and the Global Health Innovative Technology (GHIT) are committed to get funds in 2018 to produce the aminopyrazole urea lead candidate DNDi 5561, whose structure is still not disclosed, to enter in first-in-man studies [125].

Pyrazolopyrimidines (CRK-12 kinase inhibitors)

From a target-based high-throughput in vitro screening of a compound library against T. brucei GSK-3 kinase, a series of diaminothiazole carbaldehydes identified a hit, which selectively inhibited the parasite kinase, but not that of the host’s counterpart, and showed good killing effect on the parasite [126]. Encouraged with these results, University of Dundee in collaboration with Glaxo Smith Kline (GSK), Tres Cantos developed a novel series of pyrazolopyrimidines against L. donovani using the axenic amastigote platform described by De Rycker and coworkers [127]. From this lead optimization, GSK-3186899/DDD853651 showed excellent in vitro activity against L. donovani axenic amastigotes, with an EC50 value of 0.02 μM. It exhibited a more modest effect against intramacrophagic parasites – EC50 value of 1.4 μM – and good safety (SI>35) [128]. This compound was stable when incubated with liver microsomes from different sources, and displayed good bioavailability after oral administration in mice. Further characterization revealed that, when this compound was administered orally at 25 mg/kg b.i.d. for 10 consecutive days in a mouse model of visceral leishmaniasis, a reduction of 99% in the parasite load was found. Strikingly, the pull down experiments developed to deconvolute the mechanism of action of pyrazolopyrimidines, led to the identification of Cdc2-related kinase 12 (CRK12) as druggable target for further optimizations [129]. Non-clinical safety data for GSK3186899 suggests a suitable therapeutic window for further development under GLP standards. Preliminary preclinical assessment did not show remarkable undesirable effects in sub-acute toxicological study in rats. The in vivo efficacy, novel mechanism of action and safety profile of GSK3186899 (23) support progression to definitive preclinical studies and further phase I clinical trials [125].

Proteasome inhibitors

One of the latest studies of massive phenotypic screening of small molecules was carried out by Novartis in 2016, in collaboration with Wellcome Trust. Starting from a library of more than 3 million compounds, a massive screening was carried out on four kinetoplastids, including trypanosomes and leishmanias. A subpopulation of compounds with azabenzoxazole structure was isolated from the screenings. An optimization campaign was started for this type of compounds. Up to, 3000 molecules were obtained, leading to the identification of GNF6702 (24), which showed a power 400 times greater than that of the original hit in intramacrofagic L. donovani tests. Oral dosing with GNF6702 under an 8-day treatment with 10 mg/kg twice-daily, led to a steeper reduction in liver parasite burden than miltefosine, despite its modest 34% oral bioavailability. This compound showed better curative effects than the election drug against T. brucei and T. cruzi experimental infections when administered orally at 10 mg/kg, but was not able to reduce completely the parasite burden in a mouse model of L. major CL. In an attempt to disclose the mechanism of action of this type of compounds, whole-genome sequencing of a T. cruzi resistant line to a GNF6702 analogous, revealed a mutation in the locus of the proteasome β4 subunit. However, kinetic studies carried out with GNF6702 with different proteasome substrates showed that this compound blocks the chymotrypsin-like activity harbored by the β5 subunit without competing with substrate binding, and mutations in the β4 subunit, which is in direct physical contact with the β5 subunit, confer resistance to this inhibition. These results turned out to be a common feature in other kinetoplastids and pointed to a new target to be druggable against VL [130].


The interest in NTDs caused by kinetoplastids reached an inflection point at the beginning of this century. It is widely assumed that an advanced and globalized society cannot tolerate the existence of poverty-related diseases, whose incidence can be significantly reduced with preventive health measures. Thanks to this awareness-raising effort and to Non-Governmental Organizations initiatives, always in close collaboration with the Health Departments of endemic countries, the striking results achieved by Big-Pharma and Academic Institutions in recent years may represent a paradigmatic shift in the eradication of these diseases during the next decades.

In this regard, elimination campaigns promoted by Bangladesh, Nepal and India have drastically reduced the incidence by more than 79% and mortality by more than 94% in these countries, thus leaving the East African region with the highest incidence worldwide [7], [131]. Unfortunately, in addition to human suffering, the impact of VL also implies enormous economic costs, which are estimated in 2.4 million DALYs lost worldwide with a special incidence in endemic Eastern African countries. Taking into account that case detection and treatment were estimated in $11–22 per DALY averted only for VL, the future development of these countries is seriously compromised in the short and medium term [2].

During these years, several successful events have changed the pharmacological management of VL, starting with the use of miltefosine as the first oral treatment in 2002, the introduction of paromomycin in 2005, the administration of AmBisome® at single dose in India in 2010 [20], and the most recent allometric dosage of miltefosine to children in India, which has increased the effectiveness of this drug from a modest 45% to more than 90% [56], [132]. In addition to all these scientific successes, others in the commercial and political field are also remarkable. The contribution of the private-sector with donated or preferentially low-priced drugs, has reduced the costs of the treatments in endemic countries. In this regard, both the introduction of new indigenous formulations of liposomal amphothericin B – Fungisome – and the WHO/Gilead donation program of 445 000 vials in 2011 [133], (recently extended to 380 000 additional vials since 2016), has reduced in more than 90% the price of AmBisome® in India.

Unfortunately, not everything is good news during these years. The ineffectiveness of Pentostam® in north-western regions of India due to the emergence of resistant strains to pentavalent antimony is remarkable. The current unknown role played by PKDL [134], the presence of asymptomatic infections-common in endemic areas [135], [136] – as reservoirs of the disease, the high levels of arsenic in drinking water may be responsible for the cross-tolerance to antimony found in certain hyperendemic areas of Bihar state in India [28], [31], or the first cases reported of resistance to miltefosine in India [137]. Moreover, a cold chain is necessary for the proper delivery of amphotericin B formulations to target places due to the poor chemical stability of this compound. However, cold facilities are only available in clinics or hospitals of urban centers, but are absent in most villages where the incidence of VL is higher [37].

DNDi considered combination of antileishmanial drugs in clinical use adapted to the specific geographical peculiarities, a priority therapeutic initiative in order to face the more urgent challenges of VL-devastated regions. In India, until the generalized replacement with Ambisome® at a single dose in 2010, the introduction of combinations of Ambisome®+paromomycin and miltefosine+paromomycin increased the tolerability of the drugs, reduced the duration of treatment and consequently, increased direct and indirect costs [138]. Since antimony is still effective, the picture in Eastern African countries is different, and the combination of Pentostam®+paromomycin has an efficacy of more than 90% nowadays. However, repeated parenteral administration for 17 days requires hospitalization of the patient, with the subsequent loss of working days [139]. However, although it is the treatment of choice recommended by WHO in this region, its general use is not being feasible in many remote villages far from urban centers [140]. The future replacement of Pentostam®+paromomycin will greatly facilitate self-medication of patients. A validated collaborative phase III clinical study (NCT03129646) supported by EU is under way, and will allow the replacement of Pentostam+paromomycin with miltefosine+paromomycin by 2020 if, as expected, this combination has similar efficacy and complies with the TPP required for VL treatment.

To these humanitarian campaigns conduced to improve the combination and delivery of clinically active antileishmanial drugs, a further effort is also addressed in order to discover new chemical entities that can eventually replace the old ones. DNDi is promoting an integrative research in medicinal chemistry between research centers of large companies, such as GSK in Tres Cantos (Madrid) or Novartis, and academic laboratories. Thanks to this effort, myriads of small molecules from the libraries of these companies are being screened in in vitro platforms of VL. For its part, academic laboratories offer new VL models closer to reality, which have evolved rapidly in recent years due to the advances in miniaturization and robotization. On the one hand, the high content screening platforms and the use of genetically modified pathogens expressing genes that encode fluorescent reporter or luminescent proteins, are enabled to select empirically hit compounds, which will be expanded and optimized to lead candidates in a feedback process. On the other hand, academic institutions have also provided improved in vitro models of the disease. We have gone from the original studies in promastigotes and axenic amastigotes to the possibility of analyzing the effect of the drugs in intramacrophagic amastigotes or 3D cultures of infected splenocytes, which brings the in vitro model closer to the reality of the disease [119], [120].

The expansion and optimization of leads by medicinal chemistry laboratories have been guided by the principles imposed by the TPPs of the disease, where host safety, microsomal stability, oral bioavailability and low synthesis costs are prioritized. Based on all these principles, there is a plethora of new molecules, which supported by DNDi, are currently in the process of entering human clinical trials for the first time. From the collaborative consortiums, several drug candidates have overcome all the conditions stipulated in the preclinical effectiveness and safety tests under GLP standards in order to be investigated in first-to-man clinical studies. Among these new entities, those resulting from the repositioning chemical scaffolds from other diseases, such as nitroaromatic and benzoxaborole compounds, are expected to be the first molecules to provide results in humans in the coming years. These compounds could be the novel drugs replacing the current ones in the coming decades. In addition, a second line of optimized leads are waiting for entering phase I studies in a short time, in case that novel compounds had undesirable effects or begin to produce resistance soon after its commercialization.

Article note

A collection of invited papers based on presentations at the 2nd International Conference on Global Challenges in Neglected Tropical Diseases (NTD2018), San Juan, Puerto Rico, June 25–27 2018.

Award Identifier / Grant number: AGL2016-79813-C2-1R

Award Identifier / Grant number: SAF2017-83575-R

Funding statement: Financial support from the Ministerio de Economía y Competitividad (MINECO, AEI, FEDER, UE) [MINECO, Funder Id:, AGL2016-79813-C2-1R and Funder Id:, SAF2017-83575-R], and the Junta de Castilla y León co-financed by FEDER, UE [LE020P17] is gratefully acknowledged. CGC and BGA are supported by scholarships from the Junta de Castilla y León co-financed by FEDER.

List of abbreviations


Neglected tropical diseases


Cutaneous leishmaniasis


Mucocutaneous leishmaniasis


Visceral leishmaniasis


Post kala-azar dermal leishmaniasis


Human African trypanosomiasis




Drugs for neglected tropical diseases initiative


World Health Organization


Target product profile


High throughput screening


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Published Online: 2019-06-11
Published in Print: 2019-08-27

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