Pentavalent antimonials

Organometallic complexes derived from pentavalent antimony (Sb^V) 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, Sb^V-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 Sb^V fall under this denomination: meglumine antimoniate (1) (Glucantime^®) (85 mg Sb^V/100 mL) and sodium stibogluconate (2) (Pentostam^®) (100 mg Sb^V/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 Sb^V 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, Sb^V must be reduced to the more toxic form Sb^III by the reductive metabolism of Leishmania amastigotes [22], which is favored by trypanothione, the specific free-radical scavenger of trypanosomatids [23]. The reduced Sb^III 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].

Sb^V-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 Sb^V 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 This regime is currently recommended in Eastern Africa to fight VL produced by L. donovani, but it has been superseded in Southern European countries by AmBisome^® as first-line drug to fight human – but not canine VL in order to avoid the risk of emergence of resistant strains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, Sb^V are distributed by means of a two- compartment model with a first-order absorption rate [26]. Since the leishmanicidal effect of Sb^V 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 Sb^V formulations causes severe pain at the injection site as well as phlebotoxicity, muscular fibrosis and abscesses. Cardiotoxicity is a common side-effect of Sb^V formulations Arthralgia and myalgia, elevated hepatic enzymes and pancreatitis are other common adverse events [27].

A serious concern about the use of Sb^V-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

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

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

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.

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.

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.

Oxaboroles

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.

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).

Aminopyrazoles

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].