Oral Treatment For Visceral Leishmaniasis Research Articles

Leishmania donovani Dipeptidylcarboxypeptidase Inhibitor as a Potential Oral Treatment for Visceral Leishmaniasis.

Chemotherapy is the key intervention to control visceral leishmaniasis (VL), a neglected tropical disease. Current regimens include not only a few drugs but also present several drawbacks, including moderate to severe toxicity, cost, long-term administration, patient compliance, and growing drug resistance. Thus, the need for better treatment options against VL is a priority. In an endeavor to find an orally active and affordable antileishmanial agent, we evaluated the therapeutic potential of compounds belonging to the (2Z,2'Z)-3,3'-(ethane-1,2-diylbis(azanediyl))bis(1-(4-halophenyl)-6-hydroxyhex-2-en-1-ones) series, identified as inhibitor(s) of Leishmania donovani dipeptidylcarboxypeptidase, a novel drug target. Among them, compound 3c exhibited best in vivo antileishmanial efficacy via both intraperitoneal and oral routes. Therefore, the present study led to the identification of compound 3c as the lead candidate for treating VL.

Oral Efficacy of a Diselenide Compound Loaded in Nanostructured Lipid Carriers in a Murine Model of Visceral Leishmaniasis.

Leishmaniasis urgently needs new oral treatments, as it is one of the most important neglected tropical diseases that affects people with poor resources. The drug discovery pipeline for oral administration currently discards entities with poor aqueous solubility and permeability (class IV compounds in the Biopharmaceutical Classification System, BCS) such as the diselenide 2m, a trypanothione reductase (TR) inhibitor. This work was assisted by glyceryl palmitostearate and diethylene glycol monoethyl ether-based nanostructured lipid carriers (NLC) to render 2m bioavailable and effective after its oral administration. The loading of 2m in NLC drastically enhanced its intestinal permeability and provided plasmatic levels higher than its effective concentration (IC50). In L. infantum-infected BALB/c mice, 2m-NLC reduced the parasite burden in the spleen, liver, and bone marrow by at least 95% after 5 doses, demonstrating similar efficacy as intravenous Fungizone. Overall, compound 2m and its formulation merit further investigation as an oral treatment for visceral leishmaniasis.

Repositioning of Tamoxifen in Surface-Modified Nanocapsules as a Promising Oral Treatment for Visceral Leishmaniasis.

Standards of care for human visceral leishmaniasis (VL) are based on drugs used parenterally, and oral treatment options are urgently needed. In the present study, a repurposing strategy was used associating tamoxifen (TMX) with polyethylene glycol-block-polylactide nanocapsules (NC) and its anti-leishmanial efficacy was reported in vivo. Stable surface modified-NC (5 mg/mL of TMX) exhibited 200 nm in size, +42 mV of zeta potential, and 98% encapsulation efficiency. Atomic force microscopy evidenced core-shell-NC. Treatment with TMX-NC reduced parasite-DNA quantified in liver and spleen compared to free-TMX; and provided a similar reduction of parasite burden compared with meglumine antimoniate in mice and hamster models. Image-guided biodistribution showed accumulation of NC in liver and spleen after 30 min post-administration. TMX-NC reduced the number of liver granulomas and restored the aspect of capsules and trabeculae in the spleen of infected animals. TMX-NC was tested for the first time against VL models, indicating a promising formulation for oral treatment.

Structure and Antiparasitic Activity Relationship of Alkylphosphocholine Analogues against Leishmania donovani.

Miltefosine (Milt) is the only oral treatment for visceral leishmaniasis (VL) but its use is associated with adverse effects, e.g., teratogenicity, vomiting, diarrhoea. Understanding how its chemical structure induces cytotoxicity, whilst not compromising its anti-parasitic efficacy, could identify more effective compounds. Therefore, we systemically modified the compound’s head, tail and linker tested the in vitro activity of three alkylphosphocholines (APC) series against Leishmania donovani strains with different sensitivities to antimony. The analogue, APC12, with an alkyl carbon chain of 12 atoms, was also tested for anti-leishmanial in vivo activity in a murine VL model. All APCs produced had anti-leishmanial activity in the micromolar range (IC50 and IC90, 0.46– > 82.21 µM and 4.14–739.89 µM; 0.01– > 8.02 µM and 0.09–72.18 µM, respectively, against promastigotes and intracellular amastigotes). The analogue, APC12 was the most active, was 4–10 fold more effective than the parent Milt molecule (APC16), irrespective of the strain’s sensitivity to antimony. Intravenous administration of 40 mg/kg APC12 to L. donovani infected BALB/c mice reduced liver and spleen parasite burdens by 60 ± 11% and 60 ± 19%, respectively, while oral administration reduced parasite load in the bone marrow by 54 ± 34%. These studies confirm that it is possible to alter the Milt structure and produce more active anti-leishmanial compounds.

The anti-tubercular drug delamanid as a potential oral treatment for visceral leishmaniasis.

There is an urgent requirement for safe, oral and cost-effective drugs for the treatment of visceral leishmaniasis (VL). We report that delamanid (OPC-67683), an approved drug for multi-drug resistant tuberculosis, is a potent inhibitor of Leishmania donovani both in vitro and in vivo. Twice-daily oral dosing of delamanid at 30 mg kg(-1) for 5 days resulted in sterile cures in a mouse model of VL. Treatment with lower doses revealed a U-shaped (hormetic) dose-response curve with greater parasite suppression at 1 mg kg(-1) than at 3 mg kg(-1) (5 or 10 day dosing). Dosing delamanid for 10 days confirmed the hormetic dose-response and improved the efficacy at all doses investigated. Mechanistic studies reveal that delamanid is rapidly metabolised by parasites via an enzyme, distinct from the nitroreductase that activates fexinidazole. Delamanid has the potential to be repurposed as a much-needed oral therapy for VL.

Chemical and bioassay techniques to authenticate quality of the anti-leishmanial drug miltefosine.

Miltefosine, an effective oral treatment of visceral leishmaniasis (VL), was selected in May 2005, by the governments of India, Nepal, and Bangladesh for the elimination of VL. However, abnormally high treatment failure rates reported in patients in Bangladesh, given a miltefosine generic product (“Miltefos”, Popular Pharmaceuticals Ltd.) during 2008, led the World Health Organization (WHO) to procure this formulation for quality testing. Proton (1H) and phosphorous (31P) nuclear magnetic resonance (NMR) analyses of the Miltefos™ capsules did not give the peaks defined for Impavido®, the quality assured VL treatment product from Aeterna Zentaris. Contents of capsules of Impavido® yielded expected peaks for miltefosine (m/z 408.33 for the protonated parent ion and m/z 183.99 plus m/z 124.8 the fragment ions) that were absent in the Miltefos™ capsules. Furthermore, testing using an in vitro Leishmania donovani intracellular amastigote—macrophage model, yielded EC50 values of between 2.55 and 4.06 μg/mL and 3.02 to 5.92 μg/mL for extracts from the Impavido® capsules and the miltefosine standard, respectively. Lack of significant anti-leishmanial activity of Miltefos™ capsules was identified in this assay even at concentrations up to 100 μg/mL. Capsules of Miltefos™ were classified as falsified (absence of stated active pharmaceutical ingredient) by three methods—NMR and mass spectrometry analysis and bioassay.

Nitroimidazo-oxazole compound DNDI-VL-2098: an orally effective preclinical drug candidate for the treatment of visceral leishmaniasis.

The objective of this study was to identify a nitroimidazo-oxazole lead molecule for the treatment of visceral leishmaniasis (VL). A library of 72 nitroimidazo-oxazoles was evaluated in vitro for their antileishmanial activity against luciferase-transfected DD8 amastigotes of Leishmania donovani. On the basis of their in vitro potency and pharmacokinetic properties, the promising compounds were tested in acute BALB/c mouse and chronic hamster models of VL via oral administration and efficacy was evaluated by microscopic counting of amastigotes after Giemsa staining. The best antileishmanial candidates (racemate DNDI-VL-2001) and its R enantiomer (DNDI-VL-2098) were evaluated in vitro against a range of Leishmania strains. These candidates were further studied in a hamster model using various dose regimens. Cytokine and inducible nitric oxide synthase estimations by real-time PCR and nitric oxide generation by Griess assay were also carried out for DNDI-VL-2098. In vitro screening of nitroimidazo-oxazole compounds identified the racemate DNDI-VL-2001 (6-nitroimidazo-oxazole derivative) and its enantiomers as candidates for further evaluation in in vivo models of VL. DNDI-VL-2098 (IC50 of 0.03 μM for the DD8 strain) showed excellent in vivo activity in both mouse and hamster models, with an ED90 value of 3.7 and <25 mg/kg, respectively, and was also found to be very effective against high-grade infection in the hamster model. Our studies revealed that, along with leishmanicidal activity, DNDI-VL-2098 was also capable of inducing host-protective immune cells to suppress Leishmania parasites in hamsters. These studies led to the identification of compound DNDI-VL-2098 as a preclinical candidate for further drug development as an oral treatment for VL.

In vitro metabolism, disposition, preclinical pharmacokinetics and prediction of human pharmacokinetics of DNDI-VL-2098, a potential oral treatment for Visceral Leishmaniasis

The in vitro metabolism and in vivo pharmacokinetic (PK) properties of DNDI-VL-2098, a potential oral agent for Visceral Leishmaniasis (VL) were studied and used to predict its human pharmacokinetics. DNDI-VL-2098 showed a low solubility (10μM) and was highly permeable (>200nm/s) in the Caco-2 model. It was stable in vitro in liver microsomes and hepatocytes and no metabolite was detectable in circulating plasma from dosed animals suggesting very slow, if any, metabolism of the compound. DNDI-VL-2098 was moderate to highly bound to plasma proteins across the species tested (94–98%). DNDI-VL-2098 showed satisfactory PK properties in mouse, hamster, rat and dog with a low blood clearance (<15% of hepatic blood flow except hamster), a volume of distribution of about 3 times total body water, acceptable half-life (1–6h across the species) and good oral bioavailability (37–100%). Allometric scaling of the preclinical PK data to human gave a blood half-life of approximately 20h suggesting that the compound could be a once-a-day drug. Based on the above assumptions, the minimum efficacious dose predicted for a 50kg human was 150mg and 300mg, using efficacy results in the mouse and hamster, respectively.

The R Enantiomer of the Antitubercular Drug PA-824 as a Potential Oral Treatment for Visceral Leishmaniasis

The novel nitroimidazopyran agent (S)-PA-824 has potent antibacterial activity against Mycobacterium tuberculosis in vitro and in vivo and is currently in phase II clinical trials for tuberculosis (TB). In contrast to M. tuberculosis, where (R)-PA-824 is inactive, we report here that both enantiomers of PA-824 show potent parasiticidal activity against Leishmania donovani, the causative agent of visceral leishmaniasis (VL). In leishmania-infected macrophages, (R)-PA-824 is 6-fold more active than (S)-PA-824. Both des-nitro analogues are inactive, underlining the importance of the nitro group in the mechanism of action. Although the in vitro and in vivo pharmacological profiles of the two enantiomers are similar, (R)-PA-824 is more efficacious in the murine model of VL, with >99% suppression of parasite burden when administered orally at 100 mg kg of body weight−1, twice daily for 5 days. In M. tuberculosis, (S)-PA-824 is a prodrug that is activated by a deazaflavin-dependent nitroreductase (Ddn), an enzyme which is absent in Leishmania spp. Unlike the case with nifurtimox and fexinidazole, transgenic parasites overexpressing the leishmania nitroreductase are not hypersensitive to either (R)-PA-824 or (S)-PA-824, indicating that this enzyme is not the primary target of these compounds. Drug combination studies in vitro indicate that fexinidazole and (R)-PA-824 are additive whereas (S)-PA-824 and (R)-PA-824 show mild antagonistic behavior. Thus, (R)-PA-824 is a promising candidate for late lead optimization for VL and may have potential for future use in combination therapy with fexinidazole, currently in phase II clinical trials against VL.

Amphiphilic Antimony(V) Complexes for Oral Treatment of Visceral Leishmaniasis

The need for daily parenteral administration is an important limitation in the clinical use of pentavalent antimonial drugs against leishmaniasis. In this study, amphiphilic antimony(V) complexes were prepared from alkylmethylglucamides (L8 and L10, with carbon chain lengths of 8 and 10, respectively), and their potential for the oral treatment of visceral leishmaniasis (VL) was evaluated. Complexes of Sb and ligand at 1:3 (SbL8 and SbL10) were obtained from the reaction of antimony(V) with L8 and L10, as evidenced by elemental and electrospray ionization-tandem mass spectrometry (ESI-MS) analyses. Fluorescence probing of hydrophobic environment and negative-staining transmission electron microscopy showed that SbL8 forms kinetically stabilized nanoassemblies in water. Pharmacokinetic studies with mice in which the compound was administered by the oral route at 200 mg of Sb/kg of body weight indicated that the SbL8 complex promoted greater and more sustained Sb levels in serum and liver than the levels obtained for the conventional antimonial drug meglumine antimoniate (Glucantime [Glu]). The efficacy of SbL8 and SbL10 administered by the oral route was evaluated in BALB/c mice infected with Leishmania infantum after a daily dose of 200 mg of Sb/kg for 20 days. Both complexes promoted significant reduction in the liver and spleen parasite burdens in relation to those in the saline-treated control group. The extent of parasite suppression (>99.96%) was similar to that achieved after Glu given intraperitoneally at 80 mg of Sb/kg/day. As expected, there was no significant reduction in the parasitic load in the group treated orally with Glu at 200 mg of Sb/(kg day). In conclusion, amphiphilic antimony(V) complexes emerge as an innovative and promising strategy for the oral treatment of VL.

Miltefosine induces programmed cell death in Leishmania amazonensis promastigotes

In the current study, we evaluated the mechanism of action of miltefosine, which is the first effective and safe oral treatment for visceral leishmaniasis, in Leishmania amazonensis promastigotes. Miltefosine induced a process of programmed cell death, which was determined by the externalization of phosphatidylserine, the incorporation of propidium iodide, cell-cycle arrest at the sub-G0/G1 phase and DNA fragmentation into oligonucleosome-sized fragments. Despite the intrinsic variation that is detected in Leishmania spp, our results indicate that miltefosine causes apoptosis-like death in L. amazonensis promastigote cells using a similar process that is observed in Leishmania donovani.

Novel Arylimidamides for Treatment of Visceral Leishmaniasis

Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC(50)], <1 microM) against both Leishmania donovani axenic amastigotes and intracellular Leishmania, the causative agent for human visceral leishmaniasis (VL). A systematic lead discovery program was employed to characterize in vitro and in vivo antileishmanial activities, pharmacokinetics, mutagenicities, and toxicities of two novel AIAs, DB745 and DB766. They were exceptionally active (IC(50) < or = 0.12 microM) against intracellular L. donovani, Leishmania amazonensis, and Leishmania major and did not exhibit mutagenicity in an Ames screen. DB745 and DB766, given orally, produced a dose-dependent inhibition of liver parasitemia in two efficacy models, L. donovani-infected mice and hamsters. Most notably, DB766 (100 mg/kg of body weight/day for 5 days) reduced liver parasitemia in mice and hamsters by 71% and 89%, respectively. Marked reduction of parasitemia in the spleen (79%) and bone marrow (92%) of hamsters was also observed. Furthermore, these compounds distributed to target tissues (liver and spleen) and had a moderate oral bioavailability (up to 25%), a large volume of distribution, and an elimination half-life ranging from 1 to 2 days in mice. In a repeat-dose toxicity study of mice, there was no indication of liver or kidney toxicity for DB766 from serum chemistries, although mild hepatic cell eosinophilia, hypertrophy, and fatty changes were noted. These results demonstrated that arylimidamides are a promising class of molecules that possess good antileishmanial activity and desirable pharmacokinetics and should be considered for further preclinical development as an oral treatment for VL.

Advances in Chagas Disease Chemotherapy

Chagas disease is endemic from Mexico to Argentina, where it is estimated that 16 to 18 million people are infected with its causative agent, Trypanosoma cruzi, and 100 million remain at risk of infection, emphasizing the necessity to sustain and extend control measures and strategies to combat this disease. Specific chemotherapy with benznidazole or nifurtimox has been recommended for treatment of recent and congenital infection. However, clinical trials with nifurtimox and benznidazole have shown that these compounds have very low activity in preventing the development of chronic Chagas disease. Moreover, the drugs induce a number of toxic side effects. The discovery of new active, non-toxic compounds would probably expand treatment, including those patients in which clinical manifestations are absent or can only be disclosed by more elaborate medical procedures. Recent developments in the study of basic biochemical aspects of T. cruzi have allowed for the identification of new targets for chemotherapy. Like many fungi, T. cruzi has a strict requirement for specific endogenous sterol synthesis for cell viability and growth and is extremely susceptible to sterol biosynthesis inhibitors (SBI). An intensive investigation of the potential trypanocidal effect of specific SBI has been performed, and it was demonstrated that some of these compounds exhibited suppressive and curative activity in murine and dog models of acute and chronic Chagas disease. Other potential targets for anti-T. cruzi chemotherapy that include the antiproliferative lysophospholipid analogs (evaluated in clinical trials as the first oral treatment for visceral leishmaniasis), cysteine protease inhibitors and compounds that interfere with purine salvage and inositol metabolism are also discussed.

A PHASE II DOSE-INCREASING STUDY OF SITAMAQUINE FOR THE TREATMENT OF VISCERAL LEISHMANIASIS IN KENYA

Sitamaquine (WR6026) is an 8-aminoquinoline in development for the oral treatment of visceral leishmaniasis (VL). This was an open-label, dose-increasing study to determine the dose-response and safety profile for sitamaquine in Kenyan patients with VL caused by Leishmania donovani. Patients (mean age 15.9 [range = 5-47] years) received sitamaquine daily for 28 days at one of four doses: 1.75 (n = 12), 2.0 (n = 61), 2.5 (n = 12), or 3.0 (n = 12) mg/kg/day. The primary efficacy outcome was cure (absence of parasites on splenic aspirate) in the intent-to-treat population at day 180. Cure was achieved in 79 (83%) of 95 patients overall, and in 11 (92%) of 12, 49 (80%) of 61, 9 (82%) of 11, and 10 (91%) of 11 patients at sitamaquine doses of 1.75, 2.0, 2.5, or 3.0 mg/kg/day, respectively. The most frequent adverse events during active treatment were abdominal pain (12 [12%] of 97) and headache (11 [11%] of 97), and one patient in each of the 2.5 mg/kg/day and 3.0 mg/kg/day dose groups had a severe renal adverse event. The effects of sitamaquine on the kidney need further investigation. Sitamaquine was efficacious and generally well tolerated in Kenyan patients with VL.

Antiprotozoal lysophospholipid analogues: a comparison of their activity against trypanosomatid parasites and tumor cells.

Lysophospholipid analogues (LPAs), originally developed as anti-cancer agents, have shown significant activity against Leishmania spp. and Trypanosoma cruzi, both in vitro and in vivo. Miltefosine, used as a topical formulation (Miltex) for metastases, was registered in 2002 for the oral treatment of visceral leishmaniasis. LPAs interfere with lipid synthesis in T. cruzi and cancer cells, but the activity is about >20-fold higher against the parasite.

Leishmania donovani resistance to miltefosine involves a defective inward translocation of the drug.

Miltefosine (hexadecylphosphocholine [HePC]) is the first drug approved for the oral treatment of visceral leishmaniasis. As part of a study on the mechanisms of action of this drug and on the rates of resistance to this drug, we have been working in vitro with an Leishmania donovani line that was previously shown to be 15-fold more resistant to HePC. We have studied the accumulation of [(14)C]HePC by L. donovani promastigotes and have found a drastic reduction (>95%) in the ability of the resistant line to internalize the drug. Binding of HePC to the plasma membrane and drug efflux from preloaded cells were similar in both drug-sensitive and -resistant lines, and no [(14)C]HePC metabolism was evident in either line. Resistant parasites were also unable to take up other short-chain phospholipid analogs, independently of their polar head group, even though endocytosis remained unaltered. Finally, HePC uptake was temperature and energy dependent and sensitive to the thiol-reactive agent N-ethylmaleimide. We propose that inward translocation of a short-chain phospholipid across the plasma membrane may exist in Leishmania promastigotes and that such activity is defective in the resistant line.

Decrease of intestinal P-glycoprotein activity by 2 n-propylquinoline, a new oral treatment for visceral leishmaniasis

Drugs currently available for visceral leishmaniasis treatment are potentially toxic, have to be administered by parenteral route and frequently give rise to drug resistance, due to the involvement of P-glycoproteins (P-gp) in Leishmania. The purpose of this study was to investigate a possible inhibitory effect of 2 n-propylquinoline (2 nPQ) on P-gp activity. 2 nPQ is a new oral anti-leishmanial drug that has demonstrated its efficacy in BALB/c infected mice with Leishmania donovani [Antimicrob. Agents Chemother. 37 (1993) 859]. Rat everted gut sacs and human intestinal Caco-2 cell lines were used to study the effect of 2 nPQ on P-gp activity. Our results demonstrate an inhibitory effect of 2 nPQ on the P-gp activity with two P-gp substrates (rhodamine 123 and digoxin), two P-gp inhibitors (cyclosporin A and verapamil), and in two different species. Alone or associated with other active drugs, 2 nPQ would be very useful to control Leishmania Multi-Drug-Resistance and intestinal P-gp in humans with kala-azar. Index Descriptors and Abbreviations: VL, visceral leishmaniasis; 2 nPQ, 2 n-propylquinoline; P-gp, P-glycoprotein; MDR, Multi-Drug-Resistance; Caco-2 cells, human colon adenocarcinoma cell line; BALB/c, Consanguine mouse; HIV, human immunodeficiency virus; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered salt Galipea longiflora, Rutaceae plant rat; everted gut sacs; LD 50, lethal dose 50.

Antiprotozoal activities of phospholipid analogues

The antiprotozoal activity of phospholipid analogues, originally developed as anti-cancer drugs, has been determined in the past decade. The most susceptible parasites are Leishmania spp. and Trypanosoma cruzi with activity also shown against Trypanosoma brucei spp., Entamoeba histolytica and Acanthamoeba spp. Miltefosine, an alkylphosphocholine, was registered for the oral treatment of visceral leishmaniasis (VL) in India in March 2002. This review will focus on the biological activities of phospholipid analogues. Biochemical and molecular targets and mechanism(s) of action have been studied extensively in tumor cells but have not been determined in protozoa.

Oral treatment of visceral leishmaniasis with miltefosine

Oral treatment of visceral leishmaniasis with miltefosine

Trial of oral miltefosine for visceral leishmaniasis

There is no effective oral treatment for visceral leishmaniasis (kala-azar), a disseminated intracellular protozoal infection that occurs worldwide. Miltefosine, an alkyl phospholipid developed as an oral antineoplastic agent, is active against visceral infection in animal models. We tested safety, tolerance, and efficacy of miltefosine in kala-azar. Oral doses of miltefosine were given to six groups of five Indian men for 28 days: 50 mg every second day (group 1), 100 mg every second day (group 2), 100 mg/day (group 3), 150 mg/day (group 4), 200 mg/day (group 5), and 250 mg/day (group 6). Assessment for apparent cure--taken as an afebrile state with decreased spleen size and a splenic-aspirate parasite-density score of 0--was done on days 14 and 28. Definitive cure at 8 months required a parasite-free bone-marrow aspirate and no clinical evidence of relapse. 21 of 30 patients were apparently cured on day 14. Transient episodes of vomiting and diarrhoea, were common during weeks 1-2 and were seen in 22 patients. Four other patients in groups 5 and 6 had miltefosine withdrawn after 7-10 days because of vomiting. One patient in group 6 developed renal insufficiency and severe diarrhoea and died on day 21. On day 28, all 29 remaining patients were apparently cured. By 8 months, seven of ten patients in groups 1 and 2 had relapsed; however, 18 of 19 patients treated daily (groups 3-6) appeared to be cured. Among the 21 definitive cures were the four patients treated for 10 days or less and 12 for whom previous therapy with pentavalent antimony had failed. Treatment with miltefosine at 100-150 mg/day for 4 weeks has promise as an effective oral treatment of visceral leishmaniasis including antimony-resistant infection.