Leishmaniasis Chemotherapy: New Opportunities With Natural Products As Alternative Therapy

Leishmaniasis, neglected tropical diseases is caused by protozoan parasites of the genus Leishmania and causes a wide spectrum of clinical manifestations ranging from self-healing cutaneous lesions to the fatal visceral form. The use of pentavalent antimony, the mainstay of therapy of Leishmaniasis is now limited by its toxicity and alarming increase in unresponsiveness, especially in the Indian subcontinent. Furthermore, other anti-leishmanial like liposomal amphotericin B and miltefosine drugs are unaffordable in many affected countries and as vaccination based approaches have not yet proved to be effective, chemotherapy remains the only alternative, thus emphasizing the need for identifying novel drugs. In this review, we describe the current visceral leishmaniasis (VL) treatments and their limitations. We also focus on the various natural derived products that can be used for as alternative therapy for the treatment of leishmaniasis.

Keywords: visceral leishmaniasis, chemotherapeutics, natural product

INTRODUCTION

Leishmaniasis, a neglected tropical disease caused by parasite that belongs to genus Leishmania which is transmitted to mammalian host by a sand fly vector known as Phlebotomine in the old world and Lutzomyia in the new world1. Disease outcome depends upon the different species of the parasite in different geographical regions of world that results in cutaneous, mucocutaneous and visceral leishmaniasis (VL)2'3. Among these, VL is the most severe form that raises fatality rate as high as 100% within two years if left untreated (WHO). Visceral leishmaniasis also known as Kala azar affect the poorest area in; developing countries with more than 350 million people at risk. Severity of VL is primarily due to parasite migration to the vital organs like liver, spleen and bone marrow of the mammalian host that eventually causes significant morbidity and mortality. There are two types of VL, which differ in their transmission characteristics: zoonotic VL is transmitted from animal to vector to human and anthroponotic VL is transmitted from human to vector to human. Characteristics symptoms of VL include enlargement of spleen and liver, intermittent fever, weight loss, signs of anaemia, abdominal distension and pancytopenia4 that are primarily caused by three different Leishmania species in diverse regions (L. donovani in Indian subcontinent, Africa and Asia, L. infantum in Europe, North Africa and Latin America and L. chagasi in South America)5'6. In more complex situation, many cases of Leishmania and human immunodeficiency virus (HIV) co-infection have also recently increased7. Patients in the endemic area succumb with VL are completely dependent on the chemotherapeutic treatment, which is far away from promising cure rate. Though, pentavalent antimonials being the first line drugs for VL and as well CL have been used extensively for more than 60 years. However, owing to shortcomings like parasite resistance, toxicity to host cells and insensitivity shown by different Leishmania species withdraws the antimonials drugs from chemotherapeutic regime. However, more effective drugs which are currently used for treatment of VL include amphotericin B (deoxycholate formulation (Fungizone8) and its liposomal formulations like unilamellar liposomal formulation (AmBisome??), lipid complex (Abelcet??), and colloidal dispersion (AmphocilTM), paromomycin, pentamidine and the only available oral drug miltefosine9. Though several vaccine candidates have been developed against leishmaniasis, unfortunately no effective vaccine is available in market. Many compounds derived from natural sources have pharmacological activities and may, thus, be of potential utility in drug development and biomedical research.Natural products, primarily plant-derived substances of diverse structural classes, have been described in the literature showing anti-leishmanial properties.
Immunomodulation of the host by means of immunomodulator found to be an effective approach for the treatment of Leishmaniasis that works either by modulating the activity of host immune system or by direct anti-leishmanial activity. Heightened immune response can be achieved by combining a standard drug with an effective immunomodulator. As reported, CpG oligodeoxynucleotide in combination with miltefosine show potent efficacy against experimental visceral leishmaniasis by augmenting the antileishmanial activity of miltefosine10. In this review we provide a brief overview of the current treatment and the active principles of established drugs. Furthermore, we focus on the mechanisms of drug resistance and natural products that are promising leads for the development of novel chemotherapeutics.

Existing therapeutics drugs and recent advances
Antimonials
For the first time in 1905, Plimmer and Thompson showed that sodium and potassium tartrate were effective against trypanosomes. Use of the trivalent antimonials, tarter emetic was first reported for the treatment of CL by Vianna in 1913and the effective report against VL was confirmed by Di Cristina and Caronia in Sicily and Rogers in India in 1915 .However, tartar emetic exhibit side effects like chest pain, cough and great depression. This led to the discovery of pentavalent antimonials. After that, pentavalent antimony compound urea stibamine synthesized by Brahmachari, was used as an effective chemotherapeutic agent against Indian Kala-azar (KA). A homologue of mammalian aquaglyceroporin, AQP9, named AQP1 present in Leishmania is responsible for uptake of Sb (III)11. Recently it has been reported that mitogen-activated protein kinase (LmjMPK2) in Leishmania major is responsible for post translational modification of LmjAQP1, which results in the uptake of Sb (III)12. Although the exact mechanism of action of Sb (V) is still not proved. However, pentavalent antimony (Sb (V)) behaves as a prodrug, which undergoes biological reduction to much more active trivalent form of antimony (Sb (III)) that exhibits antileishmanial activity. However, the site of (amastigote or macrophage) and mechanism of reduction (enzymatic or nonenzymatic) remain controversial. Furthermore, the ability of Leishmania parasites to reduce Sb (V) to Sb (III) is stage-specific. For instance, amastigotes but not promastogotes can reduce Sb (V) to Sb(III). This explains why amastigotes are more susceptible to Sb(V) but promastigotes are not13'14'15'16'17. Other studies have suggested that reduction of Sb(V) to Sb(III) may also take place within macrophages, but level of reduction of Sb(V) to Sb(III) in macrophage cannot be that significant since Sb(III) even at a dose of ~25'??g/mL can kill 50% of the THP1 macrophages18'19.

Mechanism of action
Various mechanism of action of pentavalent antimonials against leishmania has been proposed. Numerous reports recommend that SAG either directly targets the enzyme involved in the parasite metabolic pathways or indirectly modulates the expression of host proteins involved in ROS and NO generation. Initial studies suggested that sodium stibogluconate [Sb(V)] inhibits macromolecular biosynthesis in amastigotes , possibly via perturbation of energy metabolism due to inhibition of glycolysis and fatty acid betaoxidation20. Reports also suggest an enzyme, type I DNA Topoisomerase of Leishmania donovani is also inhibited in a dose dependent manner by SAG21. It has been predicted that SAG induced generation of leishmanicidal molecules like ROS and NO in macrophage by involving signalling proteins namely Phosphoinositide 3-Kinase, mitogen-activated protein kinase and protein kinase C22.

Resistance to Antimonials

Even though antimonials are the first-line drugs, they exhibit several limitations, including parasite resistance, severe side effects and the need for daily parenteral administration. In the 1970's recommended dose regimen for antimonials was 10mg/kg/;600mg maximum for short duration (6 to 10 days).But this regimen did not show effective result in four district of Bihar23. Later on, new regimen with an interval of 10 days in two 10 day course was recommended and significant improvement was observed. A WHO expert committee revised regimen and recommended that Sb (V) dose should be as 20mg/kg/day and maximum up to 850 mg for 20 days.

One of the major causes for resistance to antimonials by parasite is irregular use and incomplete treatments, due to these practices presumably expose the parasites to drug pressure, leading to progressive tolerance of the parasite to Sb (V). On the basis of study of various genes, Leishmania manipulates its own genes to behave as a resistant entity to antimonials. SbIII resistant isolates expressed low level of AQP1 protein24. The composition of the amino acid sequence of AQP1 influences the incorporation rate of SbIII in the parasite, replacement of alanine at position 163 by serine or threonine results in less entry of the active form of antimony25.
Amphotericin B
Being an outcome of therapeutic switching, amphotericin B also display excellent antileishmanial efficacy like its inventive antifungal activity. Most important objective behind the development of amphotericin B encapsulated in the liposome is the targeted delivery into phagocytes of the reticulo- endothelial system and reduction of nephrotoxicity associated with Amphotericin B deoxycholate. Lipid formulations of amphotericin B'liposomal amphotericin B (AmBisome; Gilead Sciences), amphotericin B lipid complex (Abelcet; Enzon Pharmaceuticals), and amphotericin B cholesterol dispersion (Amphotec; InterMune)'have also been successfully applied in VL in India and elsewhere. Nevertheless, the lipid formulations have particular clinical appeal in the treatment of VL, because they have largely remedied the drawbacks of conventional amphotericin B deoxycholate26'27.
Mechanism of action
Antileishmanial activity of polyene antibiotic is due to disruption of the parasite membrane by its affinity for ergosterol, a major sterol in the membrane of parasite. The interaction of AmB with ergosterol leads to the formation of transmembrane AmB channels which induce altered permeability to cations, water and glucose which affect membrane bound enzymes28. However, amphotericin B also binds to the cholesterol present in the membrane of macrophage that leads to lesser macrophage parasite interaction and thus less parasitic infection29.
Liposomal amphotericin B is administered intravenously for a short period of time, usually 3-5 days. Recommended regimen for L-AmB in Mediterranean basin is '20 mg/kg, where as in India, single infusion of L-AmB at a dose of 7.5 or 5 mg/kg is recommended. Combination therapy of liposomal amphotericin B and miltefosine also show excellent cure rate in Indian Kala- azar30. One of the major harmful side effects of amphotericin B is the severe nephrotoxicity that might result in kidney failure31. Major issue with amphotericin B that prevent it from reaching the poor infected people is its high cost. Indeed, oral formulation had limited success due to instability at gastric pH and low solubility.

Resistance
General use of amphotericin B in liposomal formulations could not resist parasite from developing resistance because of longer half life of lipid formulations. In vitro studies claimed that resistant promastigotes reduces affinity for amphotericin B by replacing ergosterol with its precursor cholesta-5, 7,24-trien-3??-ol, which is due to loss of function of S-adenosyl-L-methionine-C24-sterolmethyltransferase (SCMT or ERG6) ,an enzyme responsible for the sterol methylation32.

Miltefosine
Miltefosine, also known as hexadecylphosphocholine is the first oral drug for treatment of visceral leishmaniasis. Miltefosine was identified and evaluated independently in the early 1980s as a potential anticancer drug in Germany and as an antileishmanial drug in the UK. In 2002, after successful phase II and phase III trials miltefosine was approved as first oral drug for the treatment of VL in India33. In vitro studies claimed that L. donovani was the most sensitive species, with ED (50) values in the range of 0.12-1.32 ??M against promastigotes and 1.2-4.6 ??M against amastigotes34.
Mechanism of action
Regarding its antileishmanial activity, various mechanisms has been proposed. Normally, lipid metabolism enzymes are targeted by miltefosine which include inhibition of glycosomal alkyl specific acyl- CoA acyltransferase. However, major factor that contribute to alter the parasite membrane composition is the inhibition of phosphatidylcholine synthesis35. In wild-type promastigotes of Leishmania donovani miltefosine induces nuclear DNA fragmentation, cell shrinkage, and phosphatidylserine exposure that lead to apoptosis like death36. Like other antileishmanial drugs, miltefosine also targets mitochondrial enzymes of L.donovani, including cytochrome C oxidase37. In India, recommended dose of miltefosine is 100mg/day for patients having body weight more than 25 kg and 50mg/day for body weight of less than 25 kg. This dose regimen is recommended for 28 days38'39. Pharmacokinetics studies revealed that first elimination half life of miltefosine is approximately 150 and 200 h ('7 days) and terminal elimination half-life of 30.9 days40, which might be the major contributing factor for resistance in nearby future. Combination chemotherapy of miltefosine with liposomal amphotericin B gave promising cure rate results than monotherapy. Patients were treated with short course, sequential regimen of single dose liposomal amphotericin B which was followed by 7-14 days treatment of miltefosine41.

Resistance
Notably, parasite resistance to miltefosine is due to less drug uptake and higher drug efflux. Resistant parasite clones express low level of L.donovani miltefosine transporter (LdMT) and LDRos3, both of which are responsible for transport of miltefosine across the membrane of parasite42. Serious side effects of miltefosine are renal dysfunction, gastrointestinal side effects, vomiting, loss of appetite and nausea43. Potential teratogenic effect induced by miltefosine contraindicated its use during pregnancy and contraception is required beyond the end of treatment in women of child-bearing age9.

Paromomycin
Paromomycin, an amino glycoside is a broad spectrum antibiotic having inhibitory activity against Gram positive, Gram negative bacteria and some protozoans44. After successful phase III clinical trial conducted in India (2006), paromomycin get licensed for treatment of VL. Being a cationic antibiotic, paromomycin binds to the negatively charged glycocalyx on the parasite membrane that leads to the uptake of paromomycin by parasite through endocytosis45.
Mechanism of action
Exact mode of action of paromomycin is still not elucidated. However, parasite killing occurs through the inhibition of respiration by interfering with the mitochondrial membrane potential46. In addition, NO level was also up regulated in paromomycin treated infected macrophages. Paromomycin is effective at lower concentration against both the stages of parasite also it was found to be effective against SAG resistant clinical isolates47. Standard dose of paromomycin sulfate for treatment of VL is 15mg/kg/day for 21 days with cure rate of 97%48. Few safety issues coupled with paromomycin include ototoxicity, renal dysfunction.

Other chemotherapeutic drugs
Pentamidine
In the early 1980's, pentamidine was used as second line drug for treatment of VL, CL and DCL. Initially with its effective activity and cure rate up to 95% in SAG resistant patients increases the intensity of chemotherapeutic regime. During the short period pentamidine was used in India as a second-line drug for Sb(V)-refractory patients, but later on there was a quick decline in the response rate from ??95% cure rate in in the early 1980s to ??70% a decade later49. Possibly its antileishmanial efficacy is due to its effect on parasite mitochondrial inner membrane potential, and polyamine biosynthesis50'51.
Sitamaquine (SQ)
Sitamaquine, an 8-aminoquinoline analogue, currently facing phase 2 clinical trials for the treatment of VL is administered by an oral route. As sitamaquine is positively charged, it binds to the anionic phospholipid of parasite membrane and then accumulates in the cytosol without interacting with sterol in membrane52. In vitro studies in Leishmania donovani promastigote proved that molecular target of SQ is succinate dehydrogenase of the respiratory chain, which ultimately causes parasite death due to lower ATP levels and altered mitochondrial membrane potential53. Like miltefosine, SQ also induces apoptosis like death in parasite. Major advantage with SQ is its shorter half life; thereby lessen the concern like parasite resistance in nearby future. But adverse effects are also associated including abdominal pain, dyspepsia, cyanosis and renal dysfunction.

Plant derived products as drug (phototherapeutics) for treatment of leishmaniasis
Since chemotherapeutic treatments have been offset by issues like parasite resistance, toxicity to host cells, long duration of parenteral therapy and more cost in developing new formulations (like liposomal Amphotericin B), to surmount these limitations alternative approach by utilizing anti leishmanial action of plant derived extracts may play a significant role in the treatment of leishmaniasis.
Plant derived active extracts has shown varied potential activity and selectivity against various species of Leishmania. Principally biological activity of plant derived extracts is attributable to chemical compounds like alkaloids, flavonoids, terpenoids, polyphenols, chalcones and saponins54.

Remedial alkaloids and their analogs for Leishmaniasis healing
Alkaloids, synthesized as secondary metabolites are pharmacologically active nitrogen containing basic compounds which also comprise anti leishmanial potential. On the way, for development of natural product based drug candidates, a broad class of alkaloids has been uncovered as a source of drug against leishmaniasis.
Quinoline alkaloids: Quinines are well known for their anti malarial activity, although their antileishmanial properties have been well documented under in vitro and in vivo conditions. Initial reports in the literature suggest that 2-n-propylquinoline, chimanine A, B, D were the first quinoline alkaloids derivatives which displayed antileishmanial activity against promastigotes of L. amazonensis55.
Indole alkaloids
Among some of the indole alkaloids, dihydrocorynantheine, corynantheine and corynantheidine showed in vitro antileishmanial activity against L. major with IC50 about 3 ??M. The mechanism of action for these metabolites was based on the inhibition of the respiratory chain of the parasite56.
Alkaloids from marine sources
Many marine sponges e.g. Amphimedon viridis, Acanthostrongylophora species, Neopetrosia species, Plakortis angulospiculatus and Pachymatisma johnstonii serve as rich sources of alkaloids with significant antileishmanial potentials. Renieramycin A shows efficient antileishmanial activity against L. amazonensis with IC50 0.2 ??g/mL. Araguspongin C isolated from a marine sponge Haliclona exigua, displays leishmanicidal activity against promastigotes as well as amastigotes at 100 ??g/mL concentrations57.

Chalcones as antileishmanial agent
A large number of Chalcones have been tested for their antileishmanial potential. Licochalcone A [3], an oxygenated chalcone isolated from Chinese liquorice Glycyrrhiza exhibits strong anti-leishmanial activity, markedly preventing the growth of L. major and L. donovani promastigotes and amastigotes58.IC50 value of licochalcone A against L. donovani intracellular amastigote was 0.9 ??g/ml (2.7 ??M) and 7.2 ??g/ml (21 ??M) against L. major promastigotes59.

Flavonoids
Flavonoids extract also exhibited noticeable activities against Leishmania donovani. Luteolin [1] and [2] isolated from Vitex negundo (Verbenaceae) and Fagopyrum esculentum (Polygonaceae) are potent anti-leishmanial compounds with IC50 values against L. donovani intracellular amastigotes of 12.5 and 45.5 ??M, respectively (Fig. 1). Both compounds are able to induce topoisomerase II-mediated kinetoplastid DNA minicircle cleavage in L. donovani promastigotes and intracellular amastigotes. Treatment of promastigotes with luteolin and quercetin leads to cell cycle arrest in the G0/G1 phase followed by apoptotic cell death. Reports also suggest that luteolin and quercetin are specific inhibitors of topoisomerase I, which is an unusual bi-subunit topoisomerase in Leishmania.

Quinones Many quinone and their derivatives exhibit broad activity against various species of leishmania. A bis-naphthoquinone, diospyrin isolated from the bark of Diospyros Montana (Ebenaceae), exhibits significant activity against promastigotes of L. donovani with an MIC of 2.67 ??M60. Anti leishmnaial activity of this metabolite is due to binding to the parasite's topoisomerase I, thus inhibits the catalytic activity of the enzyme, or by stabilizing the topoisomeraseI-DNA binary complex61.Another quinone named, plumbagin isolated from Plumbago species, shows activity against amastigotes of L. donovani and L. amazonensis with IC50 of 2.24 and 5.87 ??M, respectively62. The mechanism of the action of plumbagin is supposed to be due to their ability to perturb the electron transport chain (ETC) in the mitochondria of the parasite.

Immunomodulation
Cure of leishmaniasis, probably even during chemotherapy, appears to be dependent upon the development of an effective immune response that activates macrophages to produce toxic nitrogen and oxygen metabolites to kill the intracellular amastigotes63'64'65. This process is suppressed by the infection itself which downregulates the requisite signalling between macrophage and T cells, for example, the production of interleukin (IL)-12 or the presentation of major histocompatibility complex (MHC) and co-stimulatory molecules at the macrophage surface. Studies in the 1980s showed that biological immunomodulators such as interferon (IFN)-g can provide a missing signal and enhance the activity of antimonials in the treatment of VL and CL. Recently, a new generation of immunopotentiating drugs have shown potential for leishmaniasis treatment. The imidazoquinoline imiquimod, induces nitric oxide (NO) production in macrophages. Imiquimod was shown to have antileishmanial activity via macrophage activation in experimental models66 and in clinical studies on CL in combination with antimonials67. This sensitivity of Leishmania amastigotes to NO was also exploited in a study using the NO generator nitroso-N-pencillamine (SNAP) topically on L. brazilienisis infections68. In one approach to restore signalling, the substituted benzaldehyde tucaresol, which stimulates a signal to CD4+ T cells and promotes T helper cell (Th1) type 1 cytokine production, showed activity in mouse VL models. A 5 mg /kg oral dose for five days proved effective, resulting in a 60% reduction in the number of L. donovani liver amastigotes in mice69. On a different pathway, anisomycin restores signalling via CD40 and activates p38 mitogen-activated protein (MAP) kinase thus killing parasites in mouse models70. These results indicate that immunomodulatory drugs show promise as an adjunct to chemotherapy.

Conclusion
Leishmaniasis is a poorly investigated disease mainly affecting people in developing countries. Drug screening by the isolation of natural products seems to be an attractive approach which can result in the efficient elucidation of new lead compounds. This is a valuable option to standard screening of large compound libraries. Although a significant number of antileishmanial compounds have been investigated, the number of mechanistic studies is rather small. The actual target sites are unknown in most cases. It is important to highlight that the late discovery process, such as lead generation and optimization steps, and the drug development process, with the pre-clinical animal models for drug efficacy and PK/PD, are still in its infancy for leishmaniasis, given that more systematic approaches to develop new chemical entities for this important but neglected disease have started only relatively recently. Therefore still much about VL chemotherapy is going to be learnt on the way of development of new drugs to treat leishmaniasis, and the real measure of success will be the delivery of these future drugs to the patients in need.

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