In the recent past the standard treatment of kala-azar involved the use of pentavalent antimonials Sb(V). Because of progressive rise in treatment failure to Sb(V) was limited its use in the treatment program in the Indian subcontinent. Until now the mechanism of action of Sb(V) is not very clear. Recent studies indicated that both parasite and hosts contribute to the antimony efflux mechanism. Interestingly, antimonials show strong immunostimulatory abilities as evident from the upregulation of transplantation antigens and enhanced T cell stimulating ability of normal antigen presenting cells when treated with Sb(V)
Leishmaniasis threatens about 350 million men, women, and children in 88 countries around the world. WHO estimates the worldwide prevalence to be approximately 12 million cases, with annual mortality of about 60,000 (
Leishmaniasis is caused by a protozoan parasite of the genus
As noted above, some forms of leishmaniasis, for example, VL might be fatal for patients if left untreated. In the absence of an effective vaccine, the control of leishmaniasis is solely dependent on chemotherapy. The organoantimonial compounds have remained as the first line of treatment for all forms of leishmaniasis for more than 60 years. However, until recently, little is known about the chemical structure of these compounds and the methods used in the industry for their preparation [
Second-line drugs include pentamidine and amphotericin B, but severe side effects and high cost limit their use [
Historically, the cutaneous form of leishmaniasis is a disease of antiquity and was recognized in the Old World with various names such as oriental sore, Delhi boil, Baghdad sore, and so forth. This is an ancient disease. Descriptions of conspicuous lesions have been found on tablets in the library of King Ashurbanipal from the 7th century BC, some of which are thought to have been derived from earlier texts dating from 1500 to 2500 BC. In addition, in the 10th century Arab physicians have described the oriental sore [
Antimony has been used as therapeutics in several centuries. Some authors have suggested its earliest use in ancient Egypt for cosmetic purposes. However, it has been shown that this statement was based on a misreading of the ancient texts [
Structures of two complexes of Sb(V) with
Fast-atom bombardment mass spectrometric (FAB-MS) data of the commercially available meglumine antimoniate suggests that two molecules of meglumine (NMG) coordinate with a single Sb atom in a symmetrical geometry [
Proposed structural formula for 364 Da and 365 Da ions identified by ESI (−)-MS in aqueous solutions of meglumine antimoniate and stibogluconate, respectively. Adapted from [
Pentavalent arsenate (As(V)), a metal related to (Sb(V)), is known to enter via a phosphate transporter [
Pentavalent antimonials are in use against leishmaniases for more than six decades. However, their molecular and cellular mechanisms of action are not yet well understood. It is not even clear whether the final active form is Sb(V) or Sb(III). Three main models could be proposed regarding the mechanism of action of pentavalent antimonials.
According to this model, pentavalent antimony (Sb(V)) behaves as a prodrug, which undergoes biological reduction to much more active/toxic 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 not [
The reduction of Sb(V) to Sb(III) requires an active participation of thiol compounds of both mammalian host and parasite origin [
Interestingly, promastigotes contain higher intracellular concentrations of T(SH)2 and GSH than amastigotes [
An arsenate reductase homologue in
Trypanothione reductase (TR) and zinc-finger protein are the potential molecular targets of Sb(III). Such interaction is consistent with the modality of Cys binding of thiophilic metals such as As(III), Sb(III), and Bi(III). Metal-bound Cys systems are fully deprotonated thiolate anions, the nucleophilicity of which is greatly attenuated upon formation of metal complexes with high thermodynamic stability.
Trypanothione/TR system keeps T(SH)2 in the reduced state and thereby maintains oxidoreductive balance in Leishmania parasite. This protects the parasites from oxidative damage and toxic heavy metals, and delivers the reducing equivalents for DNA synthesis [
According to this model, Sb(V) has intrinsic antileishmanial activity. Initial studies suggested that sodium stibogluconate [Sb(V)] inhibits macromolecular biosynthesis in amastigotes [
Demicheli and coworkers have reported the formation of a complex between adenine nucleosides and Sb(V) [
According to this model antimonials clear intracellular Leishmania parasites via activation of host immune system. Action of sodium antimony gluconate (SAG) is multifaceted. SAG can activate both innate as well as adaptive immune compartments, thereby inducing effective antileishmanial immune response. This not only ameliorates existing infection but also protect from relapse.
Croft and Yardley 2002 [
We further deciphered the signaling mechanisms responsible for SAG-induced ROS and NO production and consequent killing of intracellular leishmania parasites within infected MΦ. SAG-induced ROS generation in MΦ requires phosphorylation of ERK via the PI3K-PKC-Ras/Raf pathway. On the other hand, activation of the PI3K/Akt pathway and downstream p38MAPK is essential for induction of NO production and subsequent parasite killing in
Leishmania infection has been reported to increase PTPase activity mainly that of SHP1 type [
Studies of murine VL infections (BALB/c-
Our study indicates that effect of SAG on T cell compartment is corollary to its action on antigen presenting cells like MΦ. We observed that SAG treatment enhances expression of specifically MHC I molecule on the MΦ surface and enhanced class I mediated antigen presentation, but not the presentation mediated by MHC class II (Figure
SAG increases MHC class I mediated antigen presentation and upregulates expression of MHC class I. MΦs isolated from BALB/c and C57BL/6 mice, cultured in presence or absence of SAG for 24 h. (a) To study the antigen presenting function, peritoneal MΦs from BALB/c and C57BL/6 mice either kept untreated or treated with SAG for 24 h, were used as antigen presenting cells to drive the T-cell hybridoma in presence of appropriate peptide and IL-2 secretion was tested on IL-2-dependent cell line (HT-2). The growth of HT-2 was studied using 3H-Thymidine incorporation. The studies showed that class I but not class II restricted presentation was significantly (
Interestingly stimulation of spleen cells, derived from either
SAG-induces proliferation of T-cells but not of B cells (Figure
SAG directly stimulates proliferation of T cells. 105 lymphocytes, from normal BALB/c mice (a) and 5 × 104 IL-2-dependent CD8+ cytotoxic T cell line (CTLL-2) were plated in each well and were kept either untreated or treated
Thus on the one hand SAG could activate T cell compartment (in both MHC-independent and -dependent manner), and on the other could directly activate MΦs to induce generation of microbicidal effector molecules (ROS and NO) which in concert help to potentiate both innate and cellular arms of immune system to eliminate LD parasites.
Pentavalent antimonial drugs were used worldwide for the treatment of VL and CL for over six decades with little evidence of resistance. There is a regional variation in response to antileishmanial drugs and thus recommendations for treatment of VL vary in different regions. Although the selection of resistant
Until the late 1970s, a small daily dose (10 mg/kg; 600 mg maximum of Sb(V)) for short duration (6 to 10 day) was considered adequate. In an earlier resurgence of Indian VL, which assumed epidemic proportions by 1977, an estimated 250,000 patients were affected in Bihar, when unconfirmed reports suggested a 30% treatment failure with this regimen from the four districts most severely affected, viz Muzaffarpur, Samastipur, Vaishali, and Sitamarhi [
Changing therapeutic response to pentavalent antimonials (Adapted from T. K. Jha, 2006 [
Study | Dose (mg/kg/day) | Duration (days) | No. of courses | No. of cases | Unresponsiveness (%) |
---|---|---|---|---|---|
Jha, (1980) [ | 10 | 10 | 1 | 200 | 17 |
Thakur et al., (1984) [ | 20 | 20 | 1 | 64 | 8 |
>20 | 1 | 62 | 0 | ||
Jha, (1986) [ | Child-20 | Fresh-30 | 1 | Fresh-73 | 1.1 |
Adult-10 | Relapse-60 | 1 | Relapse-17 | ||
Slow | 1 | ||||
response-42 | |||||
Thakur et al., (1988) [ | 10 | 40 | 1 | 371 | 26 |
15 | 1 | 14 | |||
20 | 1 | 3 | |||
Jha, (1992) [ | 20 | 30 | 1 | 252 | 27.1 |
Jha, (1995) [ | 20 | 30 | 1 | 32 | 25 |
Jha, (1998) [ | 20 | 30 | 1 | 30 | 37 |
Thakur et al., (1998) [ | 20 | 30 | 1 | 80 | 54 |
Sundar et al., (2001) [ | 20 | 30 | 1 | 184 | 60 |
Three years later, the same group noted a further decline in cure rate to 71% after 20 days of treatment, and recommended extended duration of treatment in nonresponders. Mishra et al. [
The reason for the emergence of resistance is widespread misuse of the drug. Sb(V) is freely available in India. Both qualified medical practitioners and unqualified quacks used the drug and this unrestricted availability of the drug led to rampant misuse. Most patients (73%) first consult unqualified medical practitioners, who might not use the drug appropriately [
The growing resistance to Sb(V) in India while it still remained sensitive all over the world could be due to the fact that leishmaniasis usually has zoonotic transmission except in the Indian subcontinent and East Africa where the transmission is largely anthroponotic. In an anthroponotic cycle, once Sb(V) resistance gets established, it spreads exponentially and organisms sensitive to the drug get eliminated quickly, whereas the drug-resistant parasites continue to circulate in the community [
In CL the response is not as predictable, because there is considerable variation in sensitivity to Sb(V) among primary isolates from untreated patients with cutaneous leishmaniasis, which correlates with patients' response to treatment [
It is evident from the above discussion that the response towards antimony treatments depends on several factors some are parasite related and some are host dependent.
Variation in clinical response to the pentavalent antimonials sodium stibogluconate, and meglumine antimonate (Glucantime) in VL, CL, and MCL has been a persistent problem in the treatment of leishmaniasis over the past 50 years. One explanation for this phenomenon is the intrinsic difference in species sensitivity to these drugs. In studies using the amastigote-macrophage model,
Role of parasites in antimony treatment failure was established using
Other reports on VL isolates from Sudan have also shown that the clinical response to sodium stibogluconate was reflected in isolates in the amastigote-macrophage model (but not in promastigotes) [
Thiol metabolism has a central role in the maintenance of an intracellular reducing environment so that the cell can defend itself against the damage caused by oxidative stress inside the macrophage, oxidants, certain heavy metals and, possibly, xenobiotics [
The efflux of a drug or its active derivative is a very common mechanism of drug resistance in bacteria, yeasts and fungi, and various pathogenic protozoa, for example,
In Leishmania, both classes of ABC transporters have also been reported to be amplified in various species in response to different drugs under laboratory conditions [
Analysis of the complete Leishmania genome (
Further, no reports are available regarding the amplification of ABC transporter gene(s) in-field isolates. Thus, it is still of great interest to determine whether or not drug-resistant field isolates adopt the same strategies to resist antimony as the laboratory mutants. A second ABC transporter protein (PRP1), involved in antimony resistance, has been isolated by functional cloning selecting for pentamidine resistance [
Dfferential gene expression study showed that expression of aquaglyceroporins AQP1, responsible for Sb(III) uptake, was downregulated at both the promastigote and the intracellular amastigote stages in antimony-resistant
Microtubules are dynamic cytoskeleton polymers consisting of repeating
The immune status of Leishmania infected patients has long been known to affect drug efficacy. This has proven to be of particular importance in relation to pentavalent antimonial treatment of DCL [
It was further shown by our group that antimonials activate important signaling pathways of host immune cells like macrophage to induce ROS and NO that ultimately leads killing of intracellular parasites [
A recent study [
The immune system performs meticulously balanced and harmonious functions and thus protects the host from any undesirable foreign insult. Despite the existence of a multifunctional immunosurveillance process, immunocompetent individuals develop cancer. Cancer induces immense local immunosuppression and global immunosuppression in late stage. Antimonials possess immunomodulatory activity, can activate multiple signaling pathways including NF
At present novel cost-effective delivery systems for antimonials using liposome and cyclodextrin are being developed by Frezard’s group and are showing enhanced efficacy. Interestingly cyclodextrin-based [
Conventional by, amphotericin B has been used as a second-line treatment for VL since the 1960s. This drug exhibits an excellent antileishmanial activity with >90%–95% cure rates in Indian VL cases. The routine scheme of conventional amhotericin B is 1 mg/kg administered on alternate days for a total of 30 days. However, a recent study in India showed 96% cure rates with a dose of 0.75 mg/kg/day for 15 days [
Unresponsiveness and relapses occur rarely, except among HIV-infected patients. In this population, secondary episodes of VL are common and are attributed mainly to relapse but also to reinfection [
Miltefosine (hexadecylphosphocholine) is the first orally administered drug for VL and the latest to enter the market. This agent is associated with high efficacy rates, including cases unresponsive to antimonials [
The exact antileishmanial mechanism of miltefosine remains largely unknown. The intracellular accumulation of the drug appears to be the critical step for its action. The intracellular accumulation of miltefosine includes the following steps: binding to plasma membrane, internalization in the parasite cell (two proteins, the miltefosine transporter LdMT and its beta subunit LdRos3, are the most significant), and intracellular targeting and metabolism [
Resistance to miltefosine may emerge easily during treatment due to single point mutations. Decrease in drug accumulation is the common denominator in all miltefosine resistant Leishmania lines studied to date, and this could be achieved through decreased uptake, increased efflux, faster metabolism, or altered plasma membrane permeability; the first two mechanisms have been already described in models of experimental miltefosine resistance [
Paromomycin (aminosidine) is an aminoglycoside with antileishmanial activity. In a phase III study of VL in India, this drug was associated with 94.6% cure rates, similar to amphotericin B [
Paromomycin inhibits protein synthesis and modifies membrane fluidity and permeability. An
The rational for using combination regimens with different resistance mechanisms over monotherapy relies on the expected enhanced efficacy (through synergy or additive activity without drug interaction), shorter treatment duration, less toxicity, improved compliance, reduced likelihood of emergence of resistance, and reduced costs. A combination policy for VL is supported by the fact that antileishmanial drugs belong to different chemical classes [
Several combination regimens with investigational agents have been tested
There are reports that peroxo- and diperoxo-vanadate compounds are potential antileishmanial agents in a number of
We tested a number of vanadium compounds, which are different from those used against experimental infection, with respect to their ancillary ligands in the coordination sphere of the compounds (Figure
Structures and formulae of the PV compounds [
Improved methods to monitor drug resistance are essential that determine either the (i) phenotypic sensitivity of parasite isolates or (ii) molecular changes that indicate alterations in either the drug target or mechanisms that alter the intraparasite level of active drug. There are problems with both approaches. First, the determination of drug sensitivity of clinical isolates is open to the criticism that pathogen adaptation from host to culture media immediately selects for a subpopulation of pathogens best suited for growth in that medium. The drug sensitivity of parasites must therefore be tested as soon as possible after isolation from the patient using defined agreed protocols. Although promastigote assays are easiest and quickest, this assay is not predictive for pentavalent antimonials, and possibly not for other antileishmanials also, for example, paromomycin, pentamidine, and miltefosine. The amastigote-macrophage assay is currently the only model able to correlate clinical response to the sensitivity of the isolate, as demonstrated in relation to pentavalent antimonials [
The introduction of an oral drug for leishmaniasis offers advantages of improved compliance, self administration, and reduced costs. In the phase IV trial for miltefosine, a 7-day supply is issued to patients who have to return to the clinic each week for examination and resupply. For drugs like miltefosine which have a long half-life and a propensity for selection of resistant forms, the monitoring of daily dosing and the completion of a course of treatment are essential. The directly observed treatment strategy for tuberculosis chemotherapy has been successfully introduced in India by the Revised National TB Control Programme in 1997 (
The approximate cost of treatment of a patient with VL in India is given in Progressive failure of antimonial drug treatment, which is the only available drug treatment in the public health program in India, has driven most of the VL patients in India towards the private sector. The drugs, including antimonials, amphotericin B, and now miltefosine, can be bought over the counter without restriction on quantity. The cash-starved population buys antileishmanial drugs in instalments, and most do not complete treatment [
The improvement in noninvasive serological diagnostic methods with high sensitivity and specificity, for example, DAT, K39, and Katex (urine dipstick), is a major advance in the control of leishmaniasis [
Drug combinations have proven to be essential features of antimicrobial treatment through design or use to (i) increase activity through use of compounds with synergistic or additive activity, (ii) prevent the emergence of drug resistance, (iii) lower required doses, reducing chances of toxic side effects and cost, or (iv) increase the spectrum of activity, for example, the use of an antileishmanial with either an antiinflammatory or immunodulator in cutaneous leishmaniasis. Previous studies on drug combinations for VL, for example allopurinol plus sodium stibogluconate and paromomycin plus sodium stibogluconate [
The strategy to reverse resistance has long been discussed in relation to chloroquine resistance in
There are few better ways to avoid drug resistance than to have an adequate armory of drugs with different targets and no cross-resistance. Although miltefosine has been approved for use in the treatment of VL in India, paromomycin is moving through phase III trials in India and Africa, and sitamaquine remains in phase II development for leishmaniasis [
The control of VL globally is challenged by the widespread emergence of antimonial resistance in India. In the last decade new formulations of conventional antileishmanial drugs as well as new agents became available. The wide use of the oral agent miltefosine was hampered by the potential for teratogenicity and emergence of resistance. Combination regimens should be evaluated in large trials. During last few years several mechanisms of in-field antileishmanial resistance were identified. Understanding their molecular and biochemical characteristics will lead to the design of new drugs and also the molecular surveillance of resistance. In order not to jeopardize the life span of available antileishmanials, their delivery, clinical response, and resistance should be monitored. Overall the development of antileishmanials has been generally slow; new drugs are needed.
The authors gratefully acknowledge the financial support from The Council of Scientific and Industrial Research, New Delhi, India.