Combination of Liposomal CpG Oligodeoxynucleotide 2006 and Miltefosine Induces Strong Cell-Mediated Immunity during Experimental Visceral Leishmaniasis

<div><p>Immuno-modulators in combination with antileishmanial drug miltefosine is a better therapeutic approach for treatment of Visceral Leishmaniasis (VL) as it not only reduces the dose of miltefosine but also shortens the treatment regimen. However, immunological mechanisms behind the perceived benefits of this combination therapy have not been investigated in detail. In the present study, we hypothesized that potential use of drugs that target the host in addition to the parasite might represent an alternative strategy for combination therapy. We investigated immune responses generated in <i>Leishmania donovani</i> infected animals (hamsters and mice) treated with combination of CpG-ODN-2006 and miltefosine at short dose regimen. Infected animals were administered CpG-ODN-2006 (0.4 mg/kg, single dose), as free and liposomal form, either alone or in combination with miltefosine for 5 consecutive days and parasite clearance was evaluated at day 4 and 7 post treatment. Animals that received liposomal CpG-ODN-2006 (lipo-CpG-ODN-2006) and sub-curative miltefosine (5 mg/kg) showed the best inhibition of parasite multiplication (∼97%) which was associated with a biased Th1 immune response in. Moreover, compared to all the other treated groups, we observed increased mRNA expression levels of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-12) and significantly suppressed levels of Th2 cytokines (IL-10 and TGF-β) on day 4 post treatment in animals that underwent combination therapy with lipo-CpG-ODN-2006 and sub-curative miltefosine. Additionally, same therapy also induced heightened iNOS mRNA levels and NO generation, increased IgG2 antibody level and strong T-cell response in these hamsters compared with all the other treated groups. Collectively, our results suggest that combination of lipo-CpG-ODN-2006 and sub-curative miltefosine generates protective T-cell response in an animal model of visceral leishmaniasis which is characterized by strong Th1 biased immune response thereby underlining our hypothesis that combination therapy, at short dose regimen can be used as a novel way of treating visceral leishmaniasis.</p></div

Effect of different formulation/combination of CpG-ODN-2006 and miltefosine on parasite burden.

<p>(A) <i>L. donovani</i>-infected mice and (B) hamsters were treated with various combinations of CpG-ODN-2006 and miltefosine as described in materials and methods section. At day 4 and day 7 post treatment (A) hamsters and (B) mice of different experimental groups were sacrificed and the hepatic as well as the splenic parasite load was determined by stamps-smear method. Total parasite load in each organ is expressed in LDU unit. 1 LDU  =  amastigote per nucleated cell × organ weight in milligram. (C) Schematic representation of the experimental protocol for treatment of <i>Leishmania donovani</i> infected mice/hamsters with different formulation/combination of CpG-ODN-2006 and miltefosine. Data represents here are representative of three independent experiments. Each of the experiments was done a minimum of three times and data represents mean ± SD. The significance between different experimental groups was calculated by one way ANOVA followed by Tukey's post test using graph pad Prism (version 5.0). Significance: Group II vs normal and all treated groups, group V vs VI and group V vs VII (*P<0.05, **P<0.01, ***P<0.001 and ns; non-significant).</p

Effect of combination therapy on production of <i>Leishmania</i>-specific IgG and its isotypes, IgG1 and IgG2.

<p>Animals (n = 5/group) were treated with various combinations of CpG-ODN-2006 and miltefosine as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094596#pone-0094596-g001" target="_blank">Figure 1</a>. Sera collected from normal, infected and treated animals were processed by ELISA as described in materials and methods section to determine the (A) IgG, IgG1 and IgG2 production in hamsters and (B) IgG1 and IgG2a production in mice. Mean ± SD were calculated by the comparison of treated groups to its infected counterparts. The data are representative of three independent experiments. Each experiment was set in triplicates. The significance between various treated groups was calculated by one way ANOVA followed by Tukey's post test using graph pad Prism (version 5.0). Significance: Group II vs normal and all treated groups (*P<0.05, **P<0.01, ***P<0.001 and ns; non-significant). The data presented here are representative of two independent experiments.</p

Sequences of forward and reverse primers used for quantitative real time-PCR.

<p>Sequences of forward and reverse primers used for quantitative real time-PCR.</p

Effect of CpG-ODN-2006 and miltefosine combination therapy on cytokine response, iNOS expression and NO generation.

<p>Animals (mice and hamsters) were infected with <i>L. donovani</i> and treated with various combinations of CpG-ODN-2006 and miltefosine as described in figure legend 1. (A and B) RNA were isolated from isolated splenocytes on day 4 and day 7 post treatments and levels of mRNA expression of Th1 cytokines (IFN-γ, IL-12 and TNF-α) and iNOS (A) as well as Th2 cytokines (IL-10 and TGF-β) (B) were evaluated by real-time PCR. The mRNA level were normalized to HGPRT and GADPH for hamsters and Balb/c mice respectively and expressed as a fold change compared with uninfected control animals. (C) Nitrite generation by supernatant of splenocytes derived from different experimental groups of animals were stimulated with SLA (5 µg/mL for both hamsters and mice) for 72 h and assayed as described in materials and methods section. Data represents here are representative of three independent experiments. Each of the experiments was done a minimum of three times and data represents mean ± SD. The significance between different experimental groups was calculated by one way ANOVA followed by Tukey's post test using graph pad Prism (version 5.0). Significance: Group II vs normal and all treated groups, group VI vs VII and group VI vs VIII (*P<0.05, **P<0.01, ***P<0.001 and ns; non-significant).</p

Discovery of a New Class of Natural Product-Inspired Quinazolinone Hybrid as Potent Antileishmanial agents

The high potential of quinazolinone containing natural products and their derivatives in medicinal chemistry led us to discover four novel series of 53 compounds of quinazolinone based on the concept of molecular hybridization. Most of the synthesized analogues exhibited potent leishmanicidal activity against intracellular amastigotes (IC₅₀ from 0.65 ± 0.2 to 7.76 ± 2.1 μM) as compared to miltefosine (IC₅₀ = 8.4 ± 2.1 μM) and nontoxic toward the J-774A.1 cell line and Vero cells. Moreover, activation of Th1 type and suppression of Th2 type immune responses and induction in nitric oxide generation proved that <b>8a</b> and <b>8g</b> induce murine macrophages to prevent survival of parasites. Compounds <b>8a</b> and <b>8g</b> exhibited significant in vivo inhibition of parasite 73.15 ± 12.69% and 80.93 ± 10.50% against Leishmania donovani/hamster model. Our results indicate that compounds <b>8a</b>, <b>8g</b>, and <b>9f</b> represent a new structural lead for this serious and neglected disease

Antileishmanial Activity of Pyrazolopyridine Derivatives and Their Potential as an Adjunct Therapy with Miltefosine

A series of pyrazolo­(dihydro)­pyridines was synthesized and evaluated for antileishmanial efficacy against experimental visceral leishmaniasis (VL). Among all compounds, <b>6d</b> and <b>6j</b> exhibited better activity than miltefosine against intracellular amastigotes. Compound <b>6j</b> (50 mg/kg/day) was further studied against Leishmania donovani/BALB/c mice via the intraperitoneal route for 5 days and displayed >91 and >93% clearance of splenic and liver parasitic burden, respectively. Combination treatment of <b>6j</b> with a subcurative dose of miltefosine (5 mg/kg) in BALB/c mice almost completely ameliorated the disease (>97% inhibition) by augmenting nitric oxide generation and shifting the immune response toward Th1. Furthermore, investigating the effect of <b>6j</b> on <i>Leishmania</i> promastigotes revealed that it induced molecular events, such as a loss in mitochondrial membrane potential, externalization of phosphatidylserine, and DNA fragmentation, that ultimately resulted in the programmed cell death of the parasite. These results along with pharmacokinetic studies suggest that <b>6j</b> could be a promising lead for treating VL as an adjunct therapy with miltefosine

Synthesis, Structure–Activity Relationships, and Biological Studies of Chromenochalcones as Potential Antileishmanial Agents

Antileishmanial activities of a library of synthetic chalcone analogues have been examined. Among them, five compounds (<b>11</b>, <b>14</b>, <b>16</b>, <b>17</b>, <b>22</b>, and <b>24</b>) exhibited better activity than the marketed drug miltefosine in in vitro studies against the intracellular amastigotes form of Leishmania donovani. Three promising compounds, <b>16</b>, <b>17</b>, and <b>22</b>, were tested in a L. donovani/hamster model. Oral administration of chalcone <b>16</b>, at a concentration of 100 mg/kg of body weight per day for 5 consecutive days, resulted in >84% parasite inhibition at day 7 post-treatment and it retained the activity until day 28. The molecular and immunological studies revealed that compound <b>16</b> has a dual nature to act as a direct parasite killing agent and as a host immunostimulant. Pharmacokinetics and serum albumin binding studies also suggest that compound <b>16</b> has the potential to be a candidate for the treatment of the nonhealing form of leishmaniasis