Enhancement in therapeutic efficacy of miltefosine in combination with synthetic bacterial lipopeptide, Pam3Cys against experimental Visceral Leishmaniasis.

Existing drugs for visceral leishmaniasis (VL) are partially effective, toxic, having high cost and long term treatment. Their efficacies are also compromised due to suppression of immune function associated during the course of infection. Combination therapy including a potential and safe immunostimulant with lower doses of effective drug has proven as a significant approach which is more effective than immunotherapy or drug therapy alone. In the present study, we have used the combination of Pam3Cys (an in-built immunoadjuvant and TLR2 ligand) and miltefosine. Initially dose optimization of both the agents was carried out and after that, antileishmanial effect of their combination was evaluated. All experiments were done in BALB/c mouse model. The immunomodulatory role of Pam3Cys on the immune functions of the host receiving combination treatment was also determined using immunological and biochemical parameters viz. phagocytosis, Th1/Th2 cytokines and production of ROS, RNS and H(2)O(2). Combination group showed significant enhancement in parasitic inhibition as compared to groups receiving miltefosine and Pam3Cys separately. Enhanced production of Th1 cytokines as well as ROS, RNS and H(2)O(2) was witnessed during the study of immunological alterations. Remarkable increase in phagocytosis index was also observed. Thus, the risk of development of drug resistance against miltefosine can be resolved through using low doses of it and Pam3Cys (single-dose) in combination and also provide a promising alternative for cure of leishmaniasis, with a pronounced transformation of the host immune response.

Augmentation of antileishmanial efficacy of miltefosine in combination with tuftsin against experimental visceral leishmaniasis.

Current drugs for the treatment of visceral leishmaniasis are inadequate, and their efficacies are also compromised due to suppression of immune function during the course of infection. Miltefosine is the only promising orally active antileishmanial drug, but due to its long half-life, there is risk of development of resistance. To overcome these problems, efforts are needed to develop combination therapy of miltefosine with effective immunostimulating agents where a decrease of parasitic burden and simultaneous enhancement of adaptive immunity can be achieved. In the present study, we have explored the antileishmanial efficacy of a subcurative dose of miltefosine in combination with free as well as liposomal palmitoyl tuftsin (p-tuftsin) using a Leishmania donovani/BALB/c mouse model. When miltefosine (2.5 mg/kg for 5 days) was given with free p-tuftsin, the inhibitory effect was significantly increased from 49.6% to 66% (P < 0.01), which was further enhanced up to 81% (P < 0.001) when given after liposomal encapsulation of p-tuftsin. Significant enhancement in parasitic inhibition (93%, P < 0.01) was witnessed when animals were co-administered with liposomal p-tuftsin + 5 mg/kg × 5 days dose of miltefosine (72.1%). Enhancement in the production of Th1 cytokines (IL-12, TNF-α, and IFN-γ), reactive oxygen, and nitrogen metabolites was witnessed in the combination group. A remarkable increase in phagocytosis index was also observed indicating overall immunological enhancement to antileishmanial activity of miltefosine by p-tuftsin.

Effect of Pam3Cys induced protection on the therapeutic efficacy of miltefosine against experimental visceral leishmaniasis.

Prophylactic potential of synthetic bacterial lipopeptide and a TLR2 agonist, Pam3Cys was first evaluated against experimental visceral leishmaniasis in rodent model. After establishing the potential its effect on therapeutic efficacy of miltefosine was also studied. Pam3Cys showed 74.64% inhibition in parasitic establishment when administered by ip route at a dose of 100 µg/animal spaced at two weeks, i.e. on day -7 and +7 of challenge with Leishmania donovani amastigotes. However, when aforesaid dose of Pam3Cys was given with sub-curative dose of miltefosine (2.5 mg/kg for 5 days) its efficacy enhanced from 49.80% to 92.25%. These findings revealed that this lipopeptide has potential protective efficacy which significantly enhanced the therapeutic efficacy of miltefosine used at low dose against Leishmania infection and warrants detailed investigations on its possible immunopotentiatory actions.

Improved treatment of visceral leishmaniasis (kala-azar) by using combination of ketoconazole, miltefosine with an immunomodulator-Picroliv.

Visceral leishmaniasis (VL) caused by the parasite Leishmania donovani, is a potentially fatal disease. It is characterized by prolonged fever, enlarged spleen and liver, substantial weight loss and progressive anemia. Available drugs are toxic, costly and require prolonged treatment duration viz; 28 days of oral treatment with miltefosine, 30 days infusion with Amphotericin B and 21 days intramascular with paromomycin sulfate. Drug combination for VL clinically proved to shorten the duration of treatment. The efficacy of drugs is also compromised due to suppression of immune function during the course of infection. To combat this situation leishmanicidal efficacy of already marketed standard antifungal drug, ketoconazole under the approach of 'therapeutic switching' in combination with standard antileishmanial drug, miltefosine and a potent immunomodulator agent, picroliv were evaluated in L. donovani/hamsters model. Animals treated with combination of ketoconazole (50 mg/kg, 5 days, po)+miltefosine (5 mg/kg, 5 days, po) showed augmentation in efficacy against leishmania parasite (72%) in comparison to those treated with ketoconazole (54.67%) and miltefosine (54.77%) separately. Co-administration of picroliv (10 mg/kg, 12 days, po) has further enhanced antileishmanial efficacy from 72% to 82%. Significant generation of ROS, RNS and H(2)O(2) and increased phagocytosis was observed in animals treated with ketoconazole+miltefosine; however, addition of picroliv to this combination did not alter the level of metabolites and phagocytosis due to its antioxidative and nonleishmanicidal characteristics, respectively. Significant rise in cell mediated immunity witnessed in this group reveals the role played by the immunomodulator, picroliv and justifies the significance of enhanced cell mediated immunity in the therapy. These findings suggest a new strategy for leishmanial chemotherapy at reduced cost and toxicity.

CpG oligodeoxynucleotide 2006 and miltefosine, a potential combination for treatment of experimental visceral leishmaniasis.

In view of the severe immunosuppression in visceral leishmaniasis (VL), a rational approach to effectively combat the parasitic scourge would be to enhance the immune status of the host. Use of CpG oligodeoxynucleotide (CpG-ODN) against leishmaniasis has previously been reported, especially as an immunomodulator and adjuvant with various immunogens. In the present study, experiments were carried out with BALB/c mice and hamsters infected with Leishmania donovani. Immunostimulating class B bacterial CpG-ODN namely, ODN-2006, was administered at various doses by the intraperitoneal (i.p.) route. The dose of CpG-ODN-2006 (1 nM/single dose) showing the most antileishmanial activity was given as free and liposomal forms with different doses of miltefosine, namely, 5 and 10 mg/kg of body weight, for 5 days in mice and hamsters, respectively. Among the various groups, mice coadministered liposomal CpG-ODN and miltefosine (5 mg/kg) showed the best inhibitory effect (97% parasite inhibition) compared with free CpG-ODN plus miltefosine and miltefosine, free CpG-ODN, and liposomal CpG-ODN given separately. Similar responses were observed in the case of hamsters, where the combination of liposomal CpG-ODN with miltefosine (10 mg/kg) gave 96% parasite inhibition. Promising antileishmanial efficacy was observed in animals treated with liposomal CpG-ODN and miltefosine.

Antileishmanial efficacy of fluconazole and miltefosine in combination with an immunomodulator--picroliv.

The chemotherapy of visceral leishmaniasis (VL) has several limitations including resistance and toxicity of the existing drugs. Downregulation of immune system further aggravates the problems. To combat this situation, leishmanicidal efficacy of already marketed standard antifungal drug, fluconazole under the approach of "therapeutic switching" in combination with standard antileishmanial drug, miltefosine, and a potent immunomodulator agent, picroliv, were evaluated in hamsters infected with Leishmania donovani. Animals treated with fluconazole (50 mg/kg × 5 days, oral (p.o.)) + miltefosine (5 mg/kg × 5 days, p.o.) showed enhancement in antileishmanial efficacy (77%), reactive nitrogen species, reactive oxygen species, hydrogen peroxide, and phagocytosis index as compared to those treated with individual drugs. Addition of picroliv to this combination further increased the antileishmanial efficacy from 77% to 88%. Upregulation of cell-mediated immunity was also observed in animals of this group which strengthens the immunomodulatory role of picroliv. These findings suggest a new option for antileishmanial chemotherapy at lower cost and toxicity.

Immunomodulatory effect of picroliv on the efficacy of paromomycin and miltefosine in combination in experimental visceral leishmaniasis.

Combination therapy for the treatment of visceral leishmaniasis has increasingly been advocated as a way to increase treatment efficacy and tolerance, to reduce treatment duration and cost, and to limit the emergence of drug resistance. In the present work, we have adopted a rational approach, which can modulate the immune response to overcome the negative control systems and to boost the positive killing responses. This study was designed to investigate the immunomodulatory effect of picroliv (standardized fraction from the alcoholic extract of root and rhizome of Picrorhiza kurroa) on a combination of paromomycin and miltefosine using Leishmania donovani/hamster model. Picroliv has significantly enhanced antileishmanial efficacy and lymphocyte proliferation when given in combination with paromomycin and miltefosine. Increased toxic oxygen metabolite generation and phagocytosis were also witnessed. Present study thus establishes the possible use of picroliv as adjunct to antileishmanial chemotherapy.

Antileishmanial activity of benzocycloalkyl azole oximino ethers: the conformationally constraint analogues of oxiconazole.

Antileishmanial activities of 16 synthetic oximino benzocycloalkyl azoles against Leishmania donovani were evaluated in vitro against extracellular promastigotes and intracellular amastigotes. Based on SI (Selectivity Index), five compounds were tested further in vivo in hamster model. Out of these, three compounds have shown medium activity (53-58%) and one has shown significant inhibition of parasite multiplication (70%). Despite the fact that these compounds were better than the existing antileishmanials in respect to IC(50) and SI values, they were less active than miltefosine in vivo. The present study has helped us in identifying a new lead that could be exploited as a potential antileishmanial agent.

CpG oligodeoxynucleotide augments the antileishmanial activity of miltefosine against experimental visceral leishmaniasis.

OBJECTIVES: To evaluate the combination of CpG oligodeoxynucleotide (CpG ODN) and miltefosine for the treatment of experimental visceral leishmaniasis (VL). METHODS: The experiments were carried out using BALB/c mice and hamsters, infected with Leishmania donovani. CpG ODN was administered at various doses by the intraperitoneal (ip) route. The dose of CpG ODN (1 nM/single dose) showing best antileishmanial activity was given as free and liposomal forms with a subcurative dose of miltefosine, namely 2.5 and 5 mg/kg x 5 days in mice and hamsters, respectively. RESULTS: Among the various groups of mice, co-administered liposomal CpG ODN and miltefosine showed the best inhibitory effect (85% inhibition) compared with free CpG ODN and miltefosine, and miltefosine, free CpG ODN and liposomal CpG ODN separately. Production of Th1 cytokines, nitric oxide (NO), reactive oxygen species (ROS) and H(2)O(2) was enhanced. A remarkable increase in the phagocytosis index was also observed, indicating overall immunological support to antileishmanial activity of miltefosine by CpG ODN. Similar responses were observed in hamsters. CONCLUSIONS: Promising antileishmanial efficacy was observed in animals treated with liposomal CpG ODN and miltefosine, strongly supported by enhancement of Th1 cytokines as well as NO, ROS and H(2)O(2) levels. The correlation of experimental findings in both the models (mouse and hamster) strengthens the potential of CpG ODN as an immunomodulator in combination with miltefosine against VL.

Synthesis and molecular docking studies of 1-phenyl-4-glycosyl-dihydropyridines as potent antileishmanial agents.

A series of 1-phenyl-4-glycosyl-dihydropyridines (4-17 and 19-21) were prepared by the one pot multicomponent reaction of glcosyl aldehyde, beta-keto compounds and aniline or substituted aniline in the presence of TBAHS as catalyst. The compounds were screened in vitro and in vivo for their antileishmanial activities. Most of the compounds exhibited moderate to good activity against amastigotes and promastigotes of Leishmania donovani. The compounds 4, 11, 12, 13, and 17 exhibited potent in vivo activity with selectivity index (SI) values 7.43-18.93. Molecular docking studies with these compounds revealed L. donovani PTR1 as the possible target to show antileishmanial activities.