scholarly journals Dynamicity in Host Metabolic Adaptation Is Influenced by the Synergistic Effect of Eugenol Oleate and Amphotericin B During Leishmania donovani Infection In Vitro

2021 ◽  
Vol 11 ◽  
Author(s):  
Amrita Kar ◽  
Adithyan Jayaraman ◽  
Avanthika Kumar ◽  
Santanu Kar Mahapatra
Keyword(s):  
Amphotericin B ◽  
Leishmania Donovani ◽  
Metabolic Adaptation ◽  
Arachidonic Acid Metabolite ◽  
Metabolic Switch ◽  
Accessible Information ◽  
Intracellular Parasites ◽  
Nos2 Expression ◽  
Combinatorial Treatment

Immune metabolic adaptation in macrophages by intracellular parasites is recognized to play a crucial role during Leishmania infection. However, there is little accessible information about changes in a metabolic switch in L. donovani infected macrophages. In previous studies, we have reported on the anti-leishmanial synergic effect of eugenol oleate with amphotericin B. In the present study, we demonstrated that glycolytic enzymes were highly expressed in infected macrophages during combinatorial treatment of eugenol oleate (2.5 µM) and amphotericin B (0.3125 µM). Additionally, we found that the biphasic role in arachidonic acid metabolite, PGE2, and LTB4, is released during this treatment. In vitro data showed that COX-2 mediated PGE2 synthesis increased significantly (p<0.01) in infected macrophages. Not only was the level of prostaglandin synthesis decreased 4.38 fold in infected macrophages after treatment with eugenol oleate with amphotericin B. The mRNA expression of PTGES, MPGES, and PTGER4 were also moderately expressed in infected macrophages, and found to be decreased in combinatorial treatment. In addition, NOS2 expression was activated by the phosphorylation of p38MAPK when combination-treated macrophages were promoted to kill intracellular parasites. The findings of the present study indicate that the synergism between eugenol oleate and amphotericin B could play an important role in immune metabolism adaptation with a concomitant increase in host immune response against the intracellular pathogen, L. donovani.

scholarly journals In Vitro Susceptibilities of Leishmania donovani Promastigote and Amastigote Stages to Antileishmanial Reference Drugs: Practical Relevance of Stage-Specific Differences

2009 ◽  
Vol 53 (9) ◽  
pp. 3855-3859 ◽  
Author(s):  
Marieke Vermeersch ◽  
Raquel Inocêncio da Luz ◽  
Kim Toté ◽  
Jean-Pierre Timmermans ◽  
Paul Cos ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Leishmania Donovani ◽  
Crystalline Substance ◽  
Sodium Stibogluconate ◽  
Cellular Mechanisms ◽  
Intracellular Amastigotes ◽  
Discovery Research ◽  
Drug Discovery Research ◽  
Primary Mouse

ABSTRACT The in vitro susceptibilities of the reference strain Leishmania donovani MHOM/ET/67/L82 to sodium stibogluconate, amphotericin B, miltefosine, and the experimental compound PX-6518 were determined for extracellular log-phase promastigotes, established axenic amastigotes, fresh spleen-derived amastigotes, and intracellular amastigotes in primary mouse peritoneal macrophages. Susceptibility to amphotericin B did not differ across the various axenic models (50% inhibitory concentrations [IC50], 0.6 to 0.7 μM), and amphotericin B showed slightly higher potency against intracellular amastigotes (IC50, 0.1 to 0.4 μM). A similar trend was observed for miltefosine, with comparable efficacies against the extracellular (IC50, 0.4 to 3.8 μM) and intracellular (IC50, 0.9 to 4.3 μM) stages. Sodium stibogluconate, used either as Pentostam or as a crystalline substance, was inactive against all axenic stages (IC50, >64 μg SbV/ml) but showed good efficacy against intracellular amastigotes (IC50, 22 to 28 μg SbV/ml); the crystalline substance was about two to three times more potent (IC50, 9 to 11 μg SbV/ml). The activity profile of PX-6518 was comparable to that of sodium stibogluconate, but at a much higher potency (IC50, 0.1 μg/ml). In conclusion, the differential susceptibility determines which in vitro models are appropriate for either drug screening or resistance monitoring of clinical field isolates. Despite the more complex and labor-intensive protocol, the current results support the intracellular amastigote model as the gold standard for in vitro Leishmania drug discovery research and for evaluation of the resistance of field strains, since it also includes host cell-mediated effects. Axenic systems can be recommended only for compounds for which no cellular mechanisms are involved, for example, amphotericin B and miltefosine.

scholarly journals Studies on the Antileishmanial Mechanism of Action of the Arylimidamide DB766: Azole Interactions and Role of CYP5122A1

2014 ◽  
Vol 58 (8) ◽  
pp. 4682-4689 ◽  
Author(s):  
Trupti Pandharkar ◽  
Xiaohua Zhu ◽  
Radhika Mathur ◽  
Jinmai Jiang ◽  
Thomas D. Schmittgen ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Mechanism Of Action ◽  
Leishmania Donovani ◽  
Synergistic Activity ◽  
Wild Type ◽  
Content Type ◽  
Axenic Amastigotes ◽  
Intracellular Parasites

ABSTRACTArylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellularLeishmaniaparasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raisedLeishmania donovaniaxenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism inLeishmania, is dramatically reduced in DB766R parasites.In vitrosusceptibility assays demonstrated that CYP5122A1 half-knockoutL. donovanipromastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellularL. donovaniamastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.

scholarly journals Mechanism of Amphotericin B Resistance inLeishmania donovani Promastigotes

1998 ◽  
Vol 42 (2) ◽  
pp. 352-357 ◽  
Author(s):  
Nicolas Mbongo ◽  
Philippe M. Loiseau ◽  
Marie A. Billion ◽  
Malka Robert-Gero
Keyword(s):  
Amphotericin B ◽  
Leishmania Donovani ◽  
Saturated Fatty Acids ◽  
Major Fatty Acid ◽  
Fluorescence Measurement ◽  
Wild Type ◽  
Drug Pressure ◽  
Resistant Cells

ABSTRACT Amphotericin B (AmB)-resistant Leishmania donovanipromastigotes were selected by increasing drug pressure, and their biological features were compared with those of the wild-type parent strain. The 50% inhibitory concentration for resistant cells was 20 times higher than that for the wild-type. Resistance was stable after more than 40 passages in drug-free medium, and resistant promastigotes were infective to macrophages in vitro but lost their virulence in vivo. They had 2.5 times longer generation time, decreased AmB uptake, and increased AmB efflux in comparison to the wild type. Fluorescence measurement with a specific plasma membrane probe, 1-[4-(trimethylammonio)-1,6-diphenylhexa]-1,3,5-triene, showed increased membrane fluidity in drug-resistant promastigotes. Analysis of lipid composition showed that in resistant cells saturated fatty acids were prevalent, with stearic acid as the major fatty acid, and the major sterol was an ergosterol precursor, the cholesta-5, 7, 24-trien-3β-ol and not ergosterol as in the AmB-sensitive strain.

scholarly journals Immunoadjuvant Chemotherapy of Visceral Leishmaniasis in Hamsters Using Amphotericin B-Encapsulated Nanoemulsion Template-Based Chitosan Nanocapsules

2013 ◽  
Vol 57 (4) ◽  
pp. 1714-1722 ◽  
Author(s):  
Shalini Asthana ◽  
Anil K. Jaiswal ◽  
Pramod K. Gupta ◽  
Vivek K. Pawar ◽  
Anuradha Dube ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Amphotericin B ◽  
Leishmania Donovani ◽  
Transforming Growth Factor ◽  
Treatment Options ◽  
Interleukin 12 ◽  
Necrosis Factor Alpha ◽  
Content Type

ABSTRACTThe accessible treatment options for life-threatening neglected visceral leishmaniasis (VL) disease have problems with efficacy, stability, adverse effects, and cost, making treatment a complex issue. Here we formulated nanometric amphotericin B (AmB)-encapsulated chitosan nanocapsules (CNC-AmB) using a polymer deposition technique mediated by nanoemulsion template fabrication. CNC-AmB exhibited good steric stabilityin vitro, where the chitosan content was found to be efficient at preventing destabilization in the presence of protein and Ca2+. A toxicity study on the model cell line J774A and erythrocytes revealed that CNC-AmB was less toxic than commercialized AmB formulations such as Fungizone and AmBisome. The results ofin vitro(macrophage-amastigote system; 50% inhibitory concentration [IC50], 0.19 ± 0.04 μg AmB/ml) andin vivo(Leishmania donovani-infected hamsters; 86.1% ± 2.08% parasite inhibition) experiments in conjunction with effective internalization by macrophages illustrated the efficacy of CNC-AmB at augmenting antileishmanial properties. Quantitative mRNA analysis by real-time PCR (RT-PCR) showed that the improved effect was synergized with the upregulation of tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and inducible nitric oxide synthase and with the downregulation of transforming growth factor β (TGF-β), IL-10, and IL-4. These research findings suggest that a cost-effective CNC-AmB immunoadjuvant chemotherapeutic delivery system could be a viable alternative to the current high-cost commercial lipid-based formulations.

scholarly journals In Vitro Reversion of Amphotericin B Resistance in Leishmania donovani by Poloxamer 188

2000 ◽  
Vol 44 (8) ◽  
pp. 2190-2192 ◽  
Author(s):  
S. Espuelas ◽  
P. Legrand ◽  
P. M. Loiseau ◽  
C. Bories ◽  
G. Barratt ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Amphotericin B ◽  
Leishmania Donovani ◽  
Effective Concentration ◽  
Resistant Line ◽  
Poloxamer 188 ◽  
New Formulation

ABSTRACT A micellar formulation of amphotericin B (AmB) solubilized with poloxamer 188 was evaluated against an AmB Leishmania donovani-resistant line. A concave isobologram showed a synergistic effect of this association against promastigotes. This result was confirmed with amastigotes since the 50% effective concentration of the new formulation was 100 times less than that of the control AmB formulation.

scholarly journals Antileishmanial Activity, Uptake, and Biodistribution of an Amphotericin B and Poly(α-Glutamic Acid) Complex

2013 ◽  
Vol 57 (10) ◽  
pp. 4608-4614 ◽  
Author(s):  
Abeer H. A. Mohamed-Ahmed ◽  
Karin Seifert ◽  
Vanessa Yardley ◽  
Hollie Burrell-Saward ◽  
Stephen Brocchini ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Amphotericin B ◽  
Leishmania Donovani ◽  
Water Soluble ◽  
Molecular Weight Range ◽  
Acid Complex ◽  
Content Type ◽  
New Treatment

ABSTRACTA noncovalent, water-soluble complex of amphotericin B (AMB) and poly(α-glutamic acid) (PGA), with AMB loadings ranging from 25 to 55% (wt/wt) using PGA with a molecular weight range of 50,000 to 70,000, was prepared as a potential new treatment for visceral leishmaniasis (VL). The AMB-PGA complex was shown to be as active as Fungizone (AMB deoxycholate) against intracellularLeishmania donovaniamastigotes in differentiated THP-1 cells. Thein vitrouptake of the AMB-PGA complex by differentiated THP-1 cells was similar to that of Fungizone and higher than that of AmBisome (liposomal AMB). The AMB-PGA complex also displayed a dose-response profile similar to that of AmBisomein vivoin BALB/c mice againstL. donovani, with 50% effective doses (ED50s) of 0.24 ± 0.03 mg/kg of body weight for the AMB-PGA complex and 0.24 ± 0.06 mg/kg for AmBisome. A biodistribution study with mice indicated that the AMB-PGA complex cleared more rapidly from plasma than AmBisome, with a comparable low level of distribution to the kidneys.

scholarly journals In Vitro Low Responsiveness of Leishmania donovani Towards Sodium Antimony Gluconate

2016 ◽  
Vol 2 (1) ◽  
pp. 8-12
Author(s):  
Murshed Alam ◽  
AKM Shamsuzzaman ◽  
AKM Musa ◽  
Abul Hossain Khan ◽  
Md Chand Mahmud ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Leishmania Donovani ◽  
Standard Dose ◽  
Kala Azar ◽  
History Of ◽  
Resistant Strains ◽  
Drug Responsiveness ◽  
Drug Resistant Strains ◽  
Sodium Antimony Gluconate

Kala-azar has been uprising concomitantly with drug-resistant strains of the causatinve agent, particularly in the neighbouring India. The actual perspective of drug resistance in Leishmania donovani in Bangladesh is yet to be explored. So, this prospective study, as a preliminary one, was done to observe in vitro drug responsiveness against Sodium Antimony Gluconate (SAG) and Amphotericin B of 41 strains of L. donovani isolated from Kala-azar cases. The cases (n=41) were selected from 45 clinically suspected febrile patients those who were positive for Kala-azar by immunochromatographic test (ICT). The selected cases were subsequently confirmed as Kala-azar by detection of Leishmania Donovan (LD) bodies from bone marrow aspirates (n=38) by microscopy and/or showing promastigotes in modified McNeal, Nicole and Novy (NNN) media (n=41). Minimum Inhibitory Concentrations (MICs) of SAG and Amphotericin B were seen in relation with history of previous SAG therapy of the patients. Among 08 strains with previous SAG therapy, MICs of SAG were 500 µg in 05 (62.5%) and 250 µg in 03 (37.5%) cases. In remaining 33 strains with no previous SAG therapy, MIC of the drug was 250 µg. In all 41 strains, MIC of Amphotericin B was 05 µg irrespective of the history of previous SAG therapy. The study revealed that strains of L. donovani with low responsiveness to standard dose of pentavalent antimonials have been started to appear in our community that needs further study at community level in a larger population.Bangladesh J Med Microbiol 2008; 02 (01): 8-12DOI: http://dx.doi.org/10.3329/bjmm.v2i1.21782

scholarly journals In Vitro Susceptibility of Field Isolates of Leishmania donovani to Miltefosine and Amphotericin B: Correlation with Sodium Antimony Gluconate Susceptibility and Implications for Treatment in Areas of Endemicity

2008 ◽  
Vol 53 (2) ◽  
pp. 835-838 ◽  
Author(s):  
Dhiraj Kumar ◽  
Arpita Kulshrestha ◽  
Ruchi Singh ◽  
Poonam Salotra
Keyword(s):  
Amphotericin B ◽  
Gene Expression Analysis ◽  
Leishmania Donovani ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
In Vitro Susceptibility ◽  
Field Isolates ◽  
Antimony Resistance ◽  
Sodium Antimony Gluconate

ABSTRACT Indian Leishmania donovani isolates (n = 19) from regional zones representing various levels of antimony resistance displayed significantly (P < 0.01) correlated results with respect to in vitro susceptibility to the antileishmanial drugs sodium antimony gluconate, amphotericin B, and Miltefosine, raising the possibility of cross-resistance mechanisms operating in the field isolates. The results of gene expression analysis of LdMT and LdRos3 were suggestive of alternate mechanisms of Miltefosine susceptibility in the isolates.

scholarly journals In Vitro Evaluation of Antileishmanial Activity of Computationally Screened Compounds against Ascorbate Peroxidase To Combat Amphotericin B Drug Resistance

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Rani Mansuri ◽  
Ashish Kumar ◽  
Sindhuprava Rana ◽  
Bhavana Panthi ◽  
M. Yousuf Ansari ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Ascorbate Peroxidase ◽  
Leishmania Donovani ◽  
Harmful Effect ◽  
Annexin V ◽  
Antileishmanial Activity ◽  
New Drugs ◽  
Content Type ◽  
Lipinski’S Rule

ABSTRACT In visceral leishmaniasis (VL), the host macrophages generate oxidative stress to destroy the pathogen, while Leishmania combats the harmful effect of radicals by redox homeostasis through its unique trypanothione cascade. Leishmania donovani ascorbate peroxidase (LdAPx) is a redox enzyme that regulates the trypanothione cascade and detoxifies the effect of H2O2. The absence of an LdAPx homologue in humans makes it an excellent drug target. In this study, the homology model of LdAPx was built, including heme, and diverse compounds were prefiltered (PAINS, ADMET, and Lipinski's rule of five) and thereafter screened against the LdAPx model. Compounds having good affinity in terms of the Glide XP (extra precision) score were clustered to select diverse compounds for experimental validation. A total of 26 cluster representatives were procured and tested on promastigote culture, yielding 12 compounds with good antileishmanial activity. Out of them, six compounds were safer on the BALB/c peritoneal macrophages and were also effective against disease-causing intracellular amastigotes. Three out of six compounds inhibited recombinant LdAPx in a noncompetitive manner and also demonstrated partial reversion of the resistance property in an amphotericin B (AmB)-resistant strain, which may be due to an increased level of reactive oxygen species (ROS) and decrease of glutathione (GSH) content. However, inhibition of LdAPx in resistant parasites enhanced annexin V staining and activation of metacaspase-like protease activity, which may help in DNA fragmentation and apoptosis-like cell death. Thus, the present study will help in the search for specific hits and templates of potential therapeutic interest and therefore may facilitate the development of new drugs for combination therapy against VL.

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