Impact of an indigenously produced multi-enzyme complex from Bacillus subtilis KT004404 on growth and blood parameters in broiler chicken.

A 42-days experiment was conducted on a day old birds (n = 400) to evaluate the effect of enzyme supplements in feed on the growth, blood parameters, phosphorous content in bones, and nitrogen retention. Different treatments included: control (C) without enzyme supplement, while the other three groups included enzyme mixture T1 and T2 with two commercially available enzyme mix, and T3 with indigenously produced multi-enzyme complex from Bacillus subtilis KT004404. Birds that were fed with indigenously produced multi-enzyme complex showed significant weight gain as compared to other groups. The total feed intake of the birds fed with enzyme supplements was higher than the birds in the control group. The feed conversion ratio was significantly improved (p < 0.05) in treatment groups (T1, T2, T3) as compared to the control. The blood parameters which were analyzed included uric acid, triglycerides, total cholesterol, and serum proteins i.e. globulin and albumin. Birds fed with the enzyme in the group T1, T2 and T3 exhibited higher (p < 0.05) body weight gain. Tibia ash content was significantly higher (p < 0.05) in T1, T2, and T3 as compared to the control. The results of the current study indicate that supplementing poultry feed with the exogenous multi-enzyme produced from Bacillus subtilis KT004404 improved the growth of the birds, feed utilization, and exhibited beneficial effects on the blood parameters, phosphorous and nitrogen retention in broiler chicken.

Novel coumarin-isatin hybrids as potent antileishmanial agents: Synthesis, in silico and in vitro evaluations.

Leishmaniasis being one of the six major tropical diseases that affects nearly 0.7-1.3 million people annually, has so far limited and high toxic therapeutic options. Herein, we report the synthesis, in silico, and in vitro evaluations of novel coumarin-incorporated isatin hydrazones (Spf-1 - Spf-10) as highly potent and safe antileishmanial agents. Molecular docking was initially carried out to decipher the binding confirmation of lead molecules towards the active cavity of the target protein (Leishmanolysin gp63) of Leishmania tropica. Among all the docked compounds, only Spf-6, Spf-8, and Spf-10 showed high binding affinities due to a pattern of strong conventional hydrogen bonds and hydrophobic π-interactions. The molecular dynamics simulations showed the stable pattern of such bonding and structure-based confirmation with a time scale of 50 ns towards the top compound (Spf-10) and protein. These analyses affirmed the high stability of the system. Three out of ten compounds evaluated for their antileishmanial activity against Leishmania tropica promastigotes and amastigotes were found to be active at micromolar concentrations (IC50 range 0.1-4.13 µmol/L), and most importantly, they were also found to be highly biocompatible when screened for their toxicity in human erythrocytes.

Isolation and Characterization of Leishmanial Adenine Aminohydrolase as a Drug Target.

BACKGROUND: Search for new drug targets is becoming imperative these days, given that marketed chemotherapeutic drugs have lost their efficacy against harmful agents because of adaptability to climatic changes and co-evolving vectors to new hosts. In the wake of such a challenge, the prominence of biochemical studies is increasing by way of exploring selective enzymes and investigating their structural and functional properties through biochemical kinetic parameter Km for the application of IC50 using designed drugs. Recently, discovered Adenine Aminohydrolase (EC 3.5.4.2) in Leishmania has been found to be absent in mammalian purine salvage pathway and thus considered as a promising drug target against infectious agents. OBJECTIVES: The objective of this study is to isolate and characterize AAH by learning its kinetic mode of action using preferred substrate Adenine and additives estimated through expected product formation Hypoxanthine. Bioassays designed to measure exact Enzyme kinetic parameter Km value through establishing hyperbolic curve of an enzyme reaction with the use of exact values of cellular quantities for IC50 application under experimental conditions devised by presteady state approach for SSA validity. METHODS: Following saturation kinetic, the plot of hyperbolic equilibrium curve developed using initial rates of product formation as a function of (Si) through forward shift under circumstance dG0 the system allows product and reactant favored reactions in relation to (Ef) ≈ [E0 = KM] until complete saturation and estimates Km and Vmax of enzyme system under applied conditions. M-M equation used to assess experimental initial rate data for estimation of Km on excel using Solver and nonlinear least square coefficient correlation "R2" using logarithmic equation for nonlinear curve assessment. RESULTS: UV/Vis spectrophotometer selectively analyzed reacting components confirming Enzyme characteristic reaction constant Km equal toi15. 0 ± 2 µ mol acquired from the Hyperbolic curve developed through the use of exact (Si) ranges at selected parameter Km and Vmax. The curve assessed by Michaelis Menten equation provides Km value=14.99 µmol and non-linear least square coefficient correlation "R2" value equal to 0.9895, along with that optimized lysis buffer formulation. In the docked complexes, the interactive amino acids identified were MSE441, ALA 364, GLN363, MSE518, VAL362, GLY517, ASP538, ALA445, TYR521, and TYR444. 2D interactions revealed hydrophobic and alkyl interactions at the noncompetitive binding site of the enzyme and therefore recommended as potential inhibitors against 3ICS protein. CONCLUSION: This study encourages biochemical analysis of the novel enzymes with the use of presteady state rationale in association with the computational tools as an effective way of designing drugs in a short time against selective enzymes to meet the current challenge efficiently.

Synthesis and computational studies of highly selective inhibitors of human recombinant tissue non-specific alkaline phosphatase (h-TNAP): A therapeutic target against vascular calcification.

In this study, we have discovered small druglike molecules as selective inhibitors of human tissue-nonspecific alkaline phosphatase (h-TNAP), an enzyme critical for the regulation of extracellular matrix calcification. The upregulation of h-TNAP is associated with various pathologies particularly the vascular calcification (VC). Selective inhibition of h-TNAP over h-NPP1 may serve as a useful therapeutic strategy against vascular calcification. A series of novel triazolyl pyrazole derivatives (10a-y) in which thiol bearing triazole moiety as the zinc binding functional group was introduced to a pyrazole based pharmacophore was synthesized and evaluated as potent and selective inhibitors of h-TNAP over h-NPP1. The biological screening against h-TNAP, h-IAP, h-NPP1 and h-NPP3 showed that many of the synthesized compounds are selective inhibitors of TNAP. Particularly, the compounds 10a-h, 10j, 10m-q, 10u, 10w and 10x displayed high potency and complete selectivity towards h-TNAP over h-NPP1. Compound 10q emerged as a highly potent inhibitor (IC50 = 0.16 µM or 160 nM) against h-TNAP with 127-fold increased inhibition compared to levamisole. On the other hand, compound 10e was found to be most selective inhibitor against the tested APs and NPPs (IC50 = 1.59 ± 0.36 µM). Binding sites architecture analysis, molecular-docking and molecular dynamics simulations (MDS), revealed the basis for h-TNAP and h-IAP ligand selectivity as well as selectivity towards h-TNAP over h-NPP1. These newly discovered inhibitors are believed to represent valuable lead structures to further streamline the generation of candidate compounds to target VC.

Reactive oxygen species generating photosynthesized ferromagnetic iron oxide nanorods as promising antileishmanial agent.

Aim: To investigate the photodynamic therapeutic potential of ferromagnetic iron oxide nanorods (FIONs), using Trigonella foenum-graecum as a reducing agent, against Leishmania tropica. Materials & methods: FIONs were characterized using ultraviolet visible spectroscopy, x-ray diffraction and scanning electron microscopy. Results: FIONs showed excellent activity against L. tropica promastigotes and amastigotes (IC50 0.036 ± 0.003 and 0.072 ± 0.001 µg/ml, respectively) upon 15 min pre-incubation light-emitting diode light (84 lm/W) exposure, resulting in reactive oxygen species generation and induction of cell death via apoptosis. FIONs were found to be highly biocompatible with human erythrocytes (LD50 779 ± 21 µg/ml) and significantly selective (selectivity index >1000) against murine peritoneal macrophages (CC50 102.7 ± 2.9 µg/ml). Conclusion: Due to their noteworthy in vitro antileishmanial properties, FIONs should be further investigated in an in vivo model of the disease.

Self-Nanoemulsifying Drug Delivery System (SNEDDS) for Improved Oral Bioavailability of Chlorpromazine: In Vitro and In Vivo Evaluation.

Background and Objectives: Lipid-based self-nanoemulsifying drug delivery systems (SNEDDS) have resurged the eminence of nanoemulsions by modest adjustments and offer many valuable opportunities in drug delivery. Chlorpromazine, an antipsychotic agent with poor aqueous solubility-with extensive first-pass metabolism-can be a suitable candidate for the development of SNEDDS. The current study was designed to develop triglyceride-based SNEDDS of chlorpromazine to achieve improved solubility, stability, and oral bioavailability. Materials and Methods: Fifteen SNEDDS formulations of each short, medium, and long chain, triglycerides were synthesized and characterized to achieve optimized formulation. The optimized formulation was characterized for several in vitro and in vivo parameters. Results: Particle size, zeta potential, and drug loading of the optimized SNEDDS (LCT14) were found to be 178 ± 16, -21.4, and 85.5%, respectively. Long chain triglyceride (LCT14) showed a 1.5-fold increased elimination half-life (p < 0.01), up to 6-fold increased oral bioavailability, and 1.7-fold decreased plasma clearance rate (p < 0.01) compared to a drug suspension. Conclusion: The findings suggest that SNEDDS based on long-chain triglycerides (LCT14) formulations seem to be a promising alternative for improving the oral bioavailability of chlorpromazine.

Mannosylated thiolated paromomycin-loaded PLGA nanoparticles for the oral therapy of visceral leishmaniasis.

AIM: The present study evaluates the efficacy of paromomycin (PM)-loaded mannosylated thiomeric nanoparticles for the targeted delivery to pathological organs for the oral therapy of visceral leishmaniasis. MATERIALS & METHODS: Mannosylated thiolated chitosan (MTC)-coated PM-loaded PLGA nanoparticles (MTC-PLGA-PM) were synthesized and evaluated for morphology, drug release, permeation enhancing and antileishmanial potential. RESULTS: MTC-PLGA-PM were spherical in shape with a size of 391.24 ± 6.91 nm and an encapsulation efficiency of 67.16 ± 14%. Ex vivo permeation indicated 12.73-fold higher permeation of PM with MTC-PLGA-PM against the free PM. Flow cytometry indicated enhanced macrophage uptake and parasite killing in Leishmania donovani infected macrophage model. In vitro antileishmanial activity indicated 36-fold lower IC50 for MTC-PLGA-PM as compared with PM. The in vivo studies indicated 3.6-fold reduced parasitic burden in the L. donovani infected BALB/c mice model. CONCLUSION: The results encouraged the concept of MTC-PLGA-PM nanoparticles as promising strategy for visceral leishmaniasis.

Design of mannosylated oral amphotericin B nanoformulation: efficacy and safety in visceral leishmaniasis.

The aim of this study was to evaluate mannose-anchored thiolated chitosan (MTC) based nanocarriers (NCs) for enhanced permeability, improved oral bioavailability and anti-parasitic potential of amphotericin B (AmB). Transgenic Leishmania donovani parasites expressing red fluorescent protein DsRed2 and imaging-flow cytometry was used to investigate parasitic burdens inside bone marrow-derived macrophages ex vivo. Cytokine estimation revealed that MTC nanocarriers activated the macrophages to impart an explicit immune response by higher production of TNF-α and IL-12 as compared to control. Cells treated with MTC NCs showed a significantly higher magnitude of nitrite and propidium iodide (PI) fluorescence intensity in contrast to cells treated with AmB. Concerning to apparent permeability coefficient (Papp) results, the MTC NCs formulation displayed more specific permeation across the Caco-2 cell monolayer as compared to AmB. The half-life of MTC NCs was about 3.3-fold persistent than oral AmB used as positive control. Also, t oral bioavailability of AmB was increased to 6.4-fold for MTC NCs compared to AmB for positive control. Acute oral evaluation indicated that MTC NCs were significantly less toxic compared to the AmB. Based on these findings, MTC NCs seems to be promising for significant oral absorption and improved oral bioavailability of AmB in leishmaniasis chemotherapy.

Mannosylated thiolated polyethylenimine nanoparticles for the enhanced efficacy of antimonial drug against Leishmaniasis.

AIM: Our aim was to inhibit trypanothione reductase (TR) and P-gp efflux pump of Leishmania by the use of thiolated polymers. Thus, increasing the intracellular accumulation and therapeutic effectiveness of antimonial compounds. METHODS: Mannosylated thiolated chitosan and mannosylated thiolated chitosan-polyethyleneimine graft were synthesized and characterized. Meglumine antimoniate-loaded nanoparticles were prepared and evaluated for TR and P-gp efflux pump inhibition, biocompatibility, macrophage uptake and antileishmanial potential. RESULTS: Thiomers inhibited TR with Ki 2.021. The macrophage uptake was 33.7- and 18.9-fold higher with mannosylated thiolated chitosan-polyethyleneimine graft and mannosylated thiolated chitosan nanoparticles, respectively, as compared with the glucantime. Moreover, the in vitro antileishmanial activity showed 14.41- and 7.4-fold improved IC50 for M-TCS-g-PEI and M-TCS, respectively as compared with glucantime. CONCLUSION: These results encouraged the concept that TR and P-gp inhibition by the use of thiomers improves the therapeutic efficacy of antimonial drugs.

Redox biology of Leishmania and macrophage targeted nanoparticles for therapy.

Intramacrophage parasite 'Leishmania' has developed various mechanisms for proficient uptake into macrophages and phagosome regulation to avoid macrophage's oxidative burst induced by peroxide, hydroxyl radical, hypochlorous acid and peroxynitrite production. One major barrier for impairing the accession of old fashioned anti-Leishmanial drugs is intrinsic incapability to pass through cell membranes and limiting their abilities to ultimately destroy intracellular pathogens. Receptor-mediated targeted drug delivery to the macrophages by using nanoparticles emerges as promising strategy to improve therapeutic efficacy of old-fashioned drug. Receptor-mediated targeted nanoparticles can migrate across the cell membrane barriers and release enclosed drug cargo at sites of infection. This review is focusing on Leishmania-macrophage signaling alterations, its association with drug resistance and role of nanoparticles for receptor mediated macrophage targeting.

Development of mannose-anchored thiolated amphotericin B nanocarriers for treatment of visceral leishmaniasis.

AIM: Our goal was to improve treatment outcomes for visceral leishmaniasis by designing nanocarriers that improve drug biodistribution and half-life. Thus, long-acting mannose-anchored thiolated chitosan amphotericin B nanocarrier complexes (MTC AmB) were developed and characterized. MATERIALS & METHODS: A mannose-anchored thiolated chitosan nanocarrier was manufactured and characterized. MTC AmB was examined for cytotoxicity, biocompatibility, uptake and antimicrobial activities. RESULTS: MTC AmB was rod shaped with a size of 362 nm. MTC AmB elicited 90% macrophage viability and 71-fold enhancement in drug uptake compared with native drug. The antileishmanial IC50 for MTC AmB was 0.02 µg/ml compared with 0.26 µg/ml for native drug. CONCLUSION: These studies show that MTC can serve as a platform for clearance of Leishmania in macrophages.

In-vitro antileishmanial potential of peptide drug hirudin.

Hirudin is clinically an important drug used for the treatment of cardiac diseases, but has never been elucidated for antileishmanial potential. This study was designed to determine the therapeutic utility of hirudin against leishmaniasis. Binding affinities of 28 potent proteinase inhibitors were screened computationally against leishmanolysin (GP63), out of which hirudin exhibited higher binding affinity with GP63 and good expected IC50 values. Experimentally, hirudin showed most promising activity against promastigote and axenic amastigote forms of leishmanial parasites with IC50 values of 0.60 ± 0.36 µg/mL and 0.43 ± 0.23 µg/mL, respectively, in a dose- and time-dependent assay. The cytotoxicity assay revealed no adverse effects on human macrophages with LD50 value of 860.11 ± 53.44 µg/mL. Hirudin caused leishmanial cell death mainly by apoptosis and membrane permeability. In spite of the basic knowledge obtained, hirudin mechanism is considerably less prone to the induction of resistance than classical drugs. Collectively, this study fosters further studies for the hirudin as new antileishmania lead with a new mode of action.

Photo-induced Leishmania DNA degradation by silver-doped zinc oxide nanoparticle: an in-vitro approach.

Recently, the authors reported newly synthesised polyethylene glycol (PEG)ylated silver (9%)-doped zinc oxide nanoparticle (doped semiconductor nanoparticle (DSN)) which has high potency for killing Leishmania tropica by producing reactive oxygen species on exposure to sunlight. The current report is focused on Leishmania DNA interaction and damage caused by the DSN. Here, we showed that the damage to Leishmania DNA was indirect, as the DSN was unable to interact with the DNA in intact Leishmania cell, indicating the incapability of PEGylated DSN to cross the nucleus barrier. The DNA damage was the result of high production of singlet oxygen on exposure to sunlight. The DNA damage was successfully prevented by singlet oxygen scavenger (sodium azide) confirming involvement of the highly energetic singlet oxygen in the DNA degradation process.

Annihilation of Leishmania by daylight responsive ZnO nanoparticles: a temporal relationship of reactive oxygen species-induced lipid and protein oxidation.

Lipid and protein oxidation are well-known manifestations of free radical activity and oxidative stress. The current study investigated extermination of Leishmania tropica promastigotes induced by lipid and protein oxidation with reactive oxygen species produced by PEGylated metal-based nanoparticles. The synthesized photodynamic therapy-based doped and nondoped zinc oxide nanoparticles were activated in daylight that produced reactive oxygen species in the immediate environment. Lipid and protein oxidation did not occur in dark. The major lipid peroxidation derivatives comprised of conjugated dienes, lipid hydroperoxides, and malondialdehyde whereas water, ethane, methanol, and ethanol were found as the end products. Proteins were oxidized to carbonyls, hydroperoxides, and thiol degrading products. Interestingly, lipid hydroperoxides were produced by more than twofold of the protein hydroperoxides, indicating higher degradation of lipids compared to proteins. The in vitro evidence represented a significant contribution of the involvement of both lipid and protein oxidation in the annihilated antipromastigote effect of nanoparticles.

Structure based medicinal chemistry-driven strategy to design substituted dihydropyrimidines as potential antileishmanial agents.

In an attempt to explore novel and more potent antileishmanial compounds to diversify the current inhibitors, we pursued a medicinal chemistry-driven strategy to synthesize novel scaffolds with common pharmacophoric features of dihydropyrimidine and chalcone as current investigational antileishmanial compounds. Based on the reported X-ray structure of Pteridine reductase 1 (PTR1) from Leishmania major, we have designed a number of dihydropyrimidine-based derivatives to make specific interactions in PTR1 active site. Our lead compound 8i has shown potent in vitro antileishmanial activity against promastigotes of L. Major and Leishmania donovani with IC50 value of 0.47 µg/ml and 1.5 µg/ml respectively. The excellent in vitro activity conclusively revealed that our lead compound is efficient enough to eradicate both visceral and topical leishmaniasis. In addition, docking analysis and in silico ADMET predictions were also carried out. Predicted molecular properties supported our experimental analysis that these compounds have potential to eradicate both visceral and topical leishmaniasis.

Antileishmanial, DNA Interaction, and Docking Studies of Some Ferrocene-Based Heteroleptic Pentavalent Antimonials.

A series of ferrocenyl pentavalent antimonials (1-8) were synthesized and characterized by elemental analysis, FT-IR, and multinuclear ((1) H and (13) C) NMR spectroscopy. These antimonials were evaluated for their antileishmanial potential against Leishmania tropica KWH23, and by biocompatibility and membrane permeability assays. Moreover, mechanistic studies were carried out, mediated by DNA targeting followed by computational docking of ferrocenyl antimonials against the leishmanial trypanothione reductase enzyme. It was observed that the antimonials 1-8 were 390-fold more efficacious (IC50 ) as compared with the standard antimonial drug used. Cytotoxicity results showed that these antimonials are highly active even at low concentrations and are biocompatible with human macrophages. Antimonials 1-8 exhibited extensive intercalation with DNA and, furthermore, docking interactions highlighted the potential interactive binding of the anitimonials within the trypanothione reductase active site, with van der Waals interactions contributing significantly to the process. Hence, it is suggested that the reported antimonials demonstrate high efficacy, less toxicity, and target multiple sites of the Leishmania parasite.

Visible-light-responsive ZnCuO nanoparticles: benign photodynamic killers of infectious protozoans.

Human beings suffer from several infectious agents such as viruses, bacteria, and protozoans. Recently, there has been a great interest in developing biocompatible nanostructures to deal with infectious agents. This study investigated benign ZnCuO nanostructures that were visible-light-responsive due to the resident copper in the lattice. The nanostructures were synthesized through a size-controlled hot-injection process, which was adaptable to the surface ligation processes. The nanostructures were then characterized through transmission electron microscopy, X-ray diffraction, diffused reflectance spectroscopy, Rutherford backscattering, and photoluminescence analysis to measure crystallite nature, size, luminescence, composition, and band-gap analyses. Antiprotozoal efficiency of the current nanoparticles revealed the photodynamic killing of Leishmania protozoan, thus acting as efficient metal-based photosensitizers. The crystalline nanoparticles showed good biocompatibility when tested for macrophage toxicity and in hemolysis assays. The study opens a wide avenue for using toxic material in resident nontoxic forms as an effective antiprotozoal treatment.

PEGylated silver doped zinc oxide nanoparticles as novel photosensitizers for photodynamic therapy against Leishmania.

We describe daylight responsive silver (Ag) doped semiconductor nanoparticles of zinc oxide (DSNs) for photodynamic therapy (PDT) against Leishmania. The developed materials were characterized by X-ray diffraction analysis (XRD), Rutherford backscattering (RBS), diffused reflectance spectroscopy (DRS), and band-gap analysis. The Ag doped semiconductor nanoparticles of zinc oxide were PEGylated to enhance their biocompatibility. The DSNs demonstrated effective daylight response in the PDT of Leishmania protozoans, through the generation of reactive oxygen species (ROS) with a quantum yield of 0.13 by nondoped zinc oxide nanoparticles (NDSN) whereas 0.28 by DSNs. None of the nanoparticles have shown any antileishmanial activity in dark, confirming that only ROS produced in the daylight were involved in the killing of leishmanial cells. Furthermore, the synthesized nanoparticles were found biocompatible. Using reactive oxygen species scavengers, cell death was attributable mainly to 77-83% singlet oxygen and 18-27% hydroxyl radical. The nanoparticles caused permeability of the cell membrane, leading to the death of parasites. Further, the uptake of nanoparticles by Leishmania cells was confirmed by inductively coupled plasma atomic emission spectroscopy (ICP-AES). We believe that these DSNs are widely applicable for the PDT of leishmaniasis, cancers, and other infections due to daylight response.

Antimicrobial, crown gall tumor inhibitory and cytotoxicity assays of N-[(1-methyl-1H-indole-3-yl)methylene]amines synthesized by an improved protocol.

The present paper reports an easy preparation of imines of N-methyl-1H-indole-3-carboxaldehyde by its condensation with alkyl and aromatic amines in ethanol without using any catalyst or dehydrating agent. The compounds have been screened for their antibacterial, antifungal, crown gall tumor inhibitory, and cytotoxic activities. As a major finding some of the compounds exhibited potential biological activity. The imine containing a 4-chlorophenyl group exhibits potential antitumor activity and brine shrimp lethality against crown gall tumor and brine shrimps, respectively. Furthermore, this imine containing a 4-chlorophenyl group also exhibits significant antifungal activity against Candida albicans fungal strains. The compound containing N-diphenylmethyl group has been observed most active against the Gram-positive bacteria.

Drug resistance in leishmaniasis: current drug-delivery systems and future perspectives.

Leishmaniasis is a complex of diseases with numerous clinical manifestations for instance harshness from skin lesions to severe disfigurement and chronic systemic infection in the liver and spleen. So far, the most classical leishmaniasis therapy, despite its documented toxicities, remains pentavalent antimonial compounds. The arvailable therapeutic modalities for leishmaniasis are overwhelmed with resistance to leishmaniasis therapy. Mechanisms of classical drug resistance are often related with the lower drug uptake, increased efflux, the faster drug metabolism, drug target modifications and over-expression of drug transporters. The high prevalence of leishmaniasis and the appearance of resistance to classical drugs reveal the demand to develop and explore novel, less toxic, low cost and more promising therapeutic modalities. The review describes the mechanisms of classical drug resistance and potential drug targets in Leishmania infection. Moreover, current drug-delivery systems and future perspectives towards Leishmaniasis treatment are also covered.