Sadhna Sharma

Curcumin nanoparticles attenuate neurochemical and neurobehavioral deficits in experimental model of Huntington's disease.

Till date, an exact causative pathway responsible for neurodegeneration in Huntington’s disease (HD) remains elusive; however, mitochondrial dysfunction appears to play an important role in HD pathogenesis. Therefore, strategies to attenuate mitochondrial impairments could provide a potential therapeutic intervention. In the present study, we used curcumin encapsulated solid lipid nanoparticles (C-SLNs) to ameliorate 3-nitropropionic acid (3-NP)-induced HD in rats. Results of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay and succinate dehydrogenase (SDH) staining of striatum revealed a marked decrease in Complex II activity. However, C-SLN-treated animals showed significant increase in the activity of mitochondrial complexes and cytochrome levels. C-SLNs also restored the glutathione levels and superoxide dismutase activity. Moreover, significant reduction in mitochondrial swelling, lipid peroxidation, protein carbonyls and reactive oxygen species was observed in rats treated with C-SLNs. Quantitative PCR and Western blot results revealed the activation of nuclear factor-erythroid 2 antioxidant pathway after C-SLNs administration in 3-NP-treated animals. In addition, C-SLN-treated rats showed significant improvement in neuromotor coordination when compared with 3-NP-treated rats. Thus, the results of this study suggest that C-SLNs administration might be a promising therapeutic intervention to ameliorate mitochondrial dysfunctions in HD.

PHARMACOKINETICS AND TISSUE DISTRIBUTION STUDIES OF ORALLY ADMINISTERED NANOPARTICLES ENCAPSULATED ETHIONAMIDE USED AS POTENTIAL DRUG DELIVERY SYSTEM IN MANAGEMENT OF MULTI-DRUG RESISTANT TUBERCULOSIS

Sustained release nanoformulations of second line anti-tubercular drugs can help in reducing their dosing frequency and improve patient’s compliance in multi-drug resistant tuberculosis (MDR TB). The objective of the current study was to investigate the pharmacokinetics and tissues distribution of ethionamide encapsulated in poly (DL-lactide-co-glycolide) (PLGA) nanoparticles. The drug loaded nanoparticles were 286 ± 26 nm in size with narrow size distribution, and zeta-potential was −13 ± 2.5 mV. The drug encapsulation efficiency and loading capacity were 35.2 ± 3.1%w/w and 38.6 ± 2.3%w/w, respectively. Ethionamide-loaded nanoparticles were administered orally to mice at two different doses and the control group received free (unencapsulated) ethionamide. Ethionamide-loaded PLGA nanoparticles produced sustained release of ethionamide for 6 days in plasma against 6 h for free ethionamide. The Ethionamide was detected in organs (lung, liver, and spleen) for up to 5–7 days in the case of encapsulated ethionamide, whereas free ethionamide was cleared within 12 h. Ethionamide-loaded PLGA nanoparticles exhibited significant improvement in pharmacokinetic parameters, i.e. Cmax, tmax, AUC0–∞, AUMC0–∞, and MRT of encapsulated ethionamide as compared with free ethionamide. Drug in nanoparticles also exhibited a dose proportional increase in the AUC0–∞ values. The pharmacodynamic parameters such as AUC0–24/MIC, Cmax/MIC, and Time > MIC were also improved. PLGA nanoparticles of ethionamide have great potential in reducing dosing frequency of ethionamide in treatment of MDR TB.

Optimization, in vitro–in vivo Evaluation, and Short-term Tolerability of Novel Levofloxacin-loaded PLGA Nanoparticle Formulation

A novel poly(lactic-co-glycolic acid) (PLGA)-based nanoformulation of levofloxacin was developed for multidrug-resistant tuberculosis with the purpose of achieving sustained release in plasma. After lyophilization of levofloxacin-loaded nanoparticles, the average size, charge, and polydispersity index were 268 ± 18 nm, -10.2 ± 1.5 mV, and 0.15 ± 0.03, respectively. The maximum drug encapsulation efficiency and loading capacity were 36.9 ± 6.1% (w/w) and 7.2 ± 1.2 mg/100 mg nanopowder, respectively. Biphasic extended-release profile was produced in vitro. Scanning electron microscopy and Fourier transform infrared studies showed spherical shape of drug-loaded nanoparticles and no drug-polymer interactions were observed. After single oral administration in mice, levofloxacin-loaded PLGA nanoparticles produced sustained release of levofloxacin for 4 days in plasma against 24 h for free levofloxacin. Levofloxacin was detected in organs (lung, liver, and spleen) for up to 4-6 days in case of levofloxacin-loaded nanoparticles, whereas free levofloxacin was cleared within 24 h. This novel formulation did not show any significant adverse effects on body weight and clinical signs in mice. No treatment-related changes were found in hematological and biochemical parameters and on histopathological evaluation. These results indicate the feasibility of development of an orally efficacious safe formulation of levofloxacin with sustained-release properties.

Phenothiazines as anti-tubercular agents: mechanistic insights and clinical implications

Introduction: Tuberculosis (TB) chemotherapy has been rendered ineffective by the emergence of multi-drug resistant (MDR), extensively drug resistant (XDR) and totally drug resistant strains reinforcing the need for the development of new drugs as a global health priority. Reconsidering phenothiazines for the improvement of TB chemotherapy seems to be a rational option especially in view of their role as inhibitors of type II NADH dehydrogenase, a key component of respiratory chain of Mycobacterium tuberculosis, thus raising the speculation that they can be effective against latent TB as well. Areas covered: This article offers a detailed description of the chemotherapeutic efficacy of phenothiazine compounds against susceptible, drug resistant and latent TB. Furthermore, their clinical implications and molecular mechanisms of action have been reviewed extensively. Expert Opinion: Phenothiazines are currently being evaluated for the treatment of TB and have been shown to be effective against M. tuberculosis through a number of in vitro, ex vivo and in vivo studies. In addition, recent clinical studies have implicated their role in the treatment of MDR/XDR TB also. Therefore, phenothiazines, particularly thioridazine, hold great potential to be considered as safe and effective antimycobacterial agents in near future.

Peptide deformylase – a promising therapeutic target for tuberculosis and antibacterial drug discovery

Background: Tuberculosis (TB) remains the most important infectious disease causing morbidity and death, due to the human pathogen Mycobacterium tuberculosis. The emergence of multi-drug-resistant and extensively-drug-resistant forms of TB have resulted in an increase in the number of TB cases. Thus, there is an urgent need to identify new drugs with novel targets to ensure future therapeutic success. Studies have indicated that peptide deformylase is an interesting potential candidate for discovering antimicrobial agents. Objective: To explore the role of peptide deformylase, a highly conserved metalloprotease and an essential enzyme in bacterial life cycle, as a target for antibacterial as well as antimycobacterial drug development. Methods: This review is based on recent published literature and online resources related to peptide deformylase inhibitors and their anbacterial potential. Results/conclusion: Peptide deformylase is an emerging therapeutic target for the treatment of tuberculosis and peptide deformylase inhibitors can act as potential future antibacterial agents.

In vitro physicochemical characterization and short term in vivo tolerability study of ethionamide loaded PLGA nanoparticles: potentially effective agent for multidrug resistant tuberculosis

Purpose: To achieve prolonged drug release for the treatment of multidrug resistant tuberculosis and to improve the patient compliance, ethionamide loaded PLGA nanoparticles were developed. Material and methods: They were developed by solvent evaporation method and optimized. The optimized formulation was subjected to various physico-chemical characterization, in vitro release studies and in vivo tolerability study. Results and discussion: There was no significant drug-polymer interaction and drug was encapsulated as crystalline form in nanoparticles. In vitro release was sustained up to 15 days in various media. Ethionamide loaded nanoparticles in mice did not reveal any statistically significant treatment related effects on body weight gain and clinical signs. Likewise, no treatment-related toxic effect was found in hematology, clinical chemistry and histopathology. Our results indicate the development of an orally effective safe formulation of ethionamide with sustained release property. Conclusion: Hence, ethionamide loaded nanoparticles offer excellent potential for further preclinical and clinical studies.

Chemotherapeutic efficacy of thioridazine as an adjunct drug in a murine model of latent tuberculosis

Thioridazine, a potent phenothiazine compound was evaluated for its chemotherapeutic efficacy against experiment model of tuberculosis. Thioridazine potentiated the activities of both isoniazid and rifampicin (>1 log CFU reduction) against the in vitro latent Mycobacterium tuberculosis bacilli. Further, a murine model of latent tuberculosis was used and the standard 9-month isoniazid and 4-month rifampicin regimen along with thioridazine as an adjunct drug were evaluated. Thioridazine led to an accelerated clearance of bacilli with both the regimen, thereby leading to completion of therapy much earlier than the standard end-point. In the case of 9-month isoniazid regimen, when thioridazine was used along with isoniazid as an adjunct drug, complete clearance was observed as early as 24 weeks as compared to the 36 week standard isoniazid monotherapy regimen. Also, in the 4-month rifampicin regimen, it was observed that the bacillary clearance was more robust when rifampicin was used along with thioridazine (>3 log CFU reduction) than rifampicin alone (>2 log CFU reduction). Our findings implicate that thioridazine, when used as an adjunct drug along with isoniazid or rifampicin has the potential to augment their chemotherapeutic efficacy against experimental latency.

Purification and characterization of cAMP dependent protein kinase from Microsporum gypseum.

A cyclic AMP dependent protein kinase (PKA), its regulatory (R) and catalytic (C) subunits were purified to homogeneity from soluble extract of Microsporum gypseum. Purified enzyme showed a final specific activity of 277.9 nmol phosphate transferred min(-1) mg protein(-1) with kemptide as substrate. The enzyme preparation showed two bands with molecular masses of 76 kDa and 45 kDa on sodium dodecyl polyacrylamide gel electrophoresis. The 76 kDa subunit was found to be the regulatory (R) subunit of PKA holoenzyme as determined by its immunoreactivity and the isoelectric point of this subunit was 3.98. The 45 kDa subunit was found to be the catalytic (C) subunit by its immunoreactivity and phosphotransferase activity. Gel filtration using Sepharose CL-6B revealed the molecular mass of PKA holoenzyme to be 240 kDa, compatible with its tetrameric structure, consisting of two regulatory subunits (76 kDa) and two catalytic subunits (45 kDa). The specificity of enzyme towards protein acceptors in decreasing order of phosphorylation was found to be kemptide, casein, syntide and histone IIs. Purified enzyme had apparent K(m) values of 71 microM and 25 microM for ATP and kemptide, respectively. Phosphorylation was strongly inhibited by mammalian PKA inhibitor (PKI) but not by inhibitors of other protein kinases. The PKA showed maximum activity at pH 7.0 and enzyme activity was inhibited in the presence of N-ethylmaleimide (NEM) which shows the involvement of sulfhydryl groups for the activity of PKA. PKA phosphorylated a number of endogenous proteins suggesting the multifunctional role of cAMP dependent protein kinase in M. gypseum. Further work is under progress to identify the natural substrates of this enzyme through which it may regulate the enzymes involved in phospholipid metabolism.

A comparative proteomic study of sera in paediatric systemic lupus erythematosus patients and in healthy controls using MALDI-TOF-TOF and LC MS–A pilot study

BackgroundPaediatric systemic lupus erythematosus (pSLE) exhibits an aggressive clinical phenotype with severe complications and overall poor prognosis. The aim of this study was to analyse differential expression of low molecular weight (LMW) serum protein molecules of pSLE patients with active disease in comparison to sera of healthy age matched controls. Further, some of the differential expressed spots were characterised and identified by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and liquid chromatography (LC-MS).Methods2D-PAGE was performed using pooled sera of active pSLE and age matched healthy controls. Gels were silver-stained and differentially expressed protein spots were detected by automated image master platinum 2D software. 79 ± 17 protein spots were detected for control gels and 78 ± 17 protein spots for patient gels. Of these eleven protein spots were selected randomly and characterized by MALDI-TOF MS (five protein spots) and LC MS (six protein spots) techniques.ResultsOut of the 11 protein spots, 5 protein spots were significantly upregulated viz., leiomodin 2 (LMOD2); epidermal cytokeratin 2; immunoglobulin kappa light chain variable region; keratin 1 and transthyretin (TTR). Three protein spots were significantly down regulated e.g., apolipoprotein A1 (APOA1); chain B human complement component C3c; campath antibody antigen complex. Two protein spots (complement component C3; retinol binding protein (RBP) were found to be expressed only in disease and one protein spot cyclohydrolase 2 was only expressed in controls.ConclusionsWe conclude that 2-D maps of patients with active pSLE and controls differ significantly. In this pilot study, using proteomic approach we have identified differential expressed proteins (of LMW) e.g., RBP, LMOD 2, TTR, Component C3c Chain B and APO A1. However, in future, further studies need to confirm the physiological and pathological role of these proteins in similar cohorts of pSLE.

cAMP regulates vegetative growth and cell cycle in Candida albicans

We demonstrate here the regulatory role of cAMP in cell cycle of Candida albicans. cAMP was found to be a positive signal for growth and morphogenesis. Phosphodiesterase inhibitor aminophylline exhibited significant effects, i.e., increased growth, as well as induced morphogenesis. Atropine and trifluoperazine negatively regulated (inhibited) growth and did not induce morphogenesis. These changes were attributed to increase in cAMP levels and protein kinase A (PKA) activity in presence of aminophylline, while reduction was observed in atropine and trifluoperazine (TFP) grown cells. Alteration in cAMP signaling pathway affected the cell cycle progression in Candida albicans. Increased cAMP levels in aminophylline grown cells reduced the duration of cell cycle by inciting the cell cycle-specific expression of G1 cyclins (CLN1 and CLN2). However atropine and trifluoperazine delayed the expression of G1 cyclins and hence prolonged the cell cycle. Implication of cAMP signaling pathway in both the cell cycle and morphogenesis further opened the channels to explore the potential of this pathway to serve as a target for development of new antifungal drugs.