Gurdarshan Singh

Immune modulation and apoptosis induction: Two sides of antitumoural activity of a standardised herbal formulation of Withania somnifera

Deregulated apoptosis and suppressed tumour reactive immunity render tumour cells to grow amok in the host body. Traditionally used botanicals may offer potential anticancer chemo-immunotherapeutic leads. We report in this study a chemically standardised herbal formulation (WSF) of Withania somnifera possessing anticancer and Th1 immune up-regulatory activities. WSF produced cytotoxicity in a panel of human cancer cell lines in vitro. The molecular mechanism of cell cytotoxicity, IC(50) 48h approximately 20mug/ml, was investigated in HL-60, where it induced apoptosis by activating both intrinsic and extrinsic signalling pathways. It induced early generation of reactive nitrogen and oxygen species (RNOS), thus producing oxidative stress mediated mitochondrial membrane potential (MMP) loss leading to the release of cytochrome c, the translocation of Bax to mitochondria and apoptosis-inducing factor to the nuclei. These events paralleled the activation of caspase-9, -3 and PARP cleavage. WSF also activated caspase-8 through enhanced expression of TNF-R1 and DR-4, suggesting also the involvement of extrinsic pathway of apoptosis. WSF at 150mg/kg, i.p., inhibited >50% tumour growth in the mouse tumour models. In tumour-bearing mice, WSF inhibited the expression of pStat-3, with a selective stimulation of Th1 immunity as evidenced by enhanced secretion of IFN-gamma and IL-2. In parallel, it enhanced the proliferation of CD4(+)/CD8(+) and NK cells along with an increased expression of CD40/CD40L/CD80. In addition, WSF also enhanced T cell activation in camptothecin treated tumour-bearing mice. WSF being safe when given orally up to 1500mg/kg to rats for 6 months may be found useful in the management of malignancy by targeting at multiple pathways.

Biodegradable polymeric system for cisplatin delivery: Development, in vitro characterization and investigation of toxicity profile ☆ ☆☆

Cisplatin is one of the most potent anticancer agent used in the treatment of various solid tumors, however, its clinical use is limited due to severe adverse effects including nephrotoxicity. In this investigation cisplatin loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles were developed and characterized for various in vitro characteristics including size distribution, zeta potential, drug loading and release profile. PLGA nanoparticles were successfully developed as investigated using scanning electron microscopy and exhibited average particles size and zeta potential as 284.8 nm and -15.8 mV, respectively. Fourier transform infrared spectroscopy and differential scanning calorimetry indicated an absence of any polymer-drug interactions. Cisplatin nanoparticles exhibited in vitro anticancer activity against A549 cells comparable to that of cisplatin solution. The biodistribution study in mice indicated that the kidney cisplatin level was significantly (p<0.01) lower with cisplatin nanoparticles than cisplatin solution. Following two cycles of cisplatin treatment, a week apart, blood urea nitrogen level was found to be higher in case of cisplatin solution as compared to cisplatin nanoparticles. Further, there was a significant (p<0.01) increase in plasma creatinine level in case of cisplatin solution as compared to cisplatin nanoparticles. Histopathological examination of kidney from cisplatin nanoparticles treated group revealed no kidney damage, however, a sign of nephrotoxicity was observed in the case of cisplatin solution. The results suggest that PLGA nanoparticle based formulation could be a potential option for cisplatin delivery.

Linking GATA-3 and interleukin-13: implications in asthma

IntroductionAsthma is one of the serious global health problems and cause of huge mortality and morbidity. It is characterized by persistent airway inflammation, airway hyperresponsiveness, increased IgE levels and mucus hypersecretion. Asthma is mediated by dominant Th2 immune response, causing enhanced expression of Th2 cytokines. These cytokines are responsible for the various pathological changes associated with allergic asthma.Materials and methods The role of Th2 cells in the pathogenesis of the asthma is primarily mediated through the cytokine IL-13, also produced by type 2 innate lymphoid cells, that comes under the transcriptional regulation of GATA3. In this review we will try to explore the link between IL-13 and GATA3 in the progression and regulation of asthma and its possible role as a therapeutic target.ConclusionInhibition of GATA3 activity or blockade of GATA3 expression may attenuate the interleukin-13 mediated asthma phenotypes. So, GATA3 might be a potential therapeutic target for the treatment of allergic asthma.

Inhibition of phosphotidylinositol-3 kinase pathway by a novel naphthol derivative of betulinic acid induces cell cycle arrest and apoptosis in cancer cells of different origin

Betulinic acid (BA) is a pentacyclic triterpenoid natural product reported to inhibit cell growth in a variety of cancers. However, the further clinical development of BA got hampered because of poor solubility and pharmacological properties. Interestingly, this molecule offer several hotspots for structural modifications in order to address its associated issues. In our endeavor, we selected C-3 position for the desirable chemical modification in order to improve its cytotoxic and pharmacological potential and prepared a library of different triazoline derivatives of BA. Among them, we previously reported the identification of a potential molecule, that is, 3{1N(5-hydroxy-naphth-1yl)-1H-1,2,3-triazol-4yl}methyloxy betulinic acid (HBA) with significant inhibition of cancer cell growth and their properties. In the present study, we have shown for the first time that HBA decreased the expression of phosphotidylinositol-3 kinase (PI3K) p110α and p85α and caused significant downregulation of pAKT and of NFκB using human leukemia and breast cancer cells as in vitro models. Further it was revealed that PI3K inhibition by HBA induced cell cycle arrest via effects on different cell cycle regulatory proteins that include CDKis cyclins and pGSK3β. Also, this target-specific inhibition was associated with mitochondrial apoptosis as was reflected by the increased expression of mitochondrial bax, downregulated bcl2 and decreased mitochondrial levels of cytochrome c, together with reactive oxygen species generation and decline in mitochondrial membrane potential. The apoptotic effectors such as caspase 8, caspase 9 and caspase 3 were found to be upregulated besides DNA repair-associated enzyme, that is, PARP cleavage caused cancer cell death. Pharmacodynamic evaluation revealed that both HBA and BA were safe upto the dose of 2000 mg/kg body weight and with acceptable pharmacodynamic parameters. The in vitro data corroborated with in vivo anticancer activity wherein Ehrlich solid tumor showed that HBA as a more potent agent than BA without any body weight loss and mortality.

Involvement of P-glycoprotein and CYP 3A4 in the enhancement of etoposide bioavailability by a piperine analogue.

Etoposide, a semi-synthetic derivative of podophyllotoxin, is widely used anticancer agent. Etoposide presents low bioavailability with wide inter-, and intra-patient variability after oral dosing. In an earlier study a piperine analogue, namely, 4-ethyl 5-(3, 4-methylenedioxyphenyl)-2E,4E-pentadienoic acid piperidide (PA-1), was shown to cause 2.32-fold enhancement of the absolute bioavailability of co-dosed etoposide in mice. In the present investigation a mechanistic evaluation was undertaken using various in vitro and animal-derived models. In everted rat gut sac studies PA-1 enhanced mucosal uptake of the drug while it inhibited efflux of Rh-123, a P-glycoprotein substrate from serosal-to-mucosal direction. In a single pass in situ perfusion experiment PA-1 significantly reduced the intestinal exsorption rate, exsorption clearance and the total plasma clearance of etoposide. On the other hand PA-1 did not alter the passive diffusion pattern of the drug in PAMPA assay. PA-1 was inhibitory to NADPH-assisted deethylation and demethylation reactions catalyzed by erythromycin N-demethylase, 7-methoxycoumarin-O-demethylase (MOCD) and ethoxyresorufin-O-deethylase (EROD). PA-1 was not cytotoxic to mucosal membrane and showed no adverse effect in acute toxicity determination. The results suggested that PA-1-mediated enhancement in the oral bioavailability of etoposide could possibly be due to its ability to modify P-gp/CYP 3A4 mediated drug disposition mechanisms.

Anti-histaminic, anti-inflammatory and bronchorelaxant activities of 2, 7-dimethyl-3-nitro-4H pyrido [1,2-a] pyrimidine-4-one

An immunopharmacological profile of 2, 7-dimethyl-3-nitro-4H pyrido [1,2-a] pyrimidine-4-one (P-I) has been investigated using in vitro and in vivo models representing various features of Type I allergy. P-I prevented compound 48/80-mediated histamine release from rat peritoneal mast cells. A promising anti-inflammatory activity of P-I was evident in active paw anaphylaxis (mice) and carragenan-induced paw edema (rat). P-I inhibited eosonophil accumulation and eosinophil peroxidase activity in bronchoalveolar lavage fluid from ovalbumin challenged balb/c mice: in these animals blood levels of IL-5, and CD4+ T cells also remained attenuated. A promising bronchorelaxant effect of P-I was observed in histamine-contracted guinea pig tracheal chain via its antagonism to H1 receptor. These findings were compared with some known compounds (ketotifen, cetirizine and promethazine). The anti-histaminic, anti-inflammatory and bronchorelaxant activities of P-I has been discussed in context with its potential profile as an anti-allergic and anti-asthmatic agent.

In vivo genotoxicity evaluation of a plant based antiarthritic and anticancer therapeutic agent Boswelic acids in rodents

The genotoxic potential of anti-inflammatory/anti-arthritic and anticancer plant based drug molecule Boswelic acids (BA) was studied by in vivo system. Systematic literature survey revealed that studies on the genotoxicity of BA are not available. Although reports on genotoxicity of Boswellia serrata dry extract and modified 3-O-acetyl-11-keto-beta-boswelic acid are available and these studies were conducted in in vitro systems. The earlier general toxicity study of BA has been conducted by us, revealed it to be non toxic. The genotoxicity was carried out in Wistar rats using different cytogenetic assay system-abnormalities viz. chromosomal aberrations; sperm morphology, micronuclei and comet assays. Six groups of animals, each comprised of five rats, were taken for each study. Group1-4 received BA at 125, 250, 500 and 1000 mg/kg p.o., respectively prepared as 2% gum acacia suspension, fifth group received a positive control cyclophosphamide (CP) 40 mg/kg p.o. or metronedazole (MTZ) 130 mg/kg p.o. or mercuric chloride (HgCl(2)) 0.864 mg/kg p.o. (as per the experiment requirement) whereas the sixth group kept as vehicle control. The results on the bases of the data obtained revealed that BA is quite safe as it did not show any genotoxicity at any dose level up to 1000 mg/kg. The positive controls used in different experiments showed highly significant abnormal cytogenetic changes in comparison to the control group.

Discovery of novel pyrazolopyrimidinone analogs as potent inhibitors of phosphodiesterase type-5.

Cyclic guanosine monophosphate (cGMP) specific phosphodiesterase type-5 (PDE5), a clinically proven target to treat erectile dysfunction and diseases associated with lower cGMP levels in humans, is present in corpus cavernosum, heart, lung, platelets, prostate, urethra, bladder, liver, brain, and stomach. Sildenafil, vardenafil, tadalafil and avanafil are FDA approved drugs in market as PDE5 inhibitors for treating erectile dysfunction. In the present study a lead molecule 4-ethoxy-N-(6-hydroxyhexyl)-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)benzenesulfonamide, that is, compound-4a, an analog of pyrazolopyrimidinone scaffold has been identified as selective PDE5 inhibitor. A series of compounds was synthesized by replacing N-methylpiperazine moiety (ring-C) of sildenafil structure with different N-substitutions towards sulfonamide end. Compound-4a showed lower IC₅₀ value (1.5 nM) against PDE5 than parent sildenafil (5.6 nM) in in vitro enzyme assay. The isoform selectivity of the compound-4a against other PDE isoforms was similar to that of the Sildenafil. In corroboration with the in vitro data, this molecule showed better efficacy in in vivo studies using the conscious rabbit model. Also compound-4a exhibited good physicochemical properties like solubility, caco-2 permeability, cLogP along with optimal PK profile having no significant CYP enzyme inhibitory liabilities. Discovery of these novel bioactive compounds may open a new alternative for developing novel preclinical candidates based on this drugable scaffold.

Reduced toxicological manifestations of cisplatin following encapsulation in folate grafted albumin nanoparticles.

AIMS Cisplatin is one of the most potent chemotherapeutic agents acting against a variety of tumors, however, its use is mainly limited due to the dose limiting toxicities and acquired resistance to cisplatin. Folate functionalized albumin nanoparticles were developed for targeted delivery of drug to limit the adverse effects of cisplatin. MAIN METHODS Cisplatin loaded nanoparticles functionalized with folate (CP-FA-BSA-NPs) were developed and characterized for various parameters. In order to investigate the targeting ability of folate conjugated nanoparticles, in vitro cellular uptake study was performed in folate receptor over expressing cells (MCF-7). Further, blood urea nitrogen (BUN) level, plasma creatinine level, body weight and kidney weight of the mice were measured followed by histopathological examination of various tissues to have an insight into the potential of developed formulation in the reduction of drug associated adverse effects. KEY FINDINGS The cellular uptake studies demonstrated higher internalization of folate conjugated nanoparticles as compared to plain counterpart (CP-BSA-NPs). Following two cycles of cisplatin treatment, a week apart, BUN and plasma creatinine level were found to be significantly higher in case of free cisplatin as compared to saline, CP-BSA-NPs and CP-FA-BSA-NPs treated groups. Body weight and kidney weight of free cisplatin treated mice were significantly reduced as compared to other group. Histopathological examination of kidney from CP-BSA-NPs and CP-FA-BSA-NPs treated groups revealed no kidney damage, however, a sign of nephrotoxicity was observed in the case of free cisplatin. SIGNIFICANCE The results demonstrated the potential of developed formulation in reducing the adverse effects of cisplatin.

Anti-asthmatic activity of azepino [2, 1-b] quinazolones, synthetic analogues of vasicine, an alkaloid from Adhatoda vasica

Asthma is characterized by persistent airway inflammation caused by over expression of pro-inflammatory immune response, predominantly by eosinophils and lymphocytes. Lymphocytes (CD4+ Th2) have been documented to be responsible for the pathogenesis of asthma by secreting Th2 cytokines and activating eosinophils, leading to airway hypersensitivity. Secretion of Th2 cytokines has been shown critical for the induction of the characteristic airway inflammation in humans and animal models of asthma. These cytokines influence the inflammatory response and lead to the pathological changes associated with asthma. In the present study, 10 azepino [2,1-b] quinazolone derivatives (R1 to R10) were synthesised and evaluated for their anti-asthmatic activity using a murine model of asthma. The compounds R2, R4, R6, R7 and R8 caused a notable decrease Th2 cytokine secretion and eosinophilia in asthma-induced animals. However, the decrease was highly significant in case of R8-treated animals. Crystal structure of R8 was made by X-ray crystallography. Molecular modelling studies were done for the compound R8 with transcription factors STAT6 and GATA3 which are the main transcription factors responsible for Th2 cell differentiation. Also the pharmacokinetics of R8 was carried out in mice after oral and intravenous administrations.