Micelles entrapped microparticles technology: a novel approach to resolve dissolution and bioavailability problems of poorly water soluble drugs.

Aim: Aim of this study was to design a solid oral delivery system for a weakly basic drug such as dasatinib (DAS), so as to achieve pH-independent dissolution and improved oral bioavailability.Methods: DAS was solubilised using sodium lauryl sulphate as an aqueous micellar system and such a system containing lactose monohydrate as carrier was spray-dried to obtain a solid mass. Subsequently, the DAS-solid was converted into a tablet using conventional tableting methods.Results: The dissolution study revealed pH-independent dissolution over a wide range of pH conditions. An in vivo bioavailability testing on rats revealed an improved Cmax and AUC0-24. Similarly, viability assay showed a better inhibitory effect of spray-dried dasatinib over the DAS.Conclusions: Micellar solubilisation and spray-drying technology can be approached to resolve poor dissolution and bioavailability of drugs belonging to biopharmaceutical classification system II and III. This technology is amenable to scale-up and has commercial potential.

Whole exome sequencing of breast cancer (TNBC) cases from India: association of MSH6 and BRIP1 variants with TNBC risk and oxidative DNA damage.

Whole exome sequencing in triple negative breast cancer cases (n = 8) and targeted sequencing in healthy controls (n = 48) revealed BRIP1 rs552752779 (MAF: 75% vs. 6.25%, OR 45.00, 95% CI 9.43-243.32), ERBB2 rs527779103 (MAF: 62.5% vs. 7.29%, OR 21.19, 95% CI 5.11-94.32), ERCC2 rs121913016 (MAF: 56.25% vs. 7.29%, OR 16.34, 95% CI 4.02-70.41), MSH6 rs2020912 (MAF: 56.25% vs. 1.04%, OR 122.13, 95% CI 12.29-2985.48) as risk factors for triple negative breast cancer. Construction of classification and regression tree followed by smart pruning identified MSH6 and BRIP1 variants as the major determinants of TNBC (Triple Negative Breast Cancer) risk. Except for ERBB2, all other genes regulate DNA repair and chromosomal integrity. In TNBC cases, two likely pathogenic variations i.e. NCOR1 rs562300336 and PIM1 rs746748226 were observed at frequencies of 18.75% and 12.5%, respectively. Among the 24 variants of unknown significance, MMP9 rs199676062, SYNE1 rs368709678, AURKA rs373550419, ABCC4 rs11568694 have variant allele frequency ≥ 62.5%. These genes regulate metastasis, nuclear modeling, cell cycle and cellular detoxification, respectively. To conclude, aberrations in DNA mismatch repair, nucleotide excision repair or BRCA1 associated genome surveillance mechanism contribute towards triple negative breast cancer.

Genome-Wide Identification and Analysis of Arabidopsis Sodium Proton Antiporter (NHX) and Human Sodium Proton Exchanger (NHE) Homologs in Sorghum bicolor.

Na⁺ transporters play an important role during salt stress and development. The present study is aimed at genome-wide identification, in silico analysis of sodium-proton antiporter (NHX) and sodium-proton exchanger (NHE)-type transporters in Sorghum bicolor and their expression patterns under varied abiotic stress conditions. In Sorghum, seven NHX and nine NHE homologs were identified. Amiloride (a known inhibitor of Na⁺/H⁺ exchanger activity) binding motif was noticed in both types of the transporters. Chromosome 2 was found to be a hotspot region with five sodium transporters. Phylogenetic analysis inferred six ortholog and three paralog groups. To gain an insight into functional divergence of SbNHX/NHE transporters, real-time gene expression was performed under salt, drought, heat, and cold stresses in embryo, root, stem, and leaf tissues. Expression patterns revealed that both SbNHXs and SbNHEs are responsive either to single or multiple abiotic stresses. The predicted protein⁻protein interaction networks revealed that only SbNHX7 is involved in the calcineurin B-like proteins (CBL)- CBL interacting protein kinases (CIPK) pathway. The study provides insights into the functional divergence of SbNHX/NHE transporter genes with tissue specific expressions in Sorghum under different abiotic stress conditions.

Microarray-based SNP genotyping to identify genetic risk factors of triple-negative breast cancer (TNBC) in South Indian population.

In the view of aggressive nature of Triple-Negative Breast cancer (TNBC) due to the lack of receptors (ER, PR, HER2) and high incidence of drug resistance associated with it, a case-control association study was conducted to identify the contributing genetic risk factors for Triple-negative breast cancer (TNBC). A total of 30 TNBC patients and 50 age and gender-matched controls of Indian origin were screened for 9,00,000 SNP markers using microarray-based SNP genotyping approach. The initial PLINK association analysis (p < 0.01, MAF 0.14-0.44, OR 10-24) identified 28 non-synonymous SNPs and one stop gain mutation in the exonic region as possible determinants of TNBC risk. All the 29 SNPs were annotated using ANNOVAR. The interactions between these markers were evaluated using Multifactor dimensionality reduction (MDR) analysis. The interactions were in the following order: exm408776 > exm1278309 > rs316389 > rs1651654 > rs635538 > exm1292477. Recursive partitioning analysis (RPA) was performed to construct decision tree useful in predicting TNBC risk. As shown in this analysis, rs1651654 and exm585172 SNPs are found to be determinants of TNBC risk. Artificial neural network model was used to generate the Receiver operating characteristic curves (ROC), which showed high sensitivity and specificity (AUC-0.94) of these markers. To conclude, among the 9,00,000 SNPs tested, CCDC42 exm1292477, ANXA3 exm408776, SASH1 exm585172 are found to be the most significant genetic predicting factors for TNBC. The interactions among exm408776, exm1278309, rs316389, rs1651654, rs635538, exm1292477 SNPs inflate the risk for TNBC further. Targeted analysis of these SNPs and genes alone also will have similar clinical utility in predicting TNBC.

Ethanol production from sweet sorghum bagasse through process optimization using response surface methodology.

In this study, comparative evaluation of acid- and alkali pretreatment of sweet sorghum bagasse (SSB) was carried out for sugar production after enzymatic hydrolysis. Results indicated that enzymatic hydrolysis of alkali-pretreated SSB resulted in higher production of glucose, xylose and arabinose, compared to the other alkali concentrations and also acid-pretreated biomass. Response Surface Methodology (RSM) was, therefore, used to optimize parameters, such as alkali concentration, temperature and time of pretreatment prior to enzymatic hydrolysis to maximize the production of sugars. The independent variables used during RSM included alkali concentration (1.5-4%), pretreatment temperature (125-140 °C) and pretreatment time (10-30 min) were investigated. Process optimization resulted in glucose and xylose concentration of 57.24 and 10.14 g/L, respectively. Subsequently, second stage optimization was conducted using RSM for optimizing parameters for enzymatic hydrolysis, which included substrate concentration (10-15%), incubation time (24-60 h), incubation temperature (40-60 °C) and Celluclast concentration (10-20 IU/g-dwt). Substrate concentration 15%, (w/v) temperature of 60 °C, Celluclast concentration of 20 IU/g-dwt and incubation time of 58 h led to a glucose concentration of 68.58 g/l. Finally, simultaneous saccharification fermentation (SSF) as well as separated hydrolysis and fermentation (SHF) was evaluated using Pichia kudriavzevii HOP-1 for production of ethanol. Significant difference in ethanol concentration was not found using either SSF or SHF; however, ethanol productivity was higher in case of SSF, compared to SHF. This study has established a platform for conducting scale-up studies using the optimized process parameters.

Increased yield of high purity recombinant human brain natriuretic peptide by acid hydrolysis of short fusion partner in Escherichia coli.

Recombinant human B-type natriuretic peptide (rhBNP) is a 32-amino acid peptide used to treat congestive heart failure. In this paper, we report a method for the increased production of rhBNP in Escherichia coli with high purity. hBNP was cloned with a short growth hormone fusion partner coupled with a unique acid-labile dipeptide linker to cleave the fusion protein to release the rhBNP. The recombinant fusion protein was expressed as an inclusion body (IB) and the fermentation process was optimized to produce on large scale. The IBs were recovered by cell lysis, and the pure IBs were directly treated with diluted acid to get the target peptide from the fusion protein and the resultant peptide was purified by reversed phase chromatography. The final purity of the rhBNP was more than 99% with yield of 50mg per liter of culture, which is ten times higher than the previous reports. The purified rhBNP exhibited specific biological activity similar to the standard peptide in producing cyclic-guanosine monophosphate.

Purification and characterisation of intracellular alpha-galactosidases from Acinetobacter sp.

Two alpha-galactosidases (Ag-I & Ag-II) were purified from Acinetobacter sp. Both the enzymes were monomeric with pH optima of 7.0 and molecular weight of 65 kDa for Ag-I and 37 kDa for Ag-II. The temperature optima for Ag-I was between 50 and 60 °C and that of Ag-II was 40 °C. Both the enzymes were strongly inhibited by metal ions Ag2+ and Hg+, pCMB and SDS (1 %). The enzymes were found to be active on both natural and synthetic substrates. Artificial substrate, pNPGal, has shown more affinity to enzyme than natural substrate raffinose. The half-life (t 1/2) of Ag-I varied from 1.85 h at 90 °C to 7.6 h at 70 °C.

Design, synthesis and screening studies of potent thiazol-2-amine derivatives as fibroblast growth factor receptor 1 inhibitors.

Fibroblast growth factor receptor 1 (FGFR1) a tyrosine kinase receptor, plays important roles in angiogenesis, embryonic development, cell proliferation, cell differentiation, and wound healing. The FGFR isoforms and their receptors (FGFRs) considered as a potential targets and under intense research to design potential anticancer agents. Fibroblast growth factors (FGF's) and its growth factor receptors (FGFR) plays vital role in one of the critical pathway in monitoring angiogenesis. In the current study, quantitative pharmacophore models were generated and validated using known FGFR1 inhibitors. The pharmacophore models were generated using a set of 28 compounds (training). The top pharmacophore model was selected and validated using a set of 126 compounds (test set) and also using external validation. The validated pharmacophore was considered as a virtual screening query to screen a database of 400,000 virtual molecules and pharmacophore model retrieved 2800 hits. The retrieved hits were subsequently filtered based on the fit value. The selected hits were subjected for docking studies to observe the binding modes of the retrieved hits and also to reduce the false positives. One of the potential hits (thiazole-2-amine derivative) was selected based the pharmacophore fit value, dock score, and synthetic feasibility. A few analogues of the thiazole-2-amine derivative were synthesized. These compounds were screened for FGFR1 activity and anti-proliferative studies. The top active compound showed 56.87% inhibition of FGFR1 activity at 50 µM and also showed good cellular activity. Further optimization of thiazole-2-amine derivatives is in progress.

Role of feed forward neural networks coupled with genetic algorithm in capitalizing of intracellular alpha-galactosidase production by Acinetobacter sp.

Alpha-galactosidase production in submerged fermentation by Acinetobacter sp. was optimized using feed forward neural networks and genetic algorithm (FFNN-GA). Six different parameters, pH, temperature, agitation speed, carbon source (raffinose), nitrogen source (tryptone), and K2HPO4, were chosen and used to construct 6-10-1 topology of feed forward neural network to study interactions between fermentation parameters and enzyme yield. The predicted values were further optimized by genetic algorithm (GA). The predictability of neural networks was further analysed by using mean squared error (MSE), root mean squared error (RMSE), mean absolute error (MAE), mean absolute percentage error (MAPE), and R2-value for training and testing data. Using hybrid neural networks and genetic algorithm, alpha-galactosidase production was improved from 7.5 U/mL to 10.2 U/mL.

Expression of avian influenza virus (H5N1) hemagglutinin and matrix protein 1 in Pichia pastoris and evaluation of their immunogenicity in mice.

The conventional avian influenza vaccines rely on development of neutralizing antibodies against the HA and NA antigens. However, these antigens are highly variable, and hence there is a need for better vaccine candidates which would offer broader protection in animals. The M1 of avian influenza is another major structural protein that has conserved epitopes that are reported to induce CD8+ T cells and can contribute to protection against morbidity and mortality from influenza. Hence in an effort to study the immune response of rM1 either alone or in combination with rHA, the hemagglutinin (HA) and matrix protein (M1) of A/Hatay/2004/H5N1 strain of avian influenza were expressed in Pichia pastoris as his-tagged proteins and purified through Ni-NTA chromatography. The His-tag was removed using TEV protease cleavage site and the immunogenicity of purified rHA and rM1 either alone or in combination was determined in mice. One group of mice was immunized with 5 µg of purified rHA, the other group was immunized with rM1, and a third group of mice were immunized with 5 µg of rHA and rM1. All the animals were boosted twice, once on 28 days postimmunization (dpi) and the second on 42 dpi. The immune response was evaluated by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) assay. The group of mice immunized with rHA and rM1 together showed significantly higher immune response against rHA and rM1 than mice immunized with either HA or M1 antigens. The addition of rM1 with rHA resulted in increased HI titer in animals immunized with both the antigens. These results suggest that the HA and M1 expressed in P. pastoris can be utilized in combination for the development of faster and cost-effective vaccines for circulating and newer strains of avian influenza and would aid in combating the disease in a pandemic situation, in which production time matters greatly.

Recombinant approach for the production of HIV fusion inhibitor Enfuvirtide using Escherichia coli.

The use of antiretroviral drugs is gaining importance in the recent past for the treatment of human immunodeficiency virus infection. Enfuvirtide (T20) is one of the fusion inhibitors, inhibits the fusion between the virus and healthy target CD4 cells. The treatment with T20 involves very high therapeutic dose. In addition to its high dose, production of T20 by synthetic methods is expensive and cumbersome. We report an alternative recombinant approach for the production of the T20 peptide through a novel short fusion-tag expression system. This expression system consists of the hydrophobic region of growth hormone (GH) as the fusion tag, a factor Xa cleavage site upstream to the T20. The fusion protein was expressed in Escherichia coli as inclusion bodies. We also report here, a simple and an efficient down-stream strategy for the purification of recombinant T20 peptide (rT20). Our study is the first to demonstrate a novel approach using GH fusion tag, ensured the peptide stability, for the production of rT20 which yields more than 250mg/L with 98% purity. The biological activity of the rT20 is comparable to its synthetic counterpart. Thus, this novel approach could be an alternate method of choice for production of therapeutically important small peptides.

X-ray structure of PTP1B in complex with a new PTP1B inhibitor.

Protein tyrosine phosphatase 1B (PTP1B) is a prototype non receptor cytoplasmic PTPase enzyme that has been implicated in regulation of insulin and leptin signaling pathways. Studies on PTP1B knockout mice and PTP1B antisense treated mice suggested that inhibition of PTP1B would be an effective strategy for the treatment of type II diabetes and obesity. Here we report the X-ray structure of PTP1B in complex with compound IN1834-146C (PDB ID 4I8N). The crystals belong to P3121 space group with cell dimensions (a = b = 87.89 Å, c = 103.68 Å) diffracted to 2.5 Å. The crystal structure contained one molecule of protein in the asymmetric unit and was solved by molecular replacement method. The compound engages both catalytic site and allosteric sites of PTP1B protein. We described the molecular interaction of the compound with the active site residues of PTP1B in this crystal structure report.

Synthesis and biological evaluation of nimesulide based new class of triazole derivatives as potential PDE4B inhibitors against cancer cells.

A new class of 1,2,3-triazol derivatives derived from nimesulide was designed as potential inhibitors of PDE4B. Synthesis of these compounds was carried out via a multi-step sequence consisting of copper-catalyzed azide-alkyne cycloaddition (CuAAC) as a key step in aqueous media. The required azide was prepared via the reaction of aryl amine (obtained from nimesulide) with α-chloroacetyl chloride followed by displacing the α-chloro group by an azide. Some of the synthesized compounds showed encouraging PDE4B inhibitory properties in vitro that is >50% inhibition at 30 µM that were supported by the docking studies of these compounds at the active site of PDE4B enzyme (dock scores ~ -28.6 for a representative compound). Two of these PDE4 inhibitors showed promising cytotoxic properties against HCT-15 human colon cancer cells in vitro with IC50 ~ 21-22 µg/mL.

Atorvastatin inhibited Rho-associated kinase 1 (ROCK1) and focal adhesion kinase (FAK) mediated adhesion and differentiation of CD133+CD44+ prostate cancer stem cells.

Prostate cancer has become a global health concern and is one of the leading causes of cancer death of men after lung and gastric cancers. It has been suggested that the 3-hydroxy-3-methyl-glutarylcoenzyme-CoA (HMG-CoA) reductase inhibitor atorvastatin shows anticancer activity in prostate cancer cell lines. To this end, we analyzed the influence of atorvastatin on the cell adhesion and differentiation of CD133(+)CD44(+) cells derived from prostate cancer biopsies and peripheral blood. CD133(+)CD44(+) cells were treated with atorvastatin (16-64µM) for different time periods. Cell adhesion to endothelial cell monolayers and differentiation into prostate cancer cells were evaluated. α1, ß1 and α2ß1 integrins adhesion receptors and the downstream target of atorvastatin Rho-dependent kinase (ROCK) and focal adhesion kinase (FAK) were analyzed by Western blot. Further blocking studies with the ROCK inhibitor H1152, anti-FAK antibody and anti-integrin α1 and ß1 antibodies were carried out. Atorvastatin treatment inhibited dose-dependently cell attachment to endothelium and differentiation. The inhibitory effect of atorvastatin on cell adhesion was associated with decreased expression of integrins α1 and ß1 and phosphorylated MYPT1 and FAK. Furthermore, atorvastatin strongly reduced ROCK1 and FAK mediated differentiation of CD133(+)CD44(+) cells, which was confirmed by antibody treatment. Atorvastatin modified the expression of cell adhesion molecules and differentiation markers. These beneficial effects of atorvastatin may be mediated by ROCK and FAK signaling pathway. The data presented may point to novel treatment options for prostate cancer.

Diphenylether derivative as selective inhibitor of protein tyrosine phosphatase 1B (PTP1B) over t-cell protein tyrosine phosphatase (TCPTP) identified through virtual screening.

Even though protein tyrosine phosphatase has been identified as a validated therapeutic target over a decade for type II diabetes and obesity, developing a selective inhibitor to protein tyrosine phosphatase 1B (PTP1B) over other cellular PTPases has been a complicated task owing to the highly conserved and polar nature of the PTP1B catalytic site. Virtual screening study of in-house chemical depository resulted in the prioritization of few low molecular weight compounds as PTP1B inhibitors. The in-vitro pNPP assays were carried out on prioritized compounds in both PTP1B and T-cell protein tyrosine phosphatase (TCPTP). From this we identified four low molecular weight compounds as PTP1B inhibitors, of which the compound AU-2439 has shown 5 fold selectivity towards PTP1B over highly homologous TCPTP. In this short communication selectivity of AU-2439 is explained based on interaction with critical active site residues in both proteins using docking models.

Docking studies of Vitamin C, Vitamin E, Damnacanthal and Scopoletin with human lens gamma D-crystalline.

Vitamin C, Vitamin E, scopoletin and damnacanthal are the major constituents of Noni (Morinda citrifolia). These compounds are known to have good medicinal properties and they are known to act as antioxidants. Loss of vision in elderly is due to opaqueness of the lens proteins such as gamma-D-crystallin during oxidative stress conditions. Therefore, it is of importance to find the potential interaction of Vitamin C, Vitamin E, Scopoletin and Damnacanthal with the lens protein gamma-D-crystallin. Hence, their physical binding to gamma-D crystallin (PDB ID: 2G98) was evaluated using molecular and structural docking procedures. Results show the potential binding of all the above anti-oxidants to gamma-D-crystalline with equal affinity. Thus, the role of cumulative anti-oxidant effect in Noni fruit juice through their potential yet predicted interaction with the lens protein gamma-D-crystallin is implied for cataract treatment.

Microparticles-entrapped micelles: a novel delivery system to improve solubility and dissolution rate of poorly water-soluble valsartan.

Surfactants are routinely included in tablets during wet or dry granulations or along with directly compressible vehicles to improve wetting, disintegration and dissolution. Besides this micellar solubilization can improve permeability of poorly soluble drugs via gastrointestinal tract membranes thereby enhancing oral bioavailability. Microparticle-entrapped micelles (MEM) technology is a novel method of incorporating surfactants in tablets for improving in vitro and in vivo performance of poorly water-soluble drugs. Valsartan (VAL) was solubilized in cremophor EL micelles at cloud point temperature; lactose was dissolved in micellar dispersion and the dispersion was directly spray-dried to obtain solid product, which was subsequently converted into tablets using suitable excipients. VAL tablets produced by applying MEM technology improved dissolution performance of valsartan tablets. These tablets exhibited superior dissolution rate over controls and marketed tablets in all media employed irrespective of pH conditions and composition.

Recombinant haemagglutinin protein of highly pathogenic avian influenza A (H5N1) virus expressed in Pichia pastoris elicits a neutralizing antibody response in mice.

Recombinant avian influenza vaccines offer several advantages over the conventional vaccines. In this study, the haemagglutinin (HA) gene of highly pathogenic avian influenza H5N1 was cloned and expressed as His tagged protein in methylotropic yeast Pichia pastoris. The expression of recombinant HA (rHA) protein was confirmed by SDS-PAGE and western blot analysis. The rHA protein was purified using Ni-NTA affinity chromatography under denaturing conditions and the functions of the protein was assessed by the haemagglutinin assay after refolding. The immunogenicity of the rHA was evaluated by immunizing four groups of mice with different payloads (2.5, 5.0, 10 and 25µg) of purified rHA and the production of rHA specific antibodies were analysed by haemagglutinin inhibition assay (HI) and enzyme-linked immunosorbent assay (ELISA). An antigen specific immune response was observed against rHA indicating that the rHA antigen could be used as a vaccine candidate against avian influenza. These results suggest that this strategy would pave the way for the development of rapid and cost effective method for the production of an avian influenza vaccine.

Agrobacterium-mediated transformation in Alpinia galanga (Linn.) Willd. for enhanced acetoxychavicol acetate production.

Agrobacterium-mediated transformations ensure elevated amounts of secondary metabolite accumulation with genetic and biosynthetic stability. In the present study, Alpinia galanga rich in bioactive compounds was genetically transformed using different strains of Agrobacterium rhizogenes viz. LBA 9402, A(4), 532, 2364 and PRTGus. Even though a higher growth rate was obtained with the LBA 9402 strain, maximum acetoxychavicol acetate accumulation (ACA) was seen in the PRTGus transformant. PRTGus root line has shown 10.1 fold higher ACA content in comparison to the control roots. The lowest ACA production was shown by the A(4) transformant (4.9 fold). The quantification of ACA in the transformed roots was carried out by using HPLC, which was found to be in the order of PRTGus > LBA 9402 > 2364 > 532 > A(4). The fast growth rate of hairy roots, genetic stability and their ability to synthesize more than one metabolite offer a promising system for the production of valuable secondary metabolites.

Potential of thermo and alkali stable xylanases from Thielaviopsis basicola (MTCC-1467) in biobleaching of wood kraft pulp.

Thermo- and alkali-stable xylanases produced from Thielaviopsis basicola (MTCC-1467) on low-cost carbon source like rice straw were evaluated for their potential application in biobleaching of wood kraft pulp. Enzyme treatment at retention time of 240 min with 20 IU/gm of dried pulp resulted in ~85.2 % of reduction in kappa number. When compared to control, 110.8, 93, and 72.2 % of enhancement in brightness (percent International Organization of Standardization), whiteness, and fluorescence, respectively, were observed for enzyme-treated pulp. Spectroscopic analysis showed significant release of chromophoric compounds from enzyme-treated pulp. Furthermore, scanning electron microscope studies of unbleached and enzyme bleached pulp revealed the effectiveness of enzymatic treatment. The enzyme-treated pulp subjected to later stages of chemical bleaching resulted in 16 % decrease in chlorine consumption along with considerable reduction in chemical oxygen demand percentage (14.5 %) level of effluent. Various pulp properties like fiber length, fiber width, burst strength, burst index, tear strength, tear index, tensile strength, and breaking length were also significantly improved after enzyme treatment when compared to control.