A Computational Approach with Biological Evaluation: Combinatorial Treatment of Curcumin and Exemestane Synergistically Regulates DDX3 Expression in Cancer Cell Lines.

DDX3 belongs to RNA helicase family that demonstrates oncogenic properties and has gained wider attention due to its role in cancer progression, proliferation and transformation. Mounting reports have evidenced the role of DDX3 in cancers making it a promising target to abrogate DDX3 triggered cancers. Dual pharmacophore models were generated and were subsequently validated. They were used as 3D queries to screen the InterBioScreen database, resulting in the selection of curcumin that was escalated to molecular dynamics simulation studies. In vitro anti-cancer analysis was conducted on three cell lines such as MCF-7, MDA-MB-231 and HeLa, which were evaluated along with exemestane. Curcumin was docked into the active site of the protein target (PDB code 2I4I) to estimate the binding affinity. The compound has interacted with two key residues and has displayed stable molecular dynamics simulation results. In vitro analysis has demonstrated that both the candidate compounds have reduced the expression of DDX3 in three cell lines. However, upon combinatorial treatment of curcumin (10 and 20 µM) and exemestane (50 µM) a synergism was exhibited, strikingly downregulating the DDX3 expression and has enhanced apoptosis in three cell lines. The obtained results illuminate the use of curcumin as an alternative DDX3 inhibitor and can serve as a chemical scaffold to design new small molecules.

Boronic-targeted albumin-shell oily-core nanocapsules for synergistic aromatase inhibitor/herbal breast cancer therapy.

Multi-modality strategies of albumin-mediated drug accumulation in tumor, boronate-based active tumor targeting and synergistic cancer therapy were combined together for effective treatment of breast cancer. Herein we report the development of albumin-shell oily-core nanocapsules (NCs), loaded with novel combination of hydrophobic drugs, exemestane (EXE) and hesperetin (HES), for targeted breast cancer therapy. This protein-lipid nanohybrid carrier was successfully fabricated using a simple protein-coating method based on the electrostatic adsorption of negatively charged albumin shell onto the oily core containing cationic surfactant. While EXE was directly encapsulated into the oily core, HES was pre-formulated in the form of phospholipid complex before solubilization in oily phase. In addition to albumin-mediated binding to albondin and SPARC, phenylboronic acid was chemically coupled to the albumin shell to confer additional tumor targeting. The targeted nanocarrier (TNC) demonstrated enhanced internalization into MCF-7 breast cancer cells resulting in synergistic cytotoxic activity with a combination index (CI) of 0.662 and dose reduction index (DRI) of 8.22 and 1.84 for EXE and HES, respectively. In vivo, TNC displayed superior anti-cancer activity in tumor-bearing mice compared to their non-targeted counterparts and the free drug combination. A significant reduction of both tumor volume (7-folds) and Ki67 expression (3-folds) was obtained by the targeted nanocarriers compared to positive control. Overall, the boronic-targeted albumin NCs offer a promising platform for hydrophobic drug combination against cancer therapy.

Design and synthesis of long acting inhaled corticosteroids for the treatment of asthma.

In this Letter we present data for a novel series of ICS for the treatment of asthma. 'Inhalation by design' principles have been applied to a series of highly potent steroidal GR agonists, with a focus on optimising the potential therapeutic index in human. Pharmacokinetic properties were tuned with high intrinsic clearance and low oral bioavailability in mind, to minimise systemic exposure and reduce systemically driven adverse events. High CYP mediated clearance as well as glucuronidation were targeted to achieve high intrinsic clearance coupled with multiple routes of clearance to minimise drug-drug interactions. Furthermore, pharmaceutical properties such as stability, crystallinity and solubility were considered to ensure compatibility with a dry powder inhaler. This work culminated in the identification of the clinical candidate 15, which demonstrates preclinically the desired efficacy and safety profiles confirming its potential as an inhaled agent for the treatment of asthma.

Discovery of a novel hybrid from finasteride and epristeride as 5α-reductase inhibitor.

Finasteride and epristeride both inhibit 5α-reductase with high potency via competitive and non-competitive mechanism, respectively. A new hybrid of finasteride and epristeride was designed as a new 5α-reductase inhibitor based on combination principles in medicinal chemistry. Human 5ß-reductase was chosen as a plausible surrogate of 5α-reductase type II and the results indicate that although the hybrid compound possesses the main bulk of epristeride, its inhibitory mechanism is same as of finasteride. The hybrid turned out to be a potent 5α-reductase inhibitor in low IC(50) ranges.

Exemestane's 17-hydroxylated metabolite exerts biological effects as an androgen.

Aromatase inhibitors (AI) are being evaluated as long-term adjuvant therapies and chemopreventives in breast cancer. However, there are concerns about bone mineral density loss in an estrogen-free environment. Unlike nonsteroidal AIs, the steroidal AI exemestane may exert beneficial effects on bone through its primary metabolite 17-hydroexemestane. We investigated 17-hydroexemestane and observed it bound estrogen receptor alpha (ERalpha) very weakly and androgen receptor (AR) strongly. Next, we evaluated 17-hydroexemestane in MCF-7 and T47D breast cancer cells and attributed dependency of its effects on ER or AR using the antiestrogen fulvestrant or the antiandrogen bicalutamide. 17-Hydroexemestane induced proliferation, stimulated cell cycle progression and regulated transcription at high sub-micromolar and micromolar concentrations through ER in both cell lines, but through AR at low nanomolar concentrations selectively in T47D cells. Responses of each cell type to high and low concentrations of the non-aromatizable synthetic androgen R1881 paralleled those of 17-hydroexemestane. 17-Hydroexemestane down-regulated ERalpha protein levels at high concentrations in a cell type-specific manner similarly as 17beta-estradiol, and increased AR protein accumulation at low concentrations in both cell types similarly as R1881. Computer docking indicated that the 17beta-OH group of 17-hydroexemestane relative to the 17-keto group of exemestane contributed significantly more intermolecular interaction energy toward binding AR than ERalpha. Molecular modeling also indicated that 17-hydroexemestane interacted with ERalpha and AR through selective recognition motifs employed by 17beta-estradiol and R1881, respectively. We conclude that 17-hydroexemestane exerts biological effects as an androgen. These results may have important implications for long-term maintenance of patients with AIs.

Effects of aromatase inhibitors on human osteoblast and osteoblast-like cells: a possible androgenic bone protective effects induced by exemestane.

Effects of aromatase inhibitors (AIs) on the human skeletal system due to systemic estrogen depletion are becoming clinically important due to their increasing use as an adjuvant therapy in postmenopausal women with breast cancer. However, possible effects of AIs on human bone cells have remained largely unknown. We therefore studied effects of AIs including the steroidal AI, exemestane (EXE), and non-steroidal AIs, Aromatase Inhibitor I (AI-I) and aminoglutethimide (AGM), on a human osteoblast. We employed a human osteoblast cell line, hFOB, which maintains relatively physiological status of estrogen and androgen pathways of human osteoblasts, i.e., expression of aromatase, androgen receptor (AR), and estrogen receptor (ER) beta. We also employed osteoblast-like cell lines, Saos-2 and MG-63 which expressed aromatase, AR, and ERalpha/beta in order to further evaluate the mechanisms of effects of AIs on osteoblasts. There was a significant increment in the number of the cells following 72 h treatment with EXE in hFOB and Saos-2 but not in MG-63, in which the level of AR mRNA was lower than that in hFOB and Saos-2. Alkaline phosphatase activity was also increased by EXE treatment in hFOB and Saos-2. Pretreatment with the AR blocker, flutamide, partially inhibited the effect of EXE. AI-I exerted no effects on osteoblast cell proliferation and AGM diminished the number of the cells. hFOB converted androstenedione into E2 and testosterone (TST). Both EXE and AI-I decreased E2 level and increased TST level. In a microarray analysis, gene profile patterns following treatment with EXE demonstrated similar patterns as with DHT but not with E2 treatment. The genes induced by EXE treatment were related to cell proliferation, differentiation which includes genes encoding cytoskeleton proteins. We also examined the expression levels of these genes using quantitative RT-PCR in hFOB and Saos-2 treated with EXE and DHT and with/without flutamide. HOXD11 gene known as bone morphogenesis factor and osteoblast growth-related genes were induced by EXE treatment as well as DHT treatment in both hFOB and Saos-2. These results indicated that the steroidal aromatase inhibitor, EXE, stimulated hFOB cell proliferation via both AR dependent and independent pathways.

Physicochemical compatibility of fluticasone-17- propionate nebulizer suspension with ipratropium and albuterol nebulizer solutions.

The objective of this in-vitro study was to determine whether mixtures of three nebulizable drugs are physicochemically compatible. Drug combinations were prepared by mixing the content of one respule Flutide forte "ready to use" (fluticasone propionate) with 2 milliliter Atrovent LS (ipratropium bromide) and 0.5 milliliter Sultanol inhalation solution (albuterol sulfate). Test suspensions were stored at room temperature and exposed to normal laboratory light for 5 hours. Concentrations of fluticasone- 17-propionate, ipratropium bromide, and albuterol sulfate were determined by using stability-indicating high-performance liquid chromatography assays with ultraviolet detection. Physical compatibility was determined by measuring pH and osmolality. Main outcome measures were the drug concentrations of the active components of the mixtures. All drug concentrations retained nearly 100% of the initial drug concentrations after mixing and storage in glass containers at room temperature. Osmolality and pH of the mixtures exhibited no significant changes and no visible changes of the mixtures were detectable over the inspection period. Mixtures of fluticasone propionate, ipratropium bromide, and albuterol sulfate inhalation drug products were shown to be physicochemically compatible over a period of 5 hrs. In order to avoid contamination and microbiological instability, mixing should only take place immediately before administration. Further investigations are needed to determine whether or not drug delivery is affected by mixing the nebulizer suspensions and to ensure that simultaneous nebulization is recommendable.

Andrastin A and barceloneic acid metabolites, protein farnesyl transferase inhibitors from Penicillium albocoremium: chemotaxonomic significance and pathological implications.

A survey of Penicillium albocoremium was undertaken to identify potential taxonomic metabolite markers. One major and four minor metabolites were consistently produced by the 19 strains surveyed on three different media. Following purification and spectral studies, the metabolites were identified as the known protein farnesyl transferase inhibitors andrastin A (1) and barceloneic acid A (2) along with barceloneic acid B (3), barceloneic lactone (4), and methyl barceloneate (5). These compounds are significant taxonomic markers for P. albocoremium; moreover this is the first report of a methyl ester of a barceloneic acid being produced as a secondary metabolite. Tissue extracts created following pathogenicity trials involving P. albocoremium and Allium cepa confirmed the production of these five metabolites in planta. Barceloneic acid B was found to be biologically active against a P388 murine leukemia cell line.

A cell-permeable, activity-based probe for protein and lipid kinases.

Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.

Aerosol characterization of nebulized intranasal glucocorticoid formulations.

Inhaled glucocorticoids (GCs) are the mainstay of long-term therapy for asthma. The lack of suitable preparations in the United States has induced clinicians to use intranasal (IN) GC formulations as "nebulizer suspensions" for off-label therapy. However, no data are available regarding aerosol production and characteristics. The aim of this study was to characterize drug outputs and aerodynamic profiles of four nebulized IN GC formulations with further analysis of flunisolide (Flu), and to test the influence of different delivery system/formulation combinations. The aerodynamic profiles and drug outputs were determined by impaction and chemical analysis. The solution output was determined by the gravimetric technique. Triamcinole acetonide (TAA), fluticasone propionate (Flut), beclomethasone dipropionate (Bec), and Flu (550, 500, 840, and 250 microg, respectively) diluted to 4 mL with saline solution were tested with the Sidestream (SID) and Aero-Tech II (AT2) nebulizers. Subsequently, Flu was tested with four additional nebulizers (Pari LC + [PARI] Acorn II, Hudson T Up-draft II, and Raindrop). All the aerosols were heterodisperse and had a particle size range optimal for peripheral airway deposition (1.85 to 3.67 microm). Flu had the highest drug output in the respirable range (22.8 and 20.3 microg/min with the AT and SID, respectively). Flu was 5-11 times more efficiently nebulized than the other formulations tested. No differences were detected in the solution outputs (0.25 to 0.3 mL/min). In subsequent testing of Flu, the PARI, AT, and SID showed the best performances. The LC+ achieved the highest drug and solution output (27.4 microg/min and 0.89 mL/min, respectively). In conclusion, Flu showed the best aerosol performance characteristics. These data do not endorse the off-label utilization of nebulized IN GC, but underscores the importance of in vitro testing before selecting any formulation/nebulizer combinations for clinical use.