Anticancer and antimetastatic potential of enterolactone: Clinical, preclinical and mechanistic perspectives.

Currently cancer is the second leading cause of death globally and worldwide incidence and mortality rates of all cancers of males and females are rising tremendously. In spite of advances in chemotherapy and radiation, metastasis and recurrence are considered as the major causes of cancer related deaths. Hence there is a mounting need to develop new therapeutic modalities to treat metastasis and recurrence in cancers. A significant amount of substantiation from epidemiological, clinical and laboratory research highlights the importance of diet and nutrition in cancer chemoprevention. Enterolactone (EL) is a bioactive phenolic metabolite known as a mammalian lignan derived from dietary lignans. Here in we review the reported anti-cancer properties of EL at preclinical as well as clinical level. Several in-vivo and in-vitro studies have provided strong evidence that EL exhibits potent anti-cancer and/or protective properties against different cancers including breast, prostate, colo-rectal, lung, ovarian, endometrial, cervical cancers and hepatocellular carcinoma. Reported laboratory studies indicate a clear role for EL in preventing cancer progression at various stages including cancer cell proliferation, survival, angiogenesis, inflammation and metastasis. In clinical settings, EL has been reported to reduce risk, decrease mortality rate and improve overall survival particularly in breast, prostate, colon, gastric and lung cancer. Further, the in-vitro human cell culture studies provide strong evidence of the anticancer and antimetastatic mechanisms of EL in several cancers. This comprehensive review supports an idea of projecting EL as a promising candidate for developing anticancer drug or adjunct dietary supplements and nutraceuticals.

Enterolactone modulates the ERK/NF-κB/Snail signaling pathway in triple-negative breast cancer cell line MDA-MB-231 to revert the TGF-ß-induced epithelial-mesenchymal transition.

OBJECTIVE: Triple-negative breast cancer (TNBC) is highly metastatic, and there is an urgent unmet need to develop novel therapeutic strategies leading to the new drug discoveries against metastasis. The transforming growth factor-ß (TGF-ß) is known to promote the invasive and migratory potential of breast cancer cells through induction of epithelial-mesenchymal transition (EMT) via the ERK/NF-κB/Snail signaling pathway, leading to breast cancer metastasis. Targeting this pathway to revert the EMT would be an attractive, novel therapeutic strategy to halt breast cancer metastasis. METHODS: Effects of enterolactone (EL) on the cell cycle and apoptosis were investigated using flow cytometry and a cleaved caspase-3 enzyme-linked immunosorbent assay (ELISA), respectively. Effects of TGF-ß induction and EL treatment on the functional malignancy of MDA-MB-231 breast cancer cells were investigated using migration and chemo-invasion assays. The effects of EL on EMT markers and the ERK/NF-κB/Snail signaling pathway after TGF-ß induction were studied using confocal microscopy, quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and flow cytometry. RESULTS: Herein, we report that EL exhibits a significant antimetastatic effect on MDA-MB-231 cells by almost reverting the TGF-ß-induced EMT in vitro. EL downregulates the mesenchymal markers N-cadherin and vimentin, and upregulates the epithelial markers E-cadherin and occludin. It represses actin stress fiber formation via inhibition of mitogen-activated protein kinase p-38 (MAPK-p38) and cluster of differentiation 44 (CD44). EL also suppresses ERK-1/2, NF-κB, and Snail at the mRNA and protein levels. CONCLUSIONS: Briefly, EL was found to inhibit TGF-ß-induced EMT by blocking the ERK/NF-κB/Snail signaling pathway, which is a promising target for breast cancer metastasis therapy.

Enterolactone Suppresses Proliferation, Migration and Metastasis of MDA-MB-231 Breast Cancer Cells Through Inhibition of uPA Induced Plasmin Activation and MMPs-Mediated ECM Remodeling

Background: To enhance their own survival, tumor cells can manipulate their microenvironment through remodeling of the extra cellular matrix (ECM). The urokinase-type plasminogen activator (uPA) system catalyzes plasmin production which further mediates activation of matrix metalloproteinases (MMPs) and plays an important role in breast cancer invasion and metastasis through ECM remodeling. This provides a potential target for therapeutic intervention of breast cancer treatment. Enterolactone (EL) is derived from dietary flax lignans in the human body and is known to have anti-breast cancer activity. We here investigated molecular and cellular mechanisms of EL action on the uPA-plasmin- MMPs system. Methods: MTT and trypan blue dye exclusion assays, anchorage-dependent clonogenic assays and wound healing assays were carried out to study effects on cell proliferation and viability, clonogenicity and migration capacity, respectively. Real-time PCR was employed to study gene expression and gelatin zymography was used to assess MMP-2 and MMP-9 activities. All data were statistically analysed and presented as mean ± SEM values. Results: All the findings collectively demonstrated anticancer and antimetastatic potential of EL with antiproliferative, antimigratory and anticlonogenic cellular mechanisms. EL was found to exhibit multiple control of plasmin activation by down-regulating uPA expression and also up-regulating its natural inhibitor, PAI-1, at the mRNA level. Further, EL was found to down-regulate expression of MMP-2 and MMP-9 genes, and up-regulate TIMP-1 and TIMP-2; natural inhibitors of MMP-2 and MMP-9, respectively. This may be as a consequence of inhibition of plasmin activation, resulting in robust control over migration and invasion of breast cancer cells during metastasis. Conclusions: EL suppresses proliferation, migration and metastasis of MDA-MB-231 breast cancer cells by inhibiting induced ECM remodeling by the 'uPA-plasmin-MMPs system'.

Reduced ulcerogenic potential and antiarthritic effect of chitosan-naproxen sodium complexes.

The purpose of this research was to address the utility of naproxen sodium-chitosan spray-dried complexes for antiulcer and antiarthritic activities. The cold stress technique was used to examine the ulcerogenic potential of naproxen sodium (NPX) and spray-dried formulations in the different doses. The ulcerations reduced with the dose of spray-dried complexes of naproxen sodium and chitosan. The conspicuous hemorrhagic lesions were visible in the morphological features of the animal treated with naproxen 50 mg/kg (p.o.). Thus, the results suggest that the spray-dried naproxen sodium-chitosan complex (NPXF) was not corrosive to the gastric mucosa at high doses of 50, 100, and 200 mg/kg (p.o.) under stressful conditions. It is evident from the present investigation that NPXF does not possess any ulcerogenic potential in comparison to naproxen which, under stressful conditions, led to the hypersecretion of HCl, culminating to petichial hemorrhages in the gastric mucosa of the animals. The biphasic pattern was observed in the various arthritic parameters. The rise in paw volume, joint diameter, WBC count, arthritis score, and fall in body weight was significantly ameliorated in the animals treated with NPXF (5, 10, and 20 mg/kg, p.o). At the end of the study, slight erythema was visible in the naproxen-treated animals. However, no erythema, redness, or ulcers were visible in the animals treated with NPXF. Thus, the direct compression properties and reduced ulcerogenic activity, combined with the demonstrated solubilizing power and analgesic effect enhancer ability toward the drug, make naproxen sodium-chitosan spray-dried complexes particularly suitable for developing a reduced-dose, fast-release, solid oral dosage form of naproxen.

Effect of oppositely charged polymer and dissolution media on rheology of spray-dried ionic complexes.

The purpose of this research was to address the utility of rheological study in understanding the influence of oppositely charged polymers on release of naproxen sodium encapsulated in chitosan particles. The interaction between oppositely charged kappa-carrageenan (kappa-Ca) and chitosan leads to relatively higher gel strength, which is proportional to the ability to retard the drug release at acidic pH. The oscillatory tests within the linear viscoelastic range where the stress is proportional to the applied strain were performed on the hydrated sample matrices containing chitosan-naproxen sodium spray-dried complexes and k-Ca or hydroxypropyl methylcellulose (HPMC) in various ratios. It was observed that the effect of pH change on the dynamic moduli in spray-dried complexes containing kappa-Ca was much stronger than that with HPMC reflecting presence of strong ionic interaction between kappa-Ca and chitosan. The combination of oppositely charged polymers in different ratios proved to be useful in modulating the rheological properties of the hydrated formulations and their release-retarding properties. Dynamic moduli can be used to measure gel strength and are significant for the interpretation of oral sustained release spray-dried complexes.

Pharmacophore generation and atom-based 3D-QSAR of novel 2-(4-methylsulfonylphenyl)pyrimidines as COX-2 inhibitors.

Cyclooxygenase-2 (COX-2) inhibitors are widely used for the treatment of pain and inflammatory disorders such as rheumatoid arthritis and osteoarthritis. A series of novel 2-(4-methylsulfonylphenyl)pyrimidine derivatives has been reported as COX-2 inhibitors. In order to understand the structural requirement of these COX-2 inhibitors, a ligand-based pharmacophore and atom-based 3D-QSAR model have been developed. A five-point pharmacophore with four hydrogen bond acceptors (A) and one hydrogen bond donor (D) was obtained. The pharmacophore hypothesis yielded a 3D-QSAR model with good partial least-square (PLS) statistics results. The training set correlation is characterized by PLS factors (r (2) = 0.642, SD = 0.65, F = 82.7, P = 7.617 e - 12). The test set correlation is characterized by PLS factors (Q (2) (ext) = 0.841, RMSE = 0.24,Pearson-R = 0.91). A docking study revealed the binding orientations of these inhibitors at active site amino acid residues (Arg513, Val523, Phe518, Ser530, Tyr355, His90) of COX-2 enzyme. The results of ligand-based pharmacophore hypothesis and atom-based 3D-QSAR give detailed structural insights as well as highlights important binding features of novel 2-(4-methylsulfonylphenyl)pyrimidine derivatives as COX-2 inhibitors which can provide guidance for the rational design of novel potent COX-2 inhibitors.

Effect of drying methods on swelling, erosion and drug release from chitosan-naproxen sodium complexes.

The purpose of this research was to explore theapplication of ionic interactions between naproxen sodium (NS) and chitosan (CH) in complexes (NSC) prepared by tray drying (TD) and spray drying (SD) methods. Drug-polymer ratio (1:1) in the NSC was optimized on the basis of dialysis studies. The particulate systems of NSC were prepared by tray drying (TD) and spray drying (SD) methods. Release retarding polymers were added to the NSC and to the physical mixtures containing NS-CH and their effects on water uptake, matrix erosion and drug release at different pH were compared. Spray dried complexes (SDC) were spherical, free flowing, light and fine amorphous particles in contrast to the crystalline, hard, tenacious, irregularly shaped, denser tray dried complexes (TDC) with poor flowability. Differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and Fourier transform infrared (FTIR) patterns confirm the conversion of crystalline to high energy amorphous phase suitable for ionic interactions in NSC. Presence of release retarding polymers, kappa carrageenan and hydroxypropylmethylcellulose (HPMC) in the NSC compacts retarded the drug release and improved the matrix integrity. Carrageenan matrices exhibited more retardation than HPMC tablets. FTIR patterns, erosion, swelling and drug release from matrices support ionic interactions between NS and CH in NSC. The reasons for retarded drug release from the chitosan matrices at acidic pH include poor solubility of drug at acidic pH, formation of a rate limiting polymer gel barrier along the periphery of matrices and the ionic interactions between oppositely charged moieties.

Development and validation of a TLC-densitometric method for the simultaneous quantitation of strychnine and brucine from Strychnos spp. and its formulations.

A simple, sensitive, and specific thin-layer chromatography densitometric method has been developed for the simultaneous quantitation of strychnine and brucine. These two marker compounds are quantitated in the seeds of Strychnos nux-vomica, Strychnos ignatii, and its formulations. The method involves densitometric evaluation of strychnine and brucine after resolving it by high-performance TLC on silica gel plate with toluene-ethyl acetate-diethyl amine-methanol (7:2:1:0.3 v/v) as the mobile phase. The method is validated for precision (interday and intraday), repeatability, and accuracy. The relationship between the concentration of standard solutions and the peak response is linear within the concentration range of 160 to 480 ng/spot for strychnine and 80 to 480 ng/spot for brucine. Instrumental precision is found to be 0.54 and 0.78 (% CV), and repeatability of the method is 1.01 and 1.06 (% CV) for strychnine and brucine, respectively. Accuracy of the method is checked by recovery study conducted at three different levels and the average percentage recovery is found to be 99.13% for strychnine and 100.16% for brucine. The proposed HPTLC method for the simultaneous quantitation of strychnine and brucine is found to be simple, precise, specific, sensitive, and accurate, and it can be used for routine quality control of raw material of Strychnos spp. It also can be applied in quantitating any of these marker compounds in other formulations.

Synthesis and antihyperlipidemic activity of some novel condensed 2-chloroalkyl-4-chloro/hydroxy-5,6-disubstituted pyrimidines.

Synthesis and antihyperlipidemic activity of a series of novel condensed 2-chloroalkyl-4-chloro/hydroxy-5,6-di-substituted pyrimidines are described. The design of these compounds is based on the earlier QSAR study on the antihyperlipidemic 2-substituted methylthienopyrimidin-4-ones. The newly synthesized condensed 4-chloro-2-chloroalkylpyrimidines (IIIa-n) have exhibited much superior antihyperlipidemic activity, compared to their earlier reported 4-hydroxy analogs. Notably, in this series, five compounds, IIIa, IIIb, IIIc, IIIi and IIIm showed good ability to reduce total cholesterol and two compounds, IIIa and IIIk exhibited better reduction in serum triglycerides. All the newly synthesized compounds have been evaluated by the Triton WR 1339 induced hyperlipidemia in albino Wistar rats model for antihyperlipidemic activity, and their activity is superior to that exhibited by the standard gemfibrozil used in the study. A 3D QSAR study has also been performed to delineate the effect of the substituents at 5 and 6 positions on the antihyperlipidemic activity of 2-chloromethyl-5,6-substituted thieno(2,3-d) pyrimidin-4(3H)-ones (IIa-e).

Human protein tyrosine phosphatase 1B inhibitors: QSAR by genetic function approximation.

Protein tyrosine phosphatase 1B (PTP 1B), a negative regulator of insulin receptor signaling system, has emerged as a highly validated, attractive target for the treatment of non-insulin dependent diabetes mellitus (NIDDM) and obesity. As a result there is a growing interest in the development of potent and specific inhibitors for this enzyme. This quantitative structure-activity relationship (QSAR) study for a series of formylchromone derivatives as PTP lB inhibitors was performed using genetic function approximation (GFA) technique. The QSAR models were developed using a training set of 29 compounds and the predictive ability of the QSAR model was evaluated against a test set of 7 compounds. The internal and external consistency of the final QSAR model was 0.766 and 0.785. The statistical quality of QSAR models was assessed by statistical parameters r2, r2 (crossvalidated r2), r2pred (predictive r2) and lack of fit (LOF) measure. The results indicate that PTP lB inhibitory activity of the formylchromone derivatives is strongly dependent on electronic, thermodynamic and shape related parameters.

Effect of oppositely charged polymer and dissolution medium on swelling, erosion, and drug release from chitosan matrices.

The reasons for retarded release of naproxen sodium from the chitosan matrices at different pH include poor aqueous solubility of drug, the formation of a rate-limiting polymer gel barrier along the periphery of matrices, the interaction of naproxen sodium with protonated amino groups of chitosan, and the interaction of ionized amino groups of chitosan with ionized sulfate groups of kappa-carrageenan.

3D-QSAR of histone deacetylase inhibitors as anticancer agents by genetic function approximation.

Histone deacetylases (HDACs) play a critical role in gene transcription and are implicated in cancer therapy and other diseases. Inhibitors of HDACs induce cell differentiation and suppress cell proliferation in the tumor cells. Although many such inhibitors have been designed and synthesized, but selective inhibitors for HDAC isoforms are lacking. Various hydroxamic acid analogues have been reported as HDAC inhibitors. Here, we report a three-dimensional quantitative structure-activity relationship (3D-QSAR) study performed using genetic function approximation (GFA) for this class of molecules. QSAR models were generated using a training set of 39 molecules and the predictive ability of final model was assessed using a test set of 17 molecules. The internal consistency of the final QSAR model was 0.712 and showed good external predictivity of 0.585. The results of the present QSAR study indicated that molecular shape analysis (MSA). thermodynamic and structural descriptors are important for inhibition of HDACs.

Chromatographic determination of itopride hydrochloride in the presence of its degradation products.

Two sensitive and reproducible methods are described for the quantitative determination of itopride hydrochloride (IH) in the presence of its degradation products. The first method is based on HPLC separation on a reversed phase Kromasil column [C18 (5-microm, 25 cm x 4.6 mm, ID)] at ambient temperature using a mobile phase consisting of methanol and water (70:30, v/v) adjusted to pH 4.0 with orthophosphoric acid with UV detection at 258 nm. The flow rate was 1.0 mL per min with an average operating pressure of 180 kg/cm2. The second method is based on HPTLC separation on silica gel 60 F254 using toluene:methanol:chloroform:10% ammonia (5.0:3.0:6.0:0.1, v/v/v/v) as mobile phase at 270 nm. The analysis of variance (ANOVA) and Student's t-test were applied to correlate the results of IH determination in dosage form by means of HPLC and HPTLC methods. The drug was subjected to acid and alkali hydrolysis, oxidation, dry heat, wet heat treatment, UV, and photodegradation. The proposed HPLC method was utilized to investigate the kinetics of the acidic, alkaline, and oxidative degradation processes at different temperatures and the apparent pseudo-first-order rate constant, half-life, and activation energy were calculated. In addition the pH-rate profile of degradation of IH in constant ionic strength buffer solutions in the pH range 2-11 was studied.

Pluronic gels for nasal delivery of Vitamin B12. Part I: preformulation study.

Thermoreversible nasal gels of Vitamin B(12) using pluronic PF 127 were aimed to improve absorption and patient compliance. In the present research work, effects of Vitamin B(12) and gel additives, viz. PF concentration, osmolarity, polyethylene glycol (PEG 15000) on thermodynamic properties of phase transitions at gelation (T(1)) and gel melting (T(2)) is reported. Aqueous PF 127 gels prepared by cold method containing pluronic (20-24%, w/w), vitamin, sorbitol, PEG, and benzalkonium chloride. T(1) decreases and T(2) increases with vitamin and PF concentration. Gelation range narrows with sorbitol and PEG. Suppression of T(2) is significantly higher than T(1) with both the additives. The linearity was observed only for semilogarithmic plot of PF concentration and 1/T(2) for sorbitol and PEG, which reveals significant interaction of both at gel melting. Enthalpy of both transitions remains unchanged with vitamin indicating no interaction with polymer. Benzalkonium chloride decreased gelation onset temperature. Thermodynamic properties of PF 127 gels are significantly altered with polymer concentration and water-soluble formulation additives.

Crystallo-co-agglomeration: a novel technique to obtain ibuprofen-paracetamol agglomerates.

The purpose of this research was to obtain directly compressible agglomerates of ibuprofen-paracetamol containing a desired ratio of drugs using a crystallo-co-agglomeration technique. Crystallo-co-agglomeration is an extension of the spherical crystallization technique, which enables simultaneous crystallization and agglomeration of 2 or more drugs or crystallization of a drug and its simultaneous agglomeration with another drug or excipient. Dichloromethane (DCM)-water system containing polyethylene glycol (PEG) 6000, polyvinyl pyrollidone, and ethylcellulose was used as the crystallization system. DCM acted as a good solvent for ibuprofen and bridging liquid for agglomeration. The process was performed at pH 5, considering the low solubility of ibuprofen and the stability of paracetamol. Loss of paracetamol was reduced by maintaining a low process temperature and by the addition of dextrose as a solubility suppressant. The agglomerates were characterized by differential scanning calorimetry, powder x-ray diffraction (PXRD), and scanning electron microscopy and were evaluated for tableting properties. The spherical agglomerates contained an ibuprofen-paracetamol ratio in the range of 1.23 to 1.36. Micromeritic, mechanical, and compressional properties of the agglomerates were affected by incorporated polymer. The PXRD data showed reduction in intensities owing to dilution and reduced crystallinity. Thermal data showed interaction between components at higher temperature. Ethylcellulose imparted mechanical strength to the agglomerates as well as compacts. The agglomerates containing PEG have better compressibility but drug release in the initial stages was affected owing to asperity melting, yielding harder compacts. The agglomeration and properties of agglomerates were influenced by the nature of polymer.