Electrosprayed mesoporous particles for improved aqueous solubility of a poorly water soluble anticancer agent: in vitro and ex vivo evaluation

open access articleEncapsulation of poorly water-soluble drugs into mesoporous materials (e.g. silica) has evolved as a favorable strategy to improve drug solubility and bioavailability. Several techniques (e.g. spray drying, solvent evaporation, microwave irradiation) have been utilized for the encapsulation of active pharmaceutical ingredients (APIs) into inorganic porous matrices. In the present work, a novel chalcone (KAZ3) with anticancer properties was successfully synthesized by Claisen-Schmidt condensation. KAZ3 was loaded into mesoporous (SBA-15 and MCM-41) and non-porous (fumed silica, FS) materials via two techniques; electrohydrodynamic atomization (EHDA) and solvent impregnation. The effect of both loading methods on the physicochemical properties of the particles (e.g. size, charge, entrapment efficiency, crystallinity, dissolution and permeability) was investigated. Results indicated that EHDA technique can load the active in a complete amorphous form within the pores of the silica particles. In contrast, reduced crystallinity (~79%) was obtained for the solvent impregnated formulations. EHDA engineered formulations significantly improved drug dissolution up to 30-fold, compared to the crystalline drug. Ex vivo studies showed EHDA formulations to exhibit higher permeability across rat intestine than their solvent impregnated counterparts. Cytocompatibility studies on Caco-2 cells demonstrated moderate toxicity at high concentrations of the anticancer agent. The findings of the present study clearly show the immense potential of EHDA as a loading technique for mesoporous materials to produce poorly water-soluble API carriers of high payload at ambient conditions. Furthermore, the scale up potential in EHDA technologies indicate a viable route to enhance drug encapsulation and dissolution rate of loaded porous inorganic materials

The Developmental Program of Murine Erythroleukemia Cells

Hematopoietic stem cells (HSCs) or early progenitors respond to external stimuli in bone marrow and differentiate into cell-restricted lineages of blood cells of limited life span. In leukemias, however, early hematopoietic progenitors self-renew themselves, fail to respond to differentiation signals, and do not undergo programmed cell death (apoptosis). The basic mechanisms of differentiation and apoptosis of leukemia cells have been the long-term objective of our work. By exploiting widely studied murine and human leukemic cell systems as models of hematopoietic cell differentiation, we explored the mechanisms by which pharmaceutical agents initiate differentiation in leukemic systems. In this article, we present the developmental program of MEL cells with emphasis given on the role of commitment to terminal maturation. Commitment is initiated via inducer-receptor-mediated processes and leads to discrete patterns of expression of several genes that contribute to growth arrest at the G1 phase, expression of differentiated phenotype, and differentiation-dependent apoptosis (DDA). Overall, MEL erythroid cell differentiation represents a developmental program with a highly coordinated set of processes that is "triggered" by an inducer and functions via a network of genes and proteins interacting with each other harmonically to give birth to lineage-restricted phenotype

miR-16-5p Promotes Erythroid Maturation of Erythroleukemia Cells by Regulating Ribosome Biogenesis

miRNAs constitute a class of non-coding RNA that act as powerful epigenetic regulators in animal and plant cells. In order to identify putative tumor-suppressor miRNAs we profiled the expression of various miRNAs during differentiation of erythroleukemia cells. RNA was purified before and after differentiation induction and subjected to quantitative RT-PCR. The majority of the miRNAs tested were found upregulated in differentiated cells with miR-16-5p showing the most significant increase. Functional studies using gain- and loss-of-function constructs proposed that miR-16-5p has a role in promoting the erythroid differentiation program of murine erythroleukemia (MEL) cells. In order to identify the underlying mechanism of action, we utilized bioinformatic in-silico platforms that incorporate predictions for the genes targeted by miR-16-5p. Interestingly, ribosome constituents, as well as ribosome biogenesis factors, were overrepresented among the miR-16-5p predicted gene targets. Accordingly, biochemical experiments showed that, indeed, miR-16-5p could modulate the levels of independent ribosomal proteins, and the overall ribosomal levels in cultured cells. In conclusion, miR-16-5p is identified as a differentiation-promoting agent in erythroleukemia cells, demonstrating antiproliferative activity, likely as a result of its ability to target the ribosomal machinery and restore any imbalanced activity imposed by the malignancy and the blockade of differentiation

Mechanisms of action of differentiation inducers: Detection of inducer binding protein(s) in murine erythroleukemia cells

We have shown previously that murine erythroleukemia (MEL) and human neuroectodermal RD/TE-671 cells are induced to differentiate by ureido derivatives of pyridine (UDPs) and may contain inducer binding protein(s). In the present study, we prepared radiolabeled [3H]UDP {2-(3-ethylureido)-6-[3H]-acetylamino-pyridine) as ligand and investigated whether it interacts selectively with novel binding proteins. MEL and RD/TE-671 cells, incubated with the inducer [3H]UDP and subsequently fractionated, yielded a radiolabeled postmitochondrial soluble fraction containing the [3H]UDP-protein complex. We purified the UDP binding protein by using UDP-sepharose affinity chromatography, gel filtration, and SDS-PAGE electrophoresis and analyzed its structure. The data presented here indicate for the first time that the inducer UDP interacts with a 38,333 ± 30 Da binding protein(s) (p38), of unknown function, in both cell lines. Microsequencing and sequence alignment search revealed that the p38 protein(s) contains at least two homologous domains, one being part of ABC-type transporters and another found in the Wingless-type (Wnt) proteins. Kinetic analysis revealed that the p38 forms a relatively stable protein complex with [3H]UDP that accumulates within the cytosol and nucleus of MEL cells during the precommitment period. This complex, however, decays later on after commitment to erythroid maturation has been initiated. De novo protein and mRNA synthesis is needed for the UDP-p38 complex to form, as shown by the use of metabolic inhibitors. Purified p38 was used to develop an anti-p38 polyclonal serum, and Western blot analysis revealed that the level of p38 was quite similar in both UDP-inducible and -resistant MEL subclones that we developed. Although only a portion of the primary structure of the p38 is known from microsequencing, the mechanism by which the UDP-p38 complex contributes to induction of differentiation in both UDP-responsive mouse MEL and human RD/TE-671 cells is discussed. Copyright © 2005 Cognizant Comm. Corp

On the Intracellular Trafficking of Mouse S5 Ribosomal Protein from Cytoplasm to Nucleoli

The non-ribosomal functions of mammalian ribosomal proteins have recently attracted worldwide attention. The mouse ribosomal protein S5 (rpS5) derived from ribosomal material is an assembled non-phosphorylated protein. The free form of rpS5 protein, however, undergoes phosphorylation. In this study, we have (a) investigated the potential role of phosphorylation in rpS5 protein transport into the nucleus an then into nucleoli and (b) determined which of the domains of rpS5 are involved in this intracellular trafficking. In vitro PCR mutagenesis of mouse rpS5 cDNA, complemented by subsequent cloning and expression of rpS5 truncated recombinant forms, produced in fusion with green fluorescent protein, permitted the investigation of rpS5 intracellular trafficking in HeLa cells using confocal microscopy complemented by Western blot analysis. Our results indicate the following: (a) rpS5 protein enters the nucleus via the region 38-50 aa that forms a random coil as revealed by molecular dynamic simulation. (b) Immunoprecipitation of rpS5 with casein kinase 11 and immobilized metal affinity chromatography analysis complemented by in vitro kinase assay revealed that phosphorylation of rpS5 seems to be indispensable for its transport from nucleus to nucleoli; upon entering the nucleus, Thr-133 phosphorylation triggers Ser-24 phosphorylation by casein kinase 11, thus promoting entrance of rpS5 into the nucleoli. Another important role of rpS5 N-terminal region is proposed to be the regulation of protein's cellular level. The repetitively co-appearance of a satellite C-terminal band below the entire rpS5 at the late stationary phase, and not at the early logarithmic phase, of cell growth suggests a specific degradation balancing probably the unassembled ribosomal protein molecules with those that are efficiently assembled to ribosomal subunits. Overall, these data provide new insights on the structural and functional domains within the rpS5 molecule that contribute to its cellular functions. (C) 2009 Elsevier Ltd. All rights reserved

Antibacterial activity of griseofulvin analogues as an example of drug repurposing

Griseofulvin is a well-known antifungal drug that was launched in 1962 by Merck & Co. for the treatment of dermatophyte infections. However, according to predictions using the Way2Drug computational drug repurposing platform, it may also have antibacterial activity. As no confirmation of this prediction was found in the published literature, this study estimated in-silico antibacterial activity for 42 griseofulvin derivatives. Antibacterial activity was predicted for 33 of the 42 compounds, which led to the conclusion that this activity might be considered as typical for this chemical series. Therefore, experimental testing of antibacterial activity was performed on a panel of Gram-positive and Gram-negative micro-organisms. Antibacterial activity was evaluated using the microdilution method detecting the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC). The tested compounds exhibited potent antibacterial activity against all the studied bacteria, with MIC and MBC values ranging from 0.0037 to 0.04 mg/mL and from 0.01 to 0.16 mg/mL, respectively. Activity was 2.5–12 times greater than that of ampicillin and 2–8 times greater than that of streptomycin, which were used as the reference drugs. Similarity analysis for all 42 compounds with the (approximately) 470,000 drug-like compounds indexed in the Clarivate Analytics Integrity database confirmed the significant novelty of the antibacterial activity for the compounds from this chemical class. Therefore, this study demonstrated that by using computer-aided prediction of biological activity spectra for a particular chemical series, it is possible to identify typical biological activities which may be used for discovery of new applications (e.g. drug repurposing)

Dissecting miRNA–Gene Networks to Map Clinical Utility Roads of Pharmacogenomics-Guided Therapeutic Decisions in Cardiovascular Precision Medicine

MicroRNAs (miRNAs) create systems networks and gene-expression circuits through molecular signaling and cell interactions that contribute to health imbalance and the emergence of cardiovascular disorders (CVDs). Because the clinical phenotypes of CVD patients present a diversity in their pathophysiology and heterogeneity at the molecular level, it is essential to establish genomic signatures to delineate multifactorial correlations, and to unveil the variability seen in therapeutic intervention outcomes. The clinically validated miRNA biomarkers, along with the relevant SNPs identified, have to be suitably implemented in the clinical setting in order to enhance patient stratification capacity, to contribute to a better understanding of the underlying pathophysiological mechanisms, to guide the selection of innovative therapeutic schemes, and to identify innovative drugs and delivery systems. In this article, the miRNA–gene networks and the genomic signatures resulting from the SNPs will be analyzed as a method of highlighting specific gene-signaling circuits as sources of molecular knowledge which is relevant to CVDs. In concordance with this concept, and as a case study, the design of the clinical trial GESS (NCT03150680) is referenced. The latter is presented in a manner to provide a direction for the improvement of the implementation of pharmacogenomics and precision cardiovascular medicine trials