Synthesis and evaluation of 2,5-substituted pyrimidines as small-molecule gankyrin binders.

Background: Gankyrin is an ankyrin-repeat protein that promotes cell proliferation, tumor development and cancer progression when overexpressed. Aim: To design and synthesize a novel series of gankyrin-binding small molecules predicated on a 2,5-pyrimidine scaffold. Materials & methods: The synthesized compounds were evaluated for their antiproliferative activity, ability to bind gankyrin and effects on cell cycle progression and the proteasomal degradation pathway. Results: Compounds 188 and 193 demonstrated the most potent antiproliferative activity against MCF7 and A549 cells, respectively. Both compounds also demonstrated the ability to effectively bind gankyrin, disrupt proteasomal degradation and inhibit cell cycle progression. Conclusion: The 2,5-pyrimidine scaffold exhibits a novel and promising strategy for binding gankyrin and inhibiting cancer cell proliferation.

Second Generation Small Molecule Inhibitors of Gankyrin for the Treatment of Pediatric Liver Cancer.

Background: Gankyrin, a member of the 26S proteasome, is an overexpressed oncoprotein in hepatoblastoma (HBL) and hepatocellular carcinoma (HCC). Cjoc42 was the first small molecule inhibitor of Gankyrin developed; however, the IC50 values of >50 µM made them unattractive for clinical use. Second-generation inhibitors demonstrate a stronger affinity toward Gankyrin and increased cytotoxicity. The aim of this study was to characterize the in vitro effects of three cjoc42 derivatives. Methods: Experiments were performed on the HepG2 (HBL) and Hep3B (pediatric HCC) cell lines. We evaluated the expression of TSPs, cell cycle markers, and stem cell markers by Western blotting and/or real-time quantitative reverse transcription PCR. We also performed apoptotic, synergy, and methylation assays. Results: The treatment with cjoc42 derivatives led to an increase in TSPs and a dose-dependent decrease in the stem cell phenotype in both cell lines. An increase in apoptosis was only seen with AFM-1 and -2 in Hep3B cells. Drug synergy was seen with doxorubicin, and antagonism was seen with cisplatin. In the presence of cjoc42 derivatives, the 20S subunit of the 26S proteasome was more available to transport doxorubicin to the nucleus, leading to synergy. Conclusion: Small-molecule inhibitors for Gankyrin are a promising therapeutic strategy, especially in combination with doxorubicin.

Small-Molecule Gankyrin Inhibition as a Therapeutic Strategy for Breast and Lung Cancer.

Gankyrin is an oncoprotein responsible for the development of numerous cancer types. It regulates the expression levels of multiple tumor suppressor proteins (TSPs) in liver cancer; however, gankyrin's regulation of these TSPs in breast and lung cancers has not been thoroughly investigated. Additionally, no small-molecule gankyrin inhibitor has been developed which demonstrates potent anti-proliferative activity against gankyrin overexpressing breast and lung cancers. Herein, we are reporting the structure-based design of gankyrin-binding small molecules which potently inhibited the proliferation of gankyrin overexpressing A549 and MDA-MB-231 cancer cells, reduced colony formation, and inhibited the growth of 3D spheroids in an in vitro tumor simulation model. Investigations demonstrated that gankyrin inhibition occurs through either stabilization or destabilization of its 3D structure. These studies shed light on the mechanism of small-molecule inhibition of gankyrin and demonstrate that gankyrin is a viable therapeutic target for the treatment of breast and lung cancer.

Bioinspired particle engineering for non-invasive inhaled drug delivery to the lungs.

Pulmonary drug delivery is governed by several biophysical parameters of delivery carriers, such as particle size, shape, density, charge, and surface modifications. Although much attention has been given to other parameters, particle shape effects have rarely been explored. In this work, we assess the influence of particle shape of inhaled delivery carriers on their aerodynamic properties and macrophage uptake by using polymeric microparticles of different geometries ranging in various sizes. Doxorubicin was conjugated to the polymer particles and the bioconjugates were characterized. Interestingly, the results of in-vitro lung deposition, performed using a next generation impactor, demonstrated a significant improvement in the aerodynamic properties of the rod-shaped particles with a high aspect ratio as compared to spherical particles with the same equivalent volume. The results of a macrophage uptake experiment demonstrate that the high aspect ratio particles were phagocytosed less than spherical particles. Furthermore, the cytotoxicity of these doxorubicin-conjugated particles was determined against murine macrophages, resulting in reduced toxicity when treated with high aspect ratio particles as compared to spherical particles. This project provides valuable insights into the influence of particle shape on aerodynamic properties and primary defense mechanisms in the peripheral lungs, while using polymeric microparticles of various sizes and geometries. Further systematic development can help translate these findings to preclinical and clinical studies for designing efficient inhalable delivery carriers.

Optimizing the aryl-triazole of cjoc42 for enhanced gankyrin binding and anti-cancer activity.

Gankyrin is an oncoprotein overexpressed in numerous cancer types and appears to play a key role in regulating cell proliferation, cell growth, and cell migration. These roles are largely due to gankyrin's protein-protein interaction with the 26S proteasome. We previously published a study exploring the aryl sulfonate ester of cjoc42 in an effort to enhance gankyrin binding and inhibit cancer cell proliferation. In order to further improve the gankyrin binding ability of the cjoc42 scaffold, an extensive SAR for the aryl-triazole moiety of cjoc42 was developed. Our cjoc42 derivatives exhibited enhanced gankyrin binding, as well as enhanced antiproliferative activity against Hep3B, HepG2, A549, and MDA-MB-231 cancer cell lines.

Cyclodextrin Complexation for Enhanced Stability and Non-invasive Pulmonary Delivery of Resveratrol-Applications in Non-small Cell Lung Cancer Treatment.

Pulmonary drug delivery is a noninvasive therapeutic approach that offers many advantages including localized drug delivery and higher patient compliance. As with all formulations, the low aqueous solubility of a drug often poses a challenge in the formulation development. Thus, strategies such as cyclodextrin (CD) complexation have been utilized to overcome this challenge. Resveratrol (RES), a natural stilbene, has shown abundant anti-cancer properties. Due to many drawbacks of conventional chemotherapeutics, RES has been proposed as an emerging alternative with promising pharmacological effects. However, RES has limited therapeutic applications due to low water solubility, chemical stability, and bioavailability. This study was aimed at developing an inhalable therapy that would increase the aqueous solubility and stability of RES by complexation with sulfobutylether-ß-cyclodextrin (SBECD). Phase solubility profiles indicated an optimal stoichiometric inclusion complex at 1:1 (SBECD:RES) ratio for formulation considerations. Physiochemical characterizations were performed to analyze CD-RES. Stability studies at pH 7.4 and in plasma indicated significant improvement in RES stability after complexation, with a much longer half-life. The mass median aerodynamic diameter (MMAD) of CD-RES was 2.6 ± 0.7 µm and fine particle fraction (FPF) of 83.4 ± 3.0% are suitable for pulmonary delivery and efficient deposition. Lung cancer was selected as the respiratory model disease, owing to its high relevance as the major cause of cancer deaths worldwide. Cell viability studies in 5 non-small-cell-lung-cancer (NSCLC) cell lines suggest CD-RES retained significant cytotoxic potential of RES. Taken together, CD-RES proves to be a promising inhalation treatment for NSCLC.

Sorafenib Loaded Inhalable Polymeric Nanocarriers against Non-Small Cell Lung Cancer.

PURPOSE: This exploration is aimed at developing sorafenib (SF)-loaded cationically-modified polymeric nanoparticles (NPs) as inhalable carriers for improving the therapeutic efficacy of SF against non-small cell lung cancer (NSCLC). METHODS: The NPs were prepared using a solvent evaporation technique while incorporating cationic agents. The optimized NPs were characterized by various physicochemical parameters and evaluated for their aerosolization properties. Several in-vitro evaluation studies were performed to determine the efficacy of our delivery carriers against NSCLC cells. RESULTS: Optimized nanoparticles exhibited an entrapment efficiency of ~40%, <200 nm particle size and a narrow poly-dispersity index. Cationically-modified nanoparticles exhibited enhanced cellular internalization and cytotoxicity (~5-fold IC50 reduction vs SF) in various lung cancer cell types. The inhalable nanoparticles displayed efficient aerodynamic properties (MMAD ~ 4 µM and FPF >80%). In-vitro evaluation also resulted in a superior ability to inhibit cancer metastasis. 3D-tumor simulation studies further established the anti-cancer efficacy of NPs as compared to just SF. CONCLUSION: The localized delivery of SF-loaded nanoparticles resulted in improved anti-tumor activity as compared to SF alone. Therefore, this strategy displays great potential as a novel treatment approach against certain lung cancers.

Structural modification of the aryl sulfonate ester of cjoc42 for enhanced gankyrin binding and anti-cancer activity.

Gankyrin is an oncogenic protein involved in various biological processes, such as cellular growth and proliferation. Its overexpression in certain cancers results in an increase of gankyrin-mediated protein-protein interactions (PPIs), leading to cancer proliferation. To date, only one small molecule (cjoc42) has been identified to bind gankyrin, which simultaneously inhibits its interaction with the 26S proteasome. Despite this advance, 2nd generation inhibitors are needed to improve gankyrin binding and cellular efficacy. To this end, an extensive SAR for the aryl sulfonate ester moiety of the cjoc42 scaffold was explored, and showed that substitutions at the 2-, 3-, and 4-positions manifested significant increases in gankyrin binding, resulting in the most potent binders of gankyrin to date. Subsequent cell-based assay evaluation of our derivatives demonstrated antiproliferative activity against pediatric liver cancer cell lines Hep3B and HepG2, which was not previously observed for cjoc42.

Cyclodextrin modified erlotinib loaded PLGA nanoparticles for improved therapeutic efficacy against non-small cell lung cancer.

This study was aimed at developing a nanoparticle strategy to overcome acquired resistance against erlotinib in non-small cell lung cancer (NSCLC). To load erlotinib on biodegradable PLGA nanoparticles, erlotinib-cyclodextrin (Erlo-CD) complex was prepared using ß-cyclodextrin sulfobutyl ether, which was in turn loaded in the core of PLGA nanoparticles using multiple emulsion solvent evaporation. Nanoparticles were characterized for size distribution, entrapment and loading efficiency, in-vitro release, and therapeutic efficacy against different lung cancer cells. Effect of formulation on cell cycle, apoptosis, and other markers was evaluated using flow cytometry and western blotting studies. The efficacy of optimized nanoformulation was evaluated using a clinically relevant in-vitro 3D-spheroid model. Results showed that Erlo-CD loaded nanoparticles (210 ±â€¯8 nm in size) demonstrated 3-fold higher entrapment (61.5 ±â€¯3.2% vs 21.9 ±â€¯3.7% of plain erlotinib loaded nanoparticles) with ~5% loading efficiency and sustained release characteristics. Developed nanoparticles demonstrated significantly improved therapeutic efficacy against NSCLC cells in terms of low IC50 values and suppressed colony forming ability of cancer cells, increased apoptosis, and autophagy inhibition. Interestingly, 3D spheroid study demonstrated better anticancer activity of Erlo-CD nanoparticles compared to plain erlotinib. Present study has shown a premise to improve therapeutic efficacy against erlotinib-resistant lung cancer using modified nanoErlo formulations.

Nintedanib-cyclodextrin complex to improve bio-activity and intestinal permeability.

Cyclodextrin complex of nintedanib was prepared aiming for increased bio-activity and improved transport across intestinal membrane with reduced p-glycoprotein (p-gp) efflux. Based on preliminary phase solubility studies and molecular modeling, sulfobutyl ether derivative of ß-cyclodextrin (SBE-ß-CD, Captisol®) was selected to prepare inclusion complex. Complexation was confirmed using FTIR, 1H NMR, DSC, and XRD. Bioactivity of the formed complex was tested using lung fibroblast cells, WI-38 for anti-proliferative activity and effect on collagen deposition and cells migration. In-vitro permeability studies were performed using epiIntestinal tissue model to assess the effect of complexation on transport and p-gp efflux. Results of the study demonstrated that cyclodextrin complexation increased stability of nintedanib in PBS (pH 7.4) and simulated intestinal fluid (SIF). Further, bioactivity of nintedanib also improved. Interestingly, complexation has increased transport of nintedanib across intestinal membrane and reduced efflux ratio, suggesting the role of cyclodextrin complexation in modulating p-gp efflux.