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.

Surface-Modified Inhaled Microparticle-Encapsulated Celastrol for Enhanced Efficacy in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer affecting the pleural lining of the lungs. Celastrol (Cela), a pentacyclic triterpenoid, has demonstrated promising therapeutic potential as an antioxidant, anti-inflammatory, neuroprotective agent, and anti-cancer agent. In this study, we developed inhaled surface-modified Cela-loaded poly(lactic-co-glycolic) acid (PLGA) microparticles (Cela MPs) for the treatment of MPM using a double emulsion solvent evaporation method. The optimized Cela MPs exhibited high entrapment efficiency (72.8 ± 6.1%) and possessed a wrinkled surface with a mean geometric diameter of ~2 µm and an aerodynamic diameter of 4.5 ± 0.1 µm, suggesting them to be suitable for pulmonary delivery. A subsequent release study showed an initial burst release up to 59.9 ± 2.9%, followed by sustained release. The therapeutic efficacy of Cela MPs was evaluated against four mesothelioma cell lines, where Cela MP exhibited significant reduction in IC50 values, and blank MPs produced no toxicity to normal cells. Additionally, a 3D-spheroid study was performed where a single dose of Cela MP at 1.0 µM significantly inhibited spheroid growth. Cela MP was also able to retain the antioxidant activity of Cela only while mechanistic studies revealed triggered autophagy and an induction of apoptosis. Therefore, these studies highlight the anti-mesothelioma activity of Cela and demonstrate that Cela MPs are a promising inhalable medicine for MPM treatment.

Solubility Enhancement and Inhalation Delivery of Cyclodextrin-Based Inclusion Complex of Delamanid for Pulmonary Tuberculosis Treatment.

Tuberculosis (TB) is a contiguous airborne disease caused by Mycobacterium tuberculosis (M.tb), primarily affecting the human lungs. The progression of drug-susceptible TB to drug-resistant strains, MDR-TB and XDR-TB, has become a global challenge toward eradicating TB. Conventional TB treatment involves frequent dosing and prolonged treatment regimens predominantly by an oral or invasive route, leading to treatment-related systemic adverse effects and patient's noncompliance. Pulmonary delivery is an attractive option as we could reduce dose, limit systemic side-effects, and achieve rapid onset of action. Delamanid (DLD), an antituberculosis drug, has poor aqueous solubility, and in this study, we aim to improve its solubility using cyclodextrin complexation. We screened different cyclodextrins and found that HP-ß-CD resulted in a 54-fold increase in solubility compared to a 27-fold and 13-fold increase by SBE-ß-CD and HP-É£-CD, respectively. The stability constant (265 ± 15 M-1) and complexation efficiency (8.5 × 10-4) suggest the formation of a stable inclusion complex of DLD and HP-ß-CD in a 2:1 ratio. Solid-state characterization studies (DSC, PXRD, and NMR) further confirmed successful complexation of DLD in HP-ß-CD. The nebulized DLD-CD complex solution showed a mass median aerodynamic diameter of 4.42 ± 0.62 µm and fine particle fraction of 82.28 ± 2.79%, suggesting deposition in the respiratory airways. In bacterial studies, minimum inhibitory concentration of DLD-CD complex was significantly reduced (four-fold) compared to free DLD in M.tb (H37Ra strain). Furthermore, accelerated stability studies confirmed that the inclusion complex was stable for 4 weeks with 90%w/w drug content. In conclusion, we increased the aqueous solubility of DLD through cyclodextrin complexation and improved its efficacy in vitro.

Repurposing an Antiepileptic Drug for the Treatment of Glioblastoma.

PURPOSE: Glioblastoma multiforme (GBM) is a grade IV, highly proliferative, and malignant form of brain tumor with a 5-year survival rate at ~ 5%. Current treatment strategies for GBM include surgery, radiation, and chemotherapy. Major challenges in GBM management include difficulties in surgical resection due to brain's vital functions and GBM metastasis, development of resistance to temozolomide (TMZ), and protection of tumor by blood brain barrier (BBB). Therefore, we aimed to discover a novel therapeutic for GBM by targeting its metabolic reprogramming. METHOD: We screened metabolic inhibitors by their effects on GBM cell viability by MTT assay. We discovered an FDA-approved drug stiripentol (STP) in our screening of metabolic inhibitors in GBM cells. STP is used for Dravet syndrome (a rare epilepsy). We further tested efficacy of STP using proliferation assay, clonogenic assay, in vitro migration assay, cell cycle assay, apoptosis assay, and in U87 3D spheroids. We also tested the toxicity of STP, and combinations used in the study on normal human dermal fibroblasts. RESULTS: STP was effective in decreasing GBM cell viability, proliferation, clonogenic ability, and migration. Moreover, cell cycle changes were involved but robust apoptosis was absent in STP's anticancer effects. STP was effective in 3D spheroid models, and in TMZ-resistant cells. STP showed additive or synergistic effect with TMZ in different anticancer assays on GBM cells and was considerably less toxic in normal cells. CONCLUSION: Our results indicate that STP can be an effective GBM therapeutic that enhances the effects of TMZ on GBM cells. Importantly, STP reduced viability of TMZ-resistant cells. Our results warrant further studies in the mechanistic basis of STP's effects on GBM cells and the preclinical potential of STP in animal models.

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.

Polypharmacology: The science of multi-targeting molecules.

Polypharmacology is a concept where a molecule can interact with two or more targets simultaneously. It offers many advantages as compared to the conventional single-targeting molecules. A multi-targeting drug is much more efficacious due to its cumulative efficacy at all of its individual targets making it much more effective in complex and multifactorial diseases like cancer, where multiple proteins and pathways are involved in the onset and development of the disease. For a molecule to be polypharmacologic in nature, it needs to possess promiscuity which is the ability to interact with multiple targets; and at the same time avoid binding to antitargets which would otherwise result in off-target adverse effects. There are certain structural features and physicochemical properties which when present would help researchers to predict if the designed molecule would possess promiscuity or not. Promiscuity can also be identified via advanced state-of-the-art computational methods. In this review, we also elaborate on the methods by which one can intentionally incorporate promiscuity in their molecules and make them polypharmacologic. The polypharmacology paradigm of "one drug-multiple targets" has numerous applications especially in drug repurposing where an already established drug is redeveloped for a new indication. Though designing a polypharmacological drug is much more difficult than designing a single-targeting drug, with the current technologies and information regarding different diseases and chemical functional groups, it is plausible for researchers to intentionally design a polypharmacological drug and unlock its advantages.

BX-795 inhibits neuroblastoma growth and enhances sensitivity towards chemotherapy.

High-risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology due to relapse of metastatic, drug-resistant disease, and treatment-related toxicities. An analysis of 1235 primary NB patient dataset revealed significant increase in AKT1 and AKT2 gene expression with cancer stage progression. Additionally, Both AKT1 and AKT2 expression inversely correlate with poor overall survival of NB patients. AKT1 and AKT2 genes code for AKT that drive a major oncogenic cell signaling pathway known in many cancers, including NB. To inhibit AKT pathway, we repurposed an antiviral inhibitor BX-795 that inhibits PDK1, an upstream activator of AKT. BX-795 potently inhibits NB cell proliferation and colony growth in a dose-dependent manner. BX-795 significantly enhances apoptosis and blocks cell cycle progression at mitosis phase in NB. Additionally, BX-795 potently inhibits tumor formation and growth in a NB spheroid tumor model. We further tested dual therapeutic approaches by combining BX-795 with either doxorubicin or crizotinib and found synergistic and significant inhibition of NB growth, in contrast to either drug alone. Overall, our data demonstrate that BX-795 inhibits AKT pathway to inhibit NB growth, and combining BX-795 with current therapies is an effective and clinically tractable therapeutic approach for NB.

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.

The diverse bioactivity of α-mangostin and its therapeutic implications.

α-Mangostin is a xanthone natural product isolated as a secondary metabolite from the mangosteen tree. It has attracted a great deal of attention due to its wide-ranging effects on certain biological activity, such as apoptosis, tumorigenesis, proliferation, metastasis, inflammation, oxidation, bacterial growth and metabolism. This review focuses on the key pathways directly affected by α-mangostin and how this varies between disease states. Insight is also provided, where investigated, into the key structural features of α-mangostin that produce these biological effects. The review then sheds light on the utility of α-mangostin as a investigational tool for certain diseases and demonstrate how future derivatives may increase selectivity and potency for specific disease states.

Repurposing Bedaquiline for Effective Non-Small Cell Lung Cancer (NSCLC) Therapy as Inhalable Cyclodextrin-Based Molecular Inclusion Complexes.

There is growing evidence that repurposed drugs demonstrate excellent efficacy against many cancers, while facilitating accelerated drug development process. In this study, bedaquiline (BDQ), an FDA approved anti-mycobacterial agent, was repurposed and an inhalable cyclodextrin complex formulation was developed to explore its anti-cancer activity in non-small cell lung cancer (NSCLC). A sulfobutyl ether derivative of ß-cyclodextrin (SBE-ß-CD) was selected based on phase solubility studies and molecular modeling to prepare an inclusion complex of BDQ and cyclodextrin. Aqueous solubility of BDQ was increased by 2.8 × 103-fold after complexation with SBE-ß-CD, as compared to its intrinsic solubility. Solid-state characterization studies confirmed the successful incorporation of BDQ in the SBE-ß-CD cavity. In vitro lung deposition study results demonstrated excellent inhalable properties (mass median aerodynamic diameter: 2.9 ± 0.6 µm (<5 µm) and fine particle fraction: 83.3 ± 3.8%) of BDQ-CD complex. Accelerated stability studies showed BDQ-CD complex to be stable up to 3 weeks. From cytotoxicity studies, a slight enhancement in the anti-cancer efficacy was observed with BDQ-cyclodextrin complex, compared to BDQ alone in H1299 cell line. The IC50 values for BDQ and BDQ-CD complex were found to be ~40 µM in case of H1299 cell line at 72 h, whereas BDQ/BDQ-CD were not found to be cytotoxic up to concentrations of 50 µM in A549 cell line. Taken together, BDQ-CD complex offers a promising inhalation strategy with efficient lung deposition and cytotoxicity for NSCLC treatment.

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.

Development of pharmaceutically scalable inhaled anti-cancer nanotherapy - Repurposing amodiaquine for non-small cell lung cancer (NSCLC).

New drug and dosage form development faces significant challenges, especially in oncology, due to longer development cycle and associated scale-up complexities. Repurposing of existing drugs with potential anti-cancer activity into new therapeutic regimens provides a feasible alternative. In this project, amodiaquine (AQ), an anti-malarial drug, has been explored for its anti-cancer efficacy through formulating inhalable nanoparticulate systems using high-pressure homogenization (HPH) with scale-up feasibility and high reproducibility. A 32 multifactorial design was employed to better understand critical processes (probe homogenization speed while formulating coarse emulsion) and formulation parameters (concentration of cationic polymer in external aqueous phase) so as to ensure product quality with improved anticancer efficacy in non-small cell lung cancer (NSCLC). Optimized AQ loaded nanoparticles (AQ NP) were evaluated for physicochemical properties, stability profile, in-vitro aerosol deposition behavior, cytotoxic potential against NSCLC cells in-vitro and in 3D simulated tumor spheroid model. The highest probe homogenization speed (25,000 rpm) resulted in lower particle size. Incorporation of cationic polymer, polyethylenimine (0.5% w/v) resulted in high drug loading efficiencies at optimal drug quantity of 5 mg. Formulated nanoparticles (liquid state) exhibited an aerodynamic diameter of 4.7 ± 0.1 µm and fine particle fraction of 81.0 ± 9.1%, indicating drug deposition in the respirable airways. Cytotoxicity studies in different NSCLC cell lines revealed significant reduction in IC50 values with AQ-loaded nanoparticles compared to plain drug, along with significant cell migration inhibition (scratch assay) and reduced % colony growth (clonogenic assay) in A549 cells with AQ NP. Moreover, 3D simulated spheroid studies revealed efficacy of nanoparticles in penetration to tumor core, and growth inhibition. AQ's autophagy inhibition ability significantly increased (increased LC3B-II levels) with nanoparticle encapsulation, along with moderate improvement in apoptosis induction (Caspase-3 levels). No impact was observed on HUVEC angiogenesis suggesting alternative anticancer mechanisms. To conclude, amodiaquine can be a promising candidate for repurposing to treat NSCLC while delivering inhalable nanoparticles developed using a scalable HPH process. Despite the involvement of complex parameters, application of DoE has simplified the process of product and process optimization.

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.

Inhalable resveratrol-cyclodextrin complex loaded biodegradable nanoparticles for enhanced efficacy against non-small cell lung cancer.

Resveratrol (RES), a natural polyphenol in fruits, has shown promising anti-cancer properties. Due to its relative low toxicity which limits the adverse effects observed for conventional chemotherapeutics, RES has been proposed as an alternative. However, the therapeutic applications of RES have been limited due to low water solubility, as well as chemical and physical instability. This study investigated enhancing the anti-cancer activity of RES against non-small-cell-lung-cancer (NSCLC) by complexing with sulfobutylether-ß-cyclodextrin (CD-RES) and loading onto polymeric nanoparticles (NPs). The physicochemical properties of the CD-RES NPs were then characterized. The CD-RES inclusion complex increased the water solubility of RES by ~66-fold. CD-RES NPs demonstrated very good aerosolization potential with a mass median aerodynamic diameter of 2.20 µm. Cell-based studies demonstrated improved therapeutic efficacy of CD-RES NPs compared to RES. This included enhanced cellular uptake, cytotoxicity, and apoptosis, while retaining antioxidant activity. The 3D spheroid study indicated an intensified anti-cancer effect of CD-RES NPs. Altogether, these findings marked CD-RES NPs as a potential inhalable delivery system of RES for the treatment 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.

Systematic Development and Optimization of Inhalable Pirfenidone Liposomes for Non-Small Cell Lung Cancer Treatment.

Non-small cell lung cancer (NSCLC) is a global disorder, treatment options for which remain limited with resistance development by cancer cells and off-target events being major roadblocks for current therapies. The discovery of new drug molecules remains time-consuming, expensive, and prone to failure in safety/efficacy studies. Drug repurposing (i.e., investigating FDA-approved drug molecules for use against new indications) provides an opportunity to shorten the drug development cycle. In this project, we propose to repurpose pirfenidone (PFD), an anti-fibrotic drug, for NSCLC treatment by encapsulation in a cationic liposomal carrier. Liposomal formulations were optimized and evaluated for their physicochemical properties, in-vitro aerosol deposition behavior, cellular internalization capability, and therapeutic potential against NSCLC cell lines in-vitro and ex-vivo. Anti-cancer activity of PFD-loaded liposomes and molecular mechanistic efficacy was determined through colony formation (1.5- to 2-fold reduction in colony growth compared to PFD treatment in H4006, A549 cell lines, respectively), cell migration, apoptosis and angiogenesis assays. Ex-vivo studies using 3D tumor spheroid models revealed superior efficacy of PFD-loaded liposomes against NSCLC, as compared to plain PFD. Hence, the potential of inhalable liposome-loaded pirfenidone in NSCLC treatment has been established in-vitro and ex-vivo, where further studies are required to determine their efficacy through in vivo preclinical studies followed by clinical studies.