Ciprofloxacin Derivative-Loaded Nanoparticles Synergize with Paclitaxel Against Type II Human Endometrial Cancer.

Combinations of chemotherapeutic agents comprise a clinically feasible approach to combat cancers that possess resistance to treatment. Type II endometrial cancer is typically associated with poor outcomes and the emergence of chemoresistance. To overcome this challenge, a combination therapy is developed comprising a novel ciprofloxacin derivative-loaded PEGylated polymeric nanoparticles (CIP2b-NPs) and paclitaxel (PTX) against human type-II endometrial cancer (Hec50co with loss of function p53). Cytotoxicity studies reveal strong synergy between CIP2b and PTX against Hec50co, and this is associated with a significant reduction in the IC50 of PTX and increased G2/M arrest. Upon formulation of CIP2b into PEGylated polymeric nanoparticles, tumor accumulation of CIP2b is significantly improved compared to its soluble counterpart; thus, enhancing the overall antitumor activity of CIP2b when co-administered with PTX. In addition, the co-delivery of CIP2b-NPs with paclitaxel results in a significant reduction in tumor progression. Histological examination of vital organs and blood chemistry was normal, confirming the absence of any apparent off-target toxicity. Thus, in a mouse model of human endometrial cancer, the combination of CIP2b-NPs and PTX exhibits superior therapeutic activity in targeting human type-II endometrial cancer.

A ciprofloxacin derivative with four mechanisms of action overcomes paclitaxel resistance in p53-mutant and MDR1 gene-expressing type II human endometrial cancer.

Dysfunction of the p53 gene and the presence of the MDR1 gene are associated with many malignant tumors including endometrial cancer and are responsible for cancer therapeutic resistance and poor survival. Thus, there is a critical need to devise novel combinatorial therapies with multiple mechanisms of action to overcome drug resistance. Here, we report a new ciprofloxacin derivative (CIP2b) tested either alone or in combination with taxanes against four human endometrial cancer cell lines. In vitro studies revealed that a combination of paclitaxel + CIP2b had synergistic cytotoxic effects against MDR1-expressing type-II human endometrial cancer cells with loss-of-function p53 (Hec50co LOFp53). Enhanced antitumor effects were confirmed by substantial increases in caspase-3 expression, cell population shifts toward the G2/M phase, and reduction of cdc2 phosphorylation. It was found that CIP2b targets multiple pathways including the inhibition of MDR1, topoisomerase I, and topoisomerase II, as well as enhancing the effects of paclitaxel (PTX) on microtubule assembly. In vivo treatment with the combination of PTX + CIP2b also led to significantly increased accumulation of PTX in tumors (compared to CIP2b alone) and reduction in tumor growth. Enhanced in vivo cytotoxic effects were confirmed by histological and immunohistochemical examination of the tumor tissues. Complete blood count and blood biochemistry data confirmed the absence of any apparent off-target toxicity. Thus, combination therapy involving PTX and CIP2b targeted multiple pathways and represents an approach that could result in improved tolerance and efficacy in patients with type-II endometrial cancer harboring the MDR1 gene and p53 mutations.

Cationic nanoparticles enhance T cell tumor infiltration and antitumor immune responses to a melanoma vaccine.

In clinical settings, cancer vaccines as monotherapies have displayed limited success compared to other cancer immunotherapeutic treatments. Nanoscale formulations have the ability to increase the efficacy of cancer vaccines by combatting the immunosuppressive nature of the tumor microenvironment. Here, we have synthesized a previously unexplored cationic polymeric nanoparticle formulation using polyamidoamine dendrimers and poly(d,l-lactic-co-glycolic acid) that demonstrate adjuvant properties in vivo. Tumor-challenged mice vaccinated with an adenovirus-based cancer vaccine [encoding tumor-associated antigen (TAA)] and subsequently treated with this nanoparticulate formulation showed significant increases in TAA-specific T cells in the peripheral blood, reduced tumor burden, protection against tumor rechallenge, and a significant increase in median survival. An investigation into cell-based pathways suggests that administration of the nanoformulation at the site of the developing tumor may have created an inflammatory environment that attracted activated TAA-specific CD8+ T cells to the vicinity of the tumor, thus enhancing the efficacy of the vaccine.

New 1,2,3-triazole linked ciprofloxacin-chalcones induce DNA damage by inhibiting human topoisomerase I& II and tubulin polymerization.

A series of novel 1,2,3-triazole-linked ciprofloxacin-chalcones 4a-j were synthesised as potential anticancer agents. Hybrids 4a-j exhibited remarkable anti-proliferative activity against colon cancer cells. Compounds 4a-j displayed IC50s ranged from 2.53-8.67 µM, 8.67-62.47 µM, and 4.19-24.37 µM for HCT116, HT29, and Caco-2 cells; respectively, whereas the doxorubicin, showed IC50 values of 1.22, 0.88, and 4.15 µM. Compounds 4a, 4b, 4e, 4i, and 4j were the most potent against HCT116 with IC50 values of 3.57, 4.81, 4.32, 4.87, and 2.53 µM, respectively, compared to doxorubicin (IC50 = 1.22 µM). Also, hybrids 4a, 4b, 4e, 4i, and 4j exhibited remarkable inhibitory activities against topoisomerase I, II, and tubulin polymerisation. They increased the protein expression level of γH2AX, indicating DNA damage, and arrested HCT116 in G2/M phase, possibly through the ATR/CHK1/Cdc25C pathway. Thus, the novel ciprofloxacin hybrids could be exploited as potential leads for further investigation as novel anticancer medicines to fight colorectal carcinoma.

Preparation and Characterization of a Liver Targeted, Poly(amidoamine) Based, Gene Delivery System.

Nonalcoholic steatohepatitis (NASH) is an aggressive liver disease that is considered a major cause of liver cirrhosis and hepatocellular carcinoma. NASH is characterized by multiple underlying genetic mutations, with no approved cure to date. Gene therapies that target those genetic mutations may play a major role in treating this disease, once delivered specifically to the hepatocytes. In this chapter we present, in detail, the synthesis and the characterization of an efficient gene delivery system capable of targeting hepatocytes by exploiting the overexpression of asialoglycoprotein receptors on their cell surface. The targeting ligand, galactose derivative, lactobionic acid (Gal), is first conjugated to bifunctional poly(ethylene glycol) (PEG), and then the formed PEG-Gal is further conjugated to the positively charged polymer, poly(amidoamine) (PAMAM) to form a PAMAM-PEG-Gal construct that can complex and deliver genetic material (e.g., pDNA, siRNA, mRNA) specifically to hepatocytes. We first synthesize PAMAM-PEG-Gal using carbodiimide click chemistry. The synthesized conjugate is characterized using 1H NMR spectroscopy and mass spectrometry. Next, nanoplexes are prepared by combining the positively charged conjugate and the negatively charged genetic material at different nitrogen to phosphate (N/P) ratios; then the size, charge, electrophoretic mobility, and surface morphology of those nanoplexes are estimated. The simplicity of complexing our conjugate with any type of genetic material, the ability of our delivery system to overcome the current limitations of delivering naked genetic material, and the efficiency of delivering its payload specifically to hepatocytes, makes our formulation a promising tool to treat any type of genetic abnormality that arises in hepatocytes, and specifically NASH.

Surface Modification of Nanoparticles Enhances Drug Delivery to the Brain and Improves Survival in a Glioblastoma Multiforme Murine Model.

Glioblastoma multiforme (GBM) is the most malignant type of brain tumor and has an extremely poor prognosis. Current treatment protocols lack favorable outcomes, and alternative treatments with superior efficacy are needed. In this study, we demonstrate that loading paclitaxel (PTX) in a polymeric, nanoparticulate delivery system is capable of improving its brain accumulation and therapeutic activity. We independently incorporated two different positively charged surface modifiers, poly(amidoamine) (PAMAM) and poly(ethylenimine) (PEI), onto poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG), PLGA-PEG, nanoparticles (NPs) using a modified nanoprecipitation technique that assures the formation of nanosized particles while exposing the positively charged polymer on the surface. The prepared NPs underwent comprehensive analyses of their size, charge, in vitro permeability against a BBB cell line, and in vivo biodistribution. Our results demonstrated the successful fabrication of positively charged NPs using PAMAM or PEI. Importantly, significant improvement in brain accumulation (in vivo) was associated with NPs containing PAMAM compared to unmodified NPs or NPs containing PEI. Finally, the efficacy of PAMAM-modified NPs loaded with PTX was evaluated with orthotopic human GBM xenografts in a mouse model, and the data demonstrated improved survival and equivalent safety compared to soluble PTX. Our data substantiate the importance of surface chemistry on the magnitude of NP accumulation in the brain and pave the way for further in vivo evaluation of chemotherapeutic drugs against GBM that have previously been overlooked because of their limited ability to cross the BBB.

The MEK 1/2 inhibitor PD98059 exhibits synergistic anti-endometrial cancer activity with paclitaxel in vitro and enhanced tissue distribution in vivo when formulated into PAMAM-coated PLGA-PEG nanoparticles.

Endometrial cancer is the most common gynecological cancer that affects the female reproductive organs. The standard therapy for EC for the past two decades has been chemotherapy and/or radiotherapy. PD98059 is a reversible MEK inhibitor that was found in these studies to increase the cytotoxicity of paclitaxel (PTX) against human endometrial cancer cells (Hec50co) in a synergistic and dose-dependent manner. Additionally, while PD98059 arrested Hec50co cells at the G0/G1 phase, and PTX increased accumulation of cells at the G2/M phase, the combination treatment increased accumulation at both the G0/G1 and G2/M phases at low PTX concentrations. We recently developed poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) modified with polyethylene glycol (PEG) and coated with polyamidoamine (PAMAM) (referred to here as PGM NPs) which have favorable biodistribution profiles in mice, compared to PD98059 solution. Here, in order to enhance tissue distribution of PD98059, PD98059-loaded PGM NPs were prepared and characterized. The average size, zeta potential, and % encapsulation efficiency (%EE) of these NPs was approximately 184 nm, + 18 mV, and 23%, respectively. The PD98059-loaded PGM NPs released ~ 25% of the total load within 3 days in vitro. In vivo murine studies revealed that the pharmacokinetics and biodistribution profile of intravenous (IV) injected PD98059 was improved when delivered as PD98059-loaded PGM NPs as opposed to soluble PD98059. Further investigation of the in vivo efficacy and safety of this formulation is expected to emphasize the potential of its clinical application in combination with commercial PTX formulations against different cancers.

Monocytes Exposed to Immune Complexes Reduce pDC Type 1 Interferon Response to Vidutolimod.

Vidutolimod, also known as CMP-001, is a virus-like particle composed of the Qß bacteriophage coat protein encasing a TLR9 agonist. Vidutolimod injected intratumorally is showing promise in early phase clinical trials based on its ability to alter the tumor microenvironment and induce an anti-tumor immune response. We previously demonstrated that the in vivo efficacy of vidutolimod is dependent on the presence of anti-Qß antibodies that enhance opsonization and uptake of vidutolimod by TLR9-expressing plasmacytoid dendritic cells (pDCs). Here, we evaluated the effect of immune complexes, including anti-Qß-coated vidutolimod, on induction of Type 1 Interferon production by peripheral blood mononuclear cells in response to vidutolimod and soluble TLR9 agonists. Immune complexes, including but not limited to anti-Qß-coated vidutolimod, indirectly suppressed TLR9-mediated Type 1 Interferon production by pDCs in a monocyte-dependent manner. These findings indicate that anti-Qß-coated vidutolimod has effects in addition to those mediated by TLR9 that could have important clinical implications for understanding the mechanism of action of this exciting new approach to in situ immunization and cancer immunotherapy.

Thiophene derivative-loaded nanoparticles mediate anticancer activity through the inhibition of kinases and microtubule assembly.

Different tetrahydrobenzo[b]thiophene derivatives were explored as new tubulin polymerization destabilizers to arrest tumor cell mitosis. A series of compounds incorporating the tetrahydrobenzo[b]thiophene scaffold were synthesized, and their biological activities were investigated. The cytotoxicity of each of the synthesized compounds was assessed against a range of cell lines. Specifically, the benzyl urea tetrahydrobenzo[b]thiophene derivative, 1-benzyl-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)urea (BU17), was identified as the most potent compound with broad-spectrum antitumor activity against several cancer cell lines. The potential mechanism(s) of action were investigated where dose-dependent G2/M accumulation and A549 cell cycle arrest were detected. Additionally, A549 cells treated with BU17 expressed enhanced levels of caspase 3 and 9, indicating the induction of apoptosis. Furthermore, it was found that BU17 inhibits WEE1 kinase and targets tubulin by blocking its polymerization. BU17 was also formulated into PLGA nanoparticles, and it was demonstrated that BU17-loaded nanoparticles could significantly enhance antitumor activity compared to the soluble counterpart.

Paclitaxel anticancer activity is enhanced by the MEK 1/2 inhibitor PD98059 in vitro and by PD98059-loaded nanoparticles in BRAFV600E melanoma-bearing mice.

Melanoma, the most malignant form of skin cancer, shows resistance to traditional anticancer drugs including paclitaxel (PTX). Furthermore, over 50% of melanoma cases express the BRAFV600E mutation which activates the MAPK pathway increasing cell proliferation and survival. In the current study, we investigated the capacity of the combination therapy of PTX and the MAPK inhibitor, PD98059, to enhance the cytotoxicity of PTX against melanoma and therefore improve treatment outcomes. Synergistic in vitro cytotoxicity was observed when soluble PTX and PD98059 were used to treat the A375 melanoma cell line as evidenced by a significant reduction in the cell viability and IC50 value for PTX. Then, in further studies, TPGS-emulsified PD98059-loaded PLGA nanoparticles (NPs) were prepared, characterized in vitro and assessed for therapeutic efficacy when used in combination with soluble PTX. The average particle size (180 nm d.), zeta potential (-34.8 mV), polydispersity index (0.081), encapsulation efficiency (20%), particle yield (90.8%), and drug loading (6.633 µg/mg) of the prepared NPs were evaluated. Also, cellular uptake and in vitro cytotoxicity studies were performed with these PD98059-loaded NPs and compared to soluble PD98059. The PD98059-loaded NPs were superior to soluble PD98059 in terms of both cellular uptake and in vitro cytotoxicity in A375 cells. In in vivo studies, using A375 challenged mice, we report improved survival in mice treated with soluble PTX and PD98059-loaded NPs. Our findings suggest the potential for using this combinatorial therapy in the management of patients with metastatic melanoma harboring the BRAF mutation as a means to improve survival outcomes.

Solubilized ubiquinol for preserving corneal function.

Defective cellular metabolism, impaired mitochondrial function, and increased cell death are major problems that adversely affect donor tissues during hypothermic preservation prior to transplantation. These problems are thought to arise from accumulated reactive oxygen species (ROS) inside cells. Oxidative stress acting on the cells of organs and tissues preserved in hypothermic conditions before surgery, as is the case for cornea transplantation, is thought to be a major reason behind cell death prior to surgery and decreased graft survival after transplantation. We have recently discovered that ubiquinol - the reduced and active form of coenzyme Q10 and a powerful antioxidant - significantly enhances mitochondrial function and reduces apoptosis in human donor corneal endothelial cells. However, ubiquinol is highly lipophilic, underscoring the need for an aqueous-based formulation of this molecule. Herein, we report a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products. Docking studies showed that γ-CD has the strongest binding affinity with ubiquinol compared to α- or ß-CD. Complexed ubiquinol showed significantly higher stability compared to free ubiquinol in different aqueous ophthalmic products including Optisol-GS® corneal storage medium, balanced salt solution for intraocular irrigation, and topical Refresh® artificial tear eye drops. Greater ROS scavenging activity was noted in a cell model with high basal metabolism and ROS generation (A549) and in HCEC-B4G12 human corneal endothelial cells after treatment with ubiquinol/γ-CD compared to free ubiquinol. Furthermore, complexed ubiquinol was more effective at lowering ROS, and at far lower concentrations, compared to free ubiquinol. Complexed ubiquinol inhibited lipid peroxidation and protected HCEC-B4G12 cells against erastin-induced ferroptosis. No evidence of cellular toxicity was detected in HCEC-B4G12 cells after treatment with complexed ubiquinol. Using a vertical diffusion system, a topically applied inclusion complex of γ-CD and a lipophilic dye (coumarin-6) demonstrated transcorneal penetrance in porcine corneas and the capacity for the γ-CD vehicle to deliver drug to the corneal endothelium. Using the same model, topically applied ubiquinol/γ-CD complex penetrated the entire thickness of human donor corneas with markedly greater ubiquinol retention in the endothelium compared to free ubiquinol. Lastly, the penetrance of ubiquinol/γ-CD complex was assayed using human donor corneas preserved for 7 days in Optisol-GS® per standard industry practices, and demonstrated higher amounts of ubiquinol retained in the corneal endothelium compared to free ubiquinol. In summary, ubiquinol complexed with γ-CD is a highly stable composition that can be incorporated into a variety of aqueous-phase products for ophthalmic use including donor corneal storage media and topical eye drops to scavenge ROS and protect corneal endothelial cells against oxidative damage.

A Large Mitral Valve Vegetation Not Visualized on Transthoracic Echocardiography: A Case Report.

BACKGROUND Immediate evaluation, diagnosis, and treatment of a patient with infective endocarditis, an infection of the endocardium and/or integral structures found within the heart, is essential to patient survival. CASE REPORT We present the case of a 41-year-old man who was brought to the Emergency Department for altered mental status and fever. He was found to have methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia complicated with severe respiratory failure and metabolic encephalopathy, necessitating intubation and mechanical ventilation. As part of the workup for persistent Staphylococcal bacteremia, 2 transthoracic echocardiograms (TTE) failed to reveal any valvular abnormalities. However, a transesophageal echocardiogram (TEE) detected a 30×30 mm large vegetation on the anterior mitral valve leaflet. Due to the overall size and risk of systemic embolization, and the fact that the patient developed new-onset heart failure, the mitral valve was replaced using an open approach. The patient tolerated the procedure well and was discharged after an extended period of hospitalization. CONCLUSIONS Although the literature emphasizes that the sensitivity of TTE significantly increases when the vegetation size is above 1 cm, it is of utmost importance for clinicians to keep in mind that this is not always true, and clinicians should consider performing a TEE to rule out infective endocarditis whenever a TTE is unable to detect any vegetation in a patient with persistent Staphylococcal bacteremia. This is clearly demonstrated by the present case, in which two TTEs failed to detect a 30×30 mm vegetation.

MTDH/AEG-1 downregulation using pristimerin-loaded nanoparticles inhibits Fanconi anemia proteins and increases sensitivity to platinum-based chemotherapy.

OBJECTIVE: Platinum compounds have been widely used as a primary treatment for many types of cancer. However, resistance is the major cause of therapeutic failure for patients with metastatic or recurrent disease, thus highlighting the need to identify novel factors driving resistance to Platinum compounds. Metadherin (MTDH, also known as AEG-1 and LYRIC), located in a frequently amplified region of chromosome 8, has been consistently associated with resistance to chemotherapeutic agents, though the precise mechanisms remain incompletely defined. METHODS: The mRNA of FANCD2 and FANCI was pulled down by RNA-binding protein immunoprecipitation. Pristimerin-loaded nanoparticles were prepared using the nanoprecipitation method. Immunocompromised mice bearing patient-derived xenograft tumors were treated with pristimerin-loaded nanoparticles, cisplatin and a combination of the two. RESULTS: MTDH, through its recently discovered role as an RNA binding protein, regulates expression of FANCD2 and FANCI, two components of the Fanconi anemia complementation group (FA) that play critical roles in interstrand crosslink damage induced by platinum compounds. Pristimerin, a quinonemethide triterpenoid extract from members of the Celastraceae family used to treat inflammation in traditional Chinese medicine, significantly decreased MTDH, FANCD2 and FANCI levels in cancer cells, thereby restoring sensitivity to platinum-based chemotherapy. Using a patient-derived xenograft model of endometrial cancer, we discovered that treatment with pristimerin in a novel nanoparticle formulation markedly inhibited tumor growth when combined with cisplatin. CONCLUSIONS: MTDH is involved in post-transcriptional regulation of FANCD2 and FANCI. Pristimerin can increase sensitivity to platinum-based agents in tumors with MTDH overexpression by inhibiting the FA pathway.

Bullfrog oil (Rana catesbeiana Shaw) induces apoptosis, in A2058 human melanoma cells by mitochondrial dysfunction triggered by oxidative stress.

Bullfrog oil, an animal oil extracted from the adipose tissue of Rana catesbeiana Shaw, showed promising cytotoxic activity against melanoma cells and, therefore, has the potential to become a pharmaceutical active compound. However, there is a lack of information regarding the pathways involved in its pharmacological activity. Thus, the aim of this study was to investigate and elucidate the cytotoxic effect of this oil against A2058 human melanoma cells. The cytotoxic potential was evaluated by the MTT assay, the cell cycle analysis and the cell death assay. In addition, the apoptotic potential was investigated by (i) the DNA fragmentation using propidium iodide staining analysis, (ii) the evaluation of mitochondrial membrane potential and (iii) the determination of intracellular Reactive Oxygen Species (ROS) level. The results showed that the bullfrog oil was able to promote a time-dependent cytotoxic effect, decreasing cell viability to 38% after 72 h of treatment without affecting the cell cycle. Additionally, the bullfrog oil induced the apoptosis in A2058 cells, increasing up to 50 ±â€¯13% of the intracellular ROS level, maintaining the DNA integrity and promoting an approximate decrease of 35 ±â€¯5% in the mitochondrial membrane potential. It can be concluded that the in vitro cytotoxic effect of the bullfrog oil in A2058 human melanoma cells is mediated by oxidative stress that induces mitochondrial dysfunction, triggering the apoptosis. These unprecedented results highlight the pharmacological potential of bullfrog oil and provide important information to support studies on the development of new pharmaceutical products for complementary and alternative treatments for melanoma.

Synthetically lethal nanoparticles for treatment of endometrial cancer.

Uterine serous carcinoma, one of the most aggressive types of endometrial cancer, is characterized by poor outcomes and mutations in the tumour suppressor p53. Our objective was to engender synthetic lethality to paclitaxel (PTX), the frontline treatment for endometrial cancer, in tumours with mutant p53 and enhance the therapeutic efficacy using polymeric nanoparticles (NPs). First, we identified the optimal NP formulation through comprehensive analyses of release profiles and cellular-uptake and cell viability studies. Not only were PTX-loaded NPs superior to PTX in solution, but the combination of PTX-loaded NPs with the antiangiogenic molecular inhibitor BIBF 1120 (BIBF) promoted synthetic lethality specifically in cells with the loss-of-function (LOF) p53 mutation. In a xenograft model of endometrial cancer, this combinatorial therapy resulted in a marked inhibition of tumour progression and extended survival. Together, our data provide compelling evidence for future studies of BIBF- and PTX-loaded NPs as a therapeutic opportunity for LOF p53 cancers.

Cationic CaMKII Inhibiting Nanoparticles Prevent Allergic Asthma.

Asthma is a common lung disease affecting over 300 million people worldwide and is associated with increased reactive oxygen species, eosinophilic airway inflammation, bronchoconstriction, and mucus production. Targeting of novel therapeutic agents to the lungs of patients with asthma may improve efficacy of treatments and minimize side effects. We previously demonstrated that Ca2+/calmodulin-dependent protein kinase (CaMKII) is expressed and activated in the bronchial epithelium of asthmatic patients. CaMKII inhibition in murine models of allergic asthma reduces key disease phenotypes, providing the rationale for targeted CaMKII inhibition as a potential therapeutic approach for asthma. Herein we developed a novel cationic nanoparticle (NP)-based system for delivery of the potent and specific CaMKII inhibitor peptide, CaMKIIN, to airways.1 CaMKIIN-loaded NPs abrogated the severity of allergic asthma in a murine model. These findings provide the basis for development of innovative, site-specific drug delivery therapies, particularly for treatment of pulmonary diseases such as asthma.

A novel role of astrocyte elevated gene-1 (AEG-1) in regulating nonalcoholic steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) is the most prevalent cause of chronic liver disease in the Western world. However, an optimum therapy for NASH is yet to be established, mandating more in-depth investigation into the molecular pathogenesis of NASH to identify novel regulatory molecules and develop targeted therapies. Here, we unravel a unique function of astrocyte elevated gene-1(AEG-1)/metadherin in NASH using a transgenic mouse with hepatocyte-specific overexpression of AEG-1 (Alb/AEG-1) and a conditional hepatocyte-specific AEG-1 knockout mouse (AEG-1ΔHEP ). Alb/AEG-1 mice developed spontaneous NASH whereas AEG-1ΔHEP mice were protected from high-fat diet (HFD)-induced NASH. Intriguingly, AEG-1 overexpression was observed in livers of NASH patients and wild-type (WT) mice that developed steatosis upon feeding HFD. In-depth molecular analysis unraveled that inhibition of peroxisome proliferator-activated receptor alpha activity resulting in decreased fatty acid ß-oxidation, augmentation of translation of fatty acid synthase resulting in de novo lipogenesis, and increased nuclear factor kappa B-mediated inflammation act in concert to mediate AEG-1-induced NASH. Therapeutically, hepatocyte-specific nanoparticle-delivered AEG-1 small interfering RNA provided marked protection from HFD-induced NASH in WT mice. CONCLUSION: AEG-1 might be a key molecule regulating initiation and progression of NASH. AEG-1 inhibitory strategies might be developed as a potential therapeutic intervention in NASH patients. (Hepatology 2017;66:466-480).

HPLC-UV method for simultaneous determination of MK-1775 and AZD-7762 in both acetonitrile-aqueous solution and mouse plasma.

A sensitive and precise method is described for the simultaneous determination of two small molecule kinase inhibitors: MK-1775 (MK) and AZD-7762 (AZD), in acetonitrile (ACN)-aqueous solution and in mouse plasma. A Nova-Pak C18 reversed phase column (3.9mm×150mm, 4µm, 60Å) was utilized in the separation using an isocratic mobile phase of 0.1% v/v triethylamine in phosphate buffer (pH=7.4): acetonitrile (ACN) (60:40, v/v), at a flow rate of 0.8mL/min. Detection wavelength was set at 310nm for both MK and AZD, and 431nm for the internal standard sunitinib (SUN). The developed method was validated following the ICH guidelines and it was shown to be accurate, precise and linear in the range of 41ng/mL to 8333ng/mL for both drugs in the ACN-aqueous solution and from 83ng/mL to 8333ng/mL for both drugs in mouse plasma samples. For the first time, the presented data suggest the suitability of this method for the simultaneous separation and quantification of MK and AZD in both ACN aqueous solution as well as in mouse plasma samples.

Combination of Nanoparticle-Delivered siRNA for Astrocyte Elevated Gene-1 (AEG-1) and All-trans Retinoic Acid (ATRA): An Effective Therapeutic Strategy for Hepatocellular Carcinoma (HCC).

Hepatocellular carcinoma (HCC) is a fatal cancer with no effective therapy. Astrocyte elevated gene-1 (AEG-1) plays a pivotal role in hepatocarcinogenesis and inhibits retinoic acid-induced gene expression and cell death. The combination of a lentivirus expressing AEG-1 shRNA and all-trans retinoic acid (ATRA) profoundly and synergistically inhibited subcutaneous human HCC xenografts in nude mice. We have now developed liver-targeted nanoplexes by conjugating poly(amidoamine) (PAMAM) dendrimers with polyethylene glycol (PEG) and lactobionic acid (Gal) (PAMAM-PEG-Gal) which were complexed with AEG-1 siRNA (PAMAM-AEG-1si). The polymer conjugate was characterized by (1)H-NMR, MALDI, and mass spectrometry; and optimal nanoplex formulations were characterized for surface charge, size, and morphology. Orthotopic xenografts of human HCC cell QGY-7703 expressing luciferase (QGY-luc) were established in the livers of athymic nude mice and tumor development was monitored by bioluminescence imaging (BLI). Tumor-bearing mice were treated with PAMAM-siCon, PAMAM-siCon+ATRA, PAMAM-AEG-1si, and PAMAM-AEG-1si+ATRA. In the control group the tumor developed aggressively. ATRA showed little effect due to high AEG-1 levels in QGY-luc cells. PAMAM-AEG-1si showed significant reduction in tumor growth, and the combination of PAMAM-AEG-1si+ATRA showed profound and synergistic inhibition so that the tumors were almost undetectable by BLI. A marked decrease in AEG-1 level was observed in tumor samples treated with PAMAM-AEG-1si. The group treated with PAMAM-AEG-1si+ATRA nanoplexes showed increased necrosis, inhibition of proliferation, and increased apoptosis when compared to other groups. Liver is an ideal organ for RNAi therapy and ATRA is an approved anticancer agent. Our exciting observations suggest that the combinatorial approach might be an effective way to combat HCC.