A cell-based drug delivery platform for treating central nervous system inflammation.

Mesenchymal stem cells (MSCs) are promising candidates for the development of cell-based drug delivery systems for autoimmune inflammatory diseases, such as multiple sclerosis (MS). Here, we investigated the effect of Ro-31-8425, an ATP-competitive kinase inhibitor, on the therapeutic properties of MSCs. Upon a simple pretreatment procedure, MSCs spontaneously took up and then gradually released significant amounts of Ro-31-8425. Ro-31-8425 (free or released by MSCs) suppressed the proliferation of CD4+ T cells in vitro following polyclonal and antigen-specific stimulation. Systemic administration of Ro-31-8425-loaded MSCs ameliorated the clinical course of experimental autoimmune encephalomyelitis (EAE), a murine model of MS, displaying a stronger suppressive effect on EAE than control MSCs or free Ro-31-8425. Ro-31-8425-MSC administration resulted in sustained levels of Ro-31-8425 in the serum of EAE mice, modulating immune cell trafficking and the autoimmune response during EAE. Collectively, these results identify MSC-based drug delivery as a potential therapeutic strategy for the treatment of autoimmune diseases. KEY MESSAGES: MSCs can spontaneously take up the ATP-competitive kinase inhibitor Ro-31-8425. Ro-31-8425-loaded MSCs gradually release Ro-31-8425 and exhibit sustained suppression of T cells. Ro-31-8425-loaded MSCs have more sustained serum levels of Ro-31-8425 than free Ro-31-8425. Ro-31-8425-loaded MSCs are more effective than MSCs and free Ro-31-8425 for EAE therapy.

Modulating TAK1 Expression Inhibits YAP and TAZ Oncogenic Functions in Pancreatic Cancer.

YAP and TAZ are central determinants of malignancy; however, their functions remain still undruggable. We identified TGFß-activated kinase 1 (TAK1) as a central hub integrating the most relevant signals sustaining pancreatic cancer aggressiveness and chemoresistance. Glycogen synthase kinase (GSK)3 is known to stabilize TAK1, and its inhibition causes a reduction in TAK1 levels. Here, we hypothesized that TAK1 could sustain YAP/TAZ program, and thus, modulation of TAK1 expression through the inhibition of GSK3 could impair YAP/TAZ functions in pancreatic cancer.Differentially expressed transcripts between pancreatic cancer cells expressing scramble or TAK1-specific shRNA were annotated for functional interrelatedness by ingenuity pathway analysis. TAK1 expression was modulated by using different GSK3 inhibitors, including LY2090314. In vivo activity of LY2090314 alone or in combination with nab-paclitaxel was evaluated in an orthotopic nude mouse model.Differential gene expression profiling revealed significant association of TAK1 expression with HIPPO and ubiquitination pathways. We measured a significant downregulation of YAP/TAZ and their regulated genes in shTAK1 cells. TAK1 prevented YAP/TAZ proteasomal degradation in a kinase independent manner, through a complex with TRAF6, thereby fostering their K63-ubiquitination versus K48-ubiquitination. Pharmacologic modulation of TAK1 by using GSK3 inhibitors significantly decreased YAP/TAZ levels and suppressed their target genes and oncogenic functions. In vivo, LY2090314 plus nab-paclitaxel significantly prolonged mice survival duration.Our study demonstrates a unique role for TAK1 in controlling YAP/TAZ in pancreatic cancer. LY2090314 is a novel agent that warrants further clinical development in combination with nab-paclitaxel for the treatment of pancreatic cancer.

Glycogen synthase kinase 3ß as a potential therapeutic target in synovial sarcoma and fibrosarcoma.

Soft tissue sarcomas (STSs) are a rare cancer type. Almost half are unresponsive to multi-pronged treatment and might therefore benefit from biologically targeted therapy. An emerging target is glycogen synthase kinase (GSK)3ß, which is implicated in various diseases including cancer. Here, we investigated the expression, activity and putative pathological role of GSK3ß in synovial sarcoma and fibrosarcoma, comprising the majority of STS that are encountered in orthopedics. Expression of the active form of GSK3ß (tyrosine 216-phosphorylated) was higher in synovial sarcoma (SYO-1, HS-SY-II, SW982) and in fibrosarcoma (HT1080) tumor cell lines than in untransformed fibroblast (NHDF) cells that are assumed to be the normal mesenchymal counterpart cells. Inhibition of GSK3ß activity by pharmacological agents (AR-A014418, SB-216763) or of its expression by RNA interference suppressed the proliferation of sarcoma cells and their invasion of collagen gel, as well as inducing their apoptosis. These effects were associated with G0/G1-phase cell cycle arrest and decreased expression of cyclin D1, cyclin-dependent kinase (CDK)4 and matrix metalloproteinase 2. Intraperitoneal injection of the GSK3ß inhibitors attenuated the growth of SYO-1 and HT1080 xenografts in athymic mice without obvious detrimental effects. It also mitigated cell proliferation and induced apoptosis in the tumors of mice. This study indicates that increased activity of GSK3ß in synovial sarcoma and fibrosarcoma sustains tumor proliferation and invasion through the cyclin D1/CDK4-mediated pathway and enhanced extracellular matrix degradation. Our results provide a biological basis for GSK3ß as a new and promising therapeutic target for these STS types.

Maleimide-functionalised PLGA-PEG nanoparticles as mucoadhesive carriers for intravesical drug delivery.

Low permeability of the urinary bladder epithelium, poor retention of the chemotherapeutic agents due to dilution and periodic urine voiding as well as intermittent catheterisations are the major limitations of intravesical drug delivery used in the treatment of bladder cancer. In this work, maleimide-functionalised poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-PEG-Mal) nanoparticles were developed. Their physicochemical characteristics, including morphology, architecture and molecular parameters have been investigated by means of dynamic light scattering, transmission electron microscopy and small-angle neutron scattering techniques. It was established that the size of nanoparticles was dependent on the solvent used in their preparation and molecular weight of PEG, for example, 105 ±â€¯1 nm and 68 ±â€¯1 nm particles were formed from PLGA20K-PEG5K in dimethyl sulfoxide and acetone, respectively. PLGA-PEG-Mal nanoparticles were explored as mucoadhesive formulations for drug delivery to the urinary bladder. The retention of fluorescein-loaded nanoparticles on freshly excised lamb bladder mucosa in vitro was evaluated and assessed using a flow-through fluorescence technique and Wash Out50 (WO50) quantitative method. PLGA-PEG-Mal nanoparticles (NPs) exhibited greater retention on urinary bladder mucosa (WO50 = 15 mL) compared to maleimide-free NPs (WO50 = 5 mL). The assessment of the biocompatibility of PEG-Mal using the slug mucosal irritation test revealed that these materials are non-irritant to mucosal surfaces.

Assessment of PKA and PKC inhibitors on force and kinetics of non-failing and failing human myocardium.

AIMS: Heart failure (HF) is a prevalent disease that is considered the foremost reason for hospitalization in the United States. Most protein kinases (PK) are activated in heart disease and their inhibition has been shown to improve cardiac function in both animal and human studies. However, little is known about the direct impact of PKA and PKC inhibitors on human cardiac contractile function. MATERIAL AND METHODS: We investigated the ex vivo effect of such inhibitors on force as well as on kinetics of left ventricular (LV) trabeculae dissected from non-failing and failing human hearts. In these experiments, we applied 0.5 µM of H-89 and GF109203X, which are PKA and PKC inhibitors, respectively, in comparison to their vehicle DMSO (0.05%). KEY FINDINGS AND CONCLUSION: Statistical analyses revealed no significant effect for H-89 and GF109203X on either contractile force or kinetics parameters of both non-failing and failing muscles even though they were used at a concentration higher than the reported IC50s and Kis. Therefore, several factors such as selectivity, concentration, and treatment time, which are related to these PK inhibitors according to previous studies require further exploration.

In situ low-immunogenic albumin-conjugating-corona guiding nanoparticles for tumor-targeting chemotherapy.

Nanoparticles (NPs) are unavoidably covered by a layer of immunogenic proteins upon injection into blood, such as immunoglobins and complements, which buries the active-targeting ligands and triggers the rapid clearance of NPs by the mononuclear phagocytic system. Low antifouling polyethylene glycol is used to inhibit the formation of the immunogenic corona but it leads to poor cellular uptake and the immunogen-related accelerated blood clearance (ABC) phenomenon in multiple administrations. Here, we develop surface maleimide-modified NPs that covalently conjugate in vivo plasma albumin in its corona upon exposure to blood. The in situ recruited low-immunogenic albumin-enriching corona is capable of protecting maleimide-decorated NPs from phagocytosis in the bloodstream, preventing the ABC phenomenon in the second administration, facilitating NP accumulation in the tumor site/cells by the passive EPR effect and albumin receptor-mediated active targeting, and finally improving the antitumor activity. Such findings suggest that the facile strategy, based on the in situ anchored albumin-enriching corona, is efficient at enabling maleimide-decorated NPs to acquire stealth and tumor-targeting ability.

In vivo tailor-made protein corona of a prodrug-based nanoassembly fabricated by redox dual-sensitive paclitaxel prodrug for the superselective treatment of breast cancer.

Prodrug self-nanoassemblies have many advantages for anticancer drug delivery, including high drug loading rate, resistance to recrystallization, and on-demand drug release. However, few studies have focused on their protein corona, which is inevitably formed after entering the blood and determines their subsequent fates in vivo. To actively tune the protein corona of prodrug nanoassemblies, three maleimide-paclitaxel prodrugs were synthesized via different redox-sensitive linkers (ester bond, thioether bond and disulfide bond). After incubation with rat plasma, the surface maleimide groups effectively captured albumins, resulting in albumin-enriched protein corona. The recruited albumin corona enabled enhanced tumor accumulation and facilitated cellular uptake, ensuring the high-efficiency delivery of nanoassemblies to tumor cells. Surprisingly, we found that the traditionally reduction-sensitive disulfide bond could also be triggered by reactive oxygen species (ROS). Such a redox dual-responsive drug release property of the disulfide bond-containing prodrug nanoassemblies further increased the selectivity in cytotoxicity between normal and tumor cells. Moreover, the disulfide bond-containing prodrug nanoassemblies exhibited the highest antitumor efficacy in vivo compared to marketed Abraxane® and other prodrug nanoassemblies. Thus, the fabrication of the maleimide-decorated disulfide bond bridged prodrug nanoassembly, integrating a tunable protein corona and on-demand drug release, is a promising strategy for improved cancer chemotherapy.

9-ING-41, a small-molecule glycogen synthase kinase-3 inhibitor, is active in neuroblastoma.

Advanced stage neuroblastoma is a very aggressive pediatric cancer with limited treatment options and a high mortality rate. Glycogen synthase kinase-3ß (GSK-3ß) is a potential therapeutic target in neuroblastoma. Using immunohistochemical staining, we observed positive GSK-3ß expression in 67% of human neuroblastomas (34 of 51 cases). Chemically distinct GSK-3 inhibitors (AR-A014418, TDZD-8, and 9-ING-41) suppressed the growth of neuroblastoma cells, whereas 9-ING-41, a clinically relevant small-molecule GSK-3ß inhibitor with broad-spectrum preclinical antitumor activity, being the most potent. Inhibition of GSK-3 resulted in a decreased expression of the antiapoptotic molecule XIAP and an increase in neuroblastoma cell apoptosis. Mouse xenograft studies showed that the combination of clinically relevant doses of CPT-11 and 9-ING-41 led to greater antitumor effect than was observed with either agent alone. These data support the inclusion of patients with advanced neuroblastoma in clinical studies of 9-ING-41, especially in combination with CPT-11.

Diels Alder-mediated release of gemcitabine from hybrid nanoparticles for enhanced pancreatic cancer therapy.

Hybrid nanoparticles (HNPs) have shown huge potential as drug delivery vehicles for pancreatic cancer. Currently, the first line treatment, gemcitabine, is only effective in 23.8% of patients. To improve this, a thermally activated system was developed by introducing a linker between HNPs and gemcitabine. Whereby, heat generation resulting from laser irradiation of the HNPs promoted linker breakdown resulting in prodrug liberation. In vitro evaluation in pancreatic adenocarcinoma cells, showed the prodrug was 4.3 times less cytotoxic than gemcitabine, but exhibited 11-fold improvement in cellular uptake. Heat activation of the formulation led to a 56% rise in cytotoxicity causing it to outperform gemcitabine by 26%. In vivo the formulation outperformed free gemcitabine with a 62% reduction in tumor weight in pancreatic xenografts. This HNP formulation is the first of its kind and has displayed superior anti-cancer activity as compared to the current first line drug gemcitabine after heat mediated controlled release.

Scaffold Hopping and Optimization of Maleimide Based Porcupine Inhibitors.

Porcupine is an O-acyltransferase that regulates Wnt secretion. Inhibiting porcupine may block the Wnt pathway which is often dysregulated in various cancers. Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt pathway inhibitors in secondary assays. We developed a pharmacophore model and used the putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting maleimide scaffold was optimized to subnanomolar potency while retaining good physical druglike properties. A preclinical development candidate was selected for which extensive in vitro and in vivo profiling is reported.

GLP-1 analogue CJC-1131 prevents amyloid ß protein-induced impirments of spatial memory and synaptic plasticity in rats.

Although amyloid ß protein (Aß) has been recognized as one of the main pathological characteristics in the brain of Alzheimer's disease (AD), the effective strategies against Aß neurotoxicity are still deficient up to now. Glucagon-like peptide 1 (GLP-1), a natural gut hormone, was found to be effective in modulating insulin signaling and neural protection, but short half-life limited its clinical application in AD treatment. CJC-1131, a newly designed GLP-1 analogue with very longer half-life, has shown good effectiveness in the treatment of type 2 diabetes mellitus (T2DM). However, it is unclear whether CJC-1131 could alleviate Aß-induced neurotoxicity in cognitive behavior and electrophysiological property. The present study investigated the effects of CJC-1131 on the Aß-induced impairments in spatial memory and synaptic plasticity of rats by using Morris water maze test and in vivo field potential recording. The results showed that Aß1-42-induced increase in the escape latency of rats in hidden platform test and decrease in swimming time percent in target quadrant were effectively reversed by CJC-1131 pretreatment. Further, CJC-1131 prevented against Aß1-42-induced suppression of hippocampal long term potentiation (LTP). In addition, Aß1-42 injection resulted in a significant decrease of p-PKA in the hippocampus, which was effectively prevented by CJC-1131 treatment. These results indicated that CJC-1131 protected the cognitive function and synaptic plasticity of rats against Aß-induced impairments, suggesting that GLP-1 analogue CJC-1131 might be potentially beneficial to the prevention and treatment of AD, especially those with T2DM or blood glucose abnormality.

Involvement of Spinal CCR5/PKCγ Signaling Pathway in the Maintenance of Cancer-Induced Bone Pain.

Cancer-induced bone pain (CIBP) is a challenging medical problem that considerably influences cancer patients' quality of life. Currently, few treatments have been developed to conquer CIBP because of a poor understanding of the potential mechanisms. Our previous work has proved that spinal RANTES (a major ligand for CCR5) was involved in the maintenance of CIBP. In this study, we attempted to investigate whether spinal CCR5 and its downstream PKCγ pathway is involved in the maintenance of CIBP. Inoculation of Walker 256 cells into the tibia could induce a marked mechanical allodynia with concomitant upregulation of spinal CCR5 and p-PKCγ expression from day 6 to day 15 after inoculation. Spinal CCR5 was prominently expressed in microglia, and mechanical allodynia was attenuated by intrathecal injection of DAPTA (a specific antagonist of CCR5) with downregulation of spinal CCR5 and p-PKCγ expression levels at day 15 in inoculated rats. Pre-intrathecal injection of RANTES could reverse the anti-allodynia effects of DAPTA. Intrathecal administration of GF109203X (an inhibitor of PKC) could alleviate mechanical allodynia as well as decrease of spinal p-PKCγ expression level, but no influence on spinal CCR5 level. Our findings suggest that CCR5/PKCγ signaling pathway in microglia may contribute to the maintenance of CIBP in rats.

Co-suppression of vitamin C composite nano-drug carrier and its drug delivery to nidus in tumor cells.

This study aimed to discuss the co-suppression of vitamin C-contained composite nano-drug carrier and its drug delivery to nidus in tumor cells. Amphiphilic polymers PLA-block-PAAA and block polymer PLA-PEG4000-Maleimide, PLA-block-PAAA and PLA-PEG4000-Maleimide composite nano-micelles were prepared, and, PLA-block-PAAA polymer-coated Nile red nano-micelle, PLA-block-PAA and PLA-PEG4000-Maleimide composite nano-micelles as well as paclitaxel-carrying composite nano-micelle in different molar ratios were given stability tests. Lastly, PLA-block-PAAA and PLA-PEG4000-Maleimide composite nano-micelle cancer cells and paclitaxel-carrying composite nano-micelle cancer cells were given toxicity tests. Stability tests showed that self stability of PLA-block-PAAA (63/8) nano-micelle was not sufficient; the stability was good when the molar ratio of PLA-block-PAAA and PLA-PEG4000-Maleimide composite nano-micelle was 3:1; paclitaxel-carrying composite nano-micelle had good stability within 48 hours; PAAA segment had an inhibiting effect on C6 cancer cells and paclitaxel-carrying composite nano-micelle had a strong inhibiting effect also on tumors. After 24 hours, with the continuous release of paclitaxel, the tumor inhibiting effect of paclitaxel-carrying composite nano-micelle enhanced gradually, and the controlled-release of drugs had continuous inhibiting effect on tumor cells. Therefore, PAAA segment and paclitaxel had time-postponed synergistic effect. In conclusion, vitamin C-contained composite nanometer drug carrier materials can deliver anti-cancer drugs to nidus and thus inhibit tumor cells.

Inhibition of glycogen synthase kinase-3 (GSK3) promotes the neural differentiation of full-term amniotic fluid-derived stem cells towards neural progenitor cells.

The amniotic fluid has a heterogeneous population of cells. Some human amniotic fluid-derived stem (hAFS) cells have been shown to harbor the potential to differentiate into neural cells. However, the neural differentiation efficiency of hAFS cells remains low. In this study, we isolated CD117-positive hAFS cells from amniotic fluid and then examined the pluripotency of these cells through the formation of embryoid bodies (EBs). Additionally, we induced the neural differentiation of these cells using neuroectodermal medium. This study revealed that the GSK3-beta inhibitor SB216763 was able to stimulate the proliferation of CD117-positive hAFS cells without influencing their undifferentiated state. Moreover, SB216763 can efficiently promote the neural differentiation of CD117-positive hAFS cells towards neural progenitor cells in the presence of DMEM/F12 and N2 supplement. These findings provide an easy and low-cost method to maintain the proliferation of hAFS cells, as well as induce an efficacious generation of neural progenitor cells from hAFS cells. Such induction of the neural commitment of hAFS cells may provide an option for the treatment of neurodegenerative diseases by hAFS cells-based therapies.

Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8(+) Cytolytic T Cell Responses.

Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and ß) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8(+) T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8(+) cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8(+) CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8(+) OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy.

Combination of long-acting HIV fusion inhibitor albuvirtide and LPV/r showed potent efficacy in HIV-1 patients.

BACKGROUND: Long acting antiretroviral drugs represent a promising approach for chronic treatment of HIV infection. Here, we study the efficacy and safety of albuvirtide (ABT), an HIV-1 fusion inhibitor with a half life of 11-12 days in human. METHODS: ABT was evaluated in a 7-week, open-label and randomized trial, combining with LPV/r. Twenty HIV-1-infected adults were assigned to two dose groups, receiving ABT (160 or 320 mg) given weekly and LPV/r given twice daily. RESULTS: At week 7, the decline of HIV-1 RNA from baseline was 1.9 (1.3-2.3) log10 and 2.2 (1.6-2.7) log10 copies/ml, and suppression of HIV-1 RNA to below 50 copies/ml was achieved in 11.1 % (1/9) and 55.6 % (5/9) patients, for the 160 and 320 mg dose group respectively. CONCLUSION: A clear dose-efficacy correlation of ABT was demonstrated. ABT combining with LPV/r is a promising two-drug regimen to be tested in larger patient population.

Emodin Inhibits the Epithelial to Mesenchymal Transition of Epithelial Ovarian Cancer Cells via ILK/GSK-3ß/Slug Signaling Pathway.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Despite the anticancer capabilities of emodin observed in many cancers, including EOC, the underlying molecular mechanism remains to be elucidated. A crucial link has been discovered between the acquisition of metastatic traits and the epithelial-mesenchymal transition (EMT). The present study aimed to determine whether emodin could inhibit the EMT of EOC cells and explore the underlying mechanism. The CCK-8 assay and transwell assay showed that emodin effectively repressed the abilities of proliferation, invasion, and migration in A2780 and SK-OV-3 cells. The Western blot showed that emodin upregulated epithelial markers (E-cadherin and Claudin) while it downregulated mesenchymal markers (N-cadherin and Vimentin) and transcription factor (Slug) in a dose-dependent fashion. After transfection of siRNA-Slug, both Slug and N-cadherin were downregulated in EOC cells while E-cadherin was upregulated, which was intensified by emodin. Besides, emodin decreased the expression of ILK, p-GSK-3ß, ß-catenin, and Slug. Transfection of siRNA-ILK also achieved the same effects, which was further strengthened by following emodin treatment. Nevertheless, SB216763, an inhibitor of GSK-3ß, could reverse the effects of emodin except for ILK expression. These findings suggest that emodin inhibited the EMT of EOC cells via ILK/GSK-3ß/Slug signaling pathway.

Administration of sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate conjugated GP100(25-33) peptide-coupled spleen cells effectively mounts antigen-specific immune response against mouse melanoma.

It remains a top research priority to develop immunotherapeutic approaches to induce potent antigen-specific immune responses against tumors. However, in spite of some promising results, most strategies are ineffective because they generate low numbers of tumor-reactive cytotoxic T lymphocytes (CTLs). Here we designed a strategy to enhance antigen-specific immune response via administering sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC)-conjugated melanoma tumor antigen GP10025-33 peptide-coupled syngeneic spleen cells in a mouse model of melanoma. We found that infusion of GP10025-33 peptide-coupled spleen cells significantly attenuated the growth of melanoma in prophylactic and therapeutic immunizations. Consistent with these findings, the adoptive transfer of spleen cells from immunized mice to naïve syngeneic mice was able to transfer anti-tumor effect, suggesting that GP10025-33 peptide-specific immune response was induced. Further studies showed that, CD8+ T cell proliferation and the frequency of interferon (IFN)-γ-producing CD8+ T cells upon ex vivo stimulation by GP10025-33 were significantly increased compared to control groups. Tumor antigen, GP10025-23 specific immune response was also confirmed by ELISpot and GP100-tetramer assays. This approach is simple, easy-handled, and efficiently delivering antigens to lymphoid tissues. Our study offers an opportunity for clinically translating this approach into tumor immunotherapy.