A neuropeptide speeds circadian entrainment by reducing intercellular synchrony.

Shift work or transmeridian travel can desynchronize the body's circadian rhythms from local light-dark cycles. The mammalian suprachiasmatic nucleus (SCN) generates and entrains daily rhythms in physiology and behavior. Paradoxically, we found that vasoactive intestinal polypeptide (VIP), a neuropeptide implicated in synchrony among SCN cells, can also desynchronize them. The degree and duration of desynchronization among SCN neurons depended on both the phase and the dose of VIP. A model of the SCN consisting of coupled stochastic cells predicted both the phase- and the dose-dependent response to VIP and that the transient phase desynchronization, or "phase tumbling", could arise from intrinsic, stochastic noise in small populations of key molecules (notably, Period mRNA near its daily minimum). The model also predicted that phase tumbling following brief VIP treatment would accelerate entrainment to shifted environmental cycles. We tested this using a prepulse of VIP during the day before a shift in either a light cycle in vivo or a temperature cycle in vitro. Although VIP during the day does not shift circadian rhythms, the VIP pretreatment approximately halved the time required for mice to reentrain to an 8-h shifted light schedule and for SCN cultures to reentrain to a 10-h shifted temperature cycle. We conclude that VIP below 100 nM synchronizes SCN cells and above 100 nM reduces synchrony in the SCN. We show that exploiting these mechanisms that transiently reduce cellular synchrony before a large shift in the schedule of daily environmental cues has the potential to reduce jet lag.

Chitosan-based hybrid nanocomplex for siRNA delivery and its application for cancer therapy.

PURPOSE: Chitosan, a natural and biocompatible cationic polymer, is an attractive carrier for small interfering RNA (siRNA) delivery. The purpose of this study was to develop a chitosan-based hybrid nanocomplex that exhibits enhanced physical stability in the bloodstream compared with conventional chitosan complexes. Hybrid nanocomplexes composed of chitosan, protamine, lecithin, and thiamine pyrophosphate were prepared for systemic delivery of survivin (SVN) siRNA. METHODS: Physicochemical properties of the nanoparticles including mean diameters and zeta potentials were characterized, and target gene silencing and cellular uptake efficiencies of the siRNA nanocomplexes in prostate cancer cells (PC-3 cells) were measured. In vivo tumor targetability and anti-tumor efficacy by systemic administration were assessed in a PC-3 tumor xenograft mouse model by near-infrared fluorescence (NIRF) imaging and tumor growth monitoring, respectively. RESULTS: Mean diameters of the SVN siRNA-loaded hybrid nanocomplex (GP-L-CT) were less than 200 nm with a positive zeta potential value in water and were maintained without aggregation in culture media and 50% fetal bovine serum. SVN expression in PC-3 cells was reduced to 21.9% after treating with GP-L-CT. The tumor targetability and growth inhibitory efficacies of GP-L-CT supported the use of this novel hybrid nanocomplex as a cancer therapeutic and as a theranostic system for systemic administration. CONCLUSIONS: A chitosan-based hybrid nanocomplex was successfully developed for the systemic delivery of SVN siRNA, which could serve as an alternative to cationic polymeric nanoparticles that are unstable in serum.

Conformation-specific Antibodies Targeting Aggregated Forms of α-synuclein Block the Propagation of Synucleinopathy.

Abnormal aggregation of α-synuclein is a key element in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. α-synuclein aggregation spreads through various brain regions during the course of disease progression, a propagation that is thought to be mediated by the secretion and subsequent uptake of extracellular α-synuclein aggregates between neuronal cells. Thus, aggregated forms of this protein have emerged as promising targets for disease-modifying therapy for PD and related diseases. Here, we generated and characterized conformation-specific antibodies that preferentially recognize aggregated forms of α-synuclein. These antibodies promoted phagocytosis of extracellular α-synuclein aggregates by microglial cells and interfered with cell-to-cell propagation of α-synuclein. In an α-synuclein transgenic model, passive immunization with aggregate-specific antibodies significantly ameliorated pathological phenotypes, reducing α-synuclein aggregation, gliosis, inflammation, and neuronal loss. These results suggest that conformation-specific antibodies targeting α-synuclein aggregates are promising therapeutic agents for PD and related synucleinopathies.

Grabody B, an IGF1 receptor-based shuttle, mediates efficient delivery of biologics across the blood-brain barrier.

Effective delivery of therapeutics to the brain is challenging. Molecular shuttles use receptors expressed on brain endothelial cells to deliver therapeutics. Antibodies targeting transferrin receptor (TfR) have been widely developed as molecular shuttles. However, the TfR-based approach raises concerns about safety and developmental burden. Here, we report insulin-like growth factor 1 receptor (IGF1R) as an ideal target for the molecular shuttle. We also describe Grabody B, an antibody against IGF1R, as a molecular shuttle. Grabody B has broad cross-species reactivity and does not interfere with IGF1R-mediated signaling. We demonstrate that administration of Grabody B-fused anti-alpha-synuclein (α-Syn) antibody induces better improvement in neuropathology and behavior in a Parkinson's disease animal model than the therapeutic antibody alone due to its superior serum pharmacokinetics and enhanced brain exposure. The results indicate that IGF1R is an ideal shuttle target and Grabody B is a safe and efficient molecular shuttle.

Vasoactive intestinal polypeptide requires parallel changes in adenylate cyclase and phospholipase C to entrain circadian rhythms to a predictable phase.

Circadian oscillations in the suprachiasmatic nucleus (SCN) depend on transcriptional repression by Period (PER)1 and PER2 proteins within single cells and on vasoactive intestinal polypeptide (VIP) signaling between cells. Because VIP is released by SCN neurons in a circadian pattern, and, after photic stimulation, it has been suggested to play a role in the synchronization to environmental light cycles. It is not known, however, if or how VIP entrains circadian gene expression or behavior. Here, we tested candidate signaling pathways required for VIP-mediated entrainment of SCN rhythms. We found that single applications of VIP reset PER2 rhythms in a time- and dose-dependent manner that differed from light. Unlike VIP-mediated signaling in other cell types, simultaneous antagonism of adenylate cyclase and phospholipase C activities was required to block the VIP-induced phase shifts of SCN rhythms. Consistent with this, VIP rapidly increased intracellular cAMP in most SCN neurons. Critically, daily VIP treatment entrained PER2 rhythms to a predicted phase angle within several days, depending on the concentration of VIP and the interval between VIP applications. We conclude that VIP entrains circadian timing among SCN neurons through rapid and parallel changes in adenylate cyclase and phospholipase C activities.

Development of antibody-based c-Met inhibitors for targeted cancer therapy.

Signaling pathways mediated by receptor tyrosine kinases (RTKs) and their ligands play important roles in the development and progression of human cancers, which makes RTK-mediated signaling pathways promising therapeutic targets in the treatment of cancer. Compared with small-molecule compounds, antibody-based therapeutics can more specifically recognize and bind to ligands and RTKs. Several antibody inhibitors of RTK-mediated signaling pathways, such as human epidermal growth factor receptor 2, vascular endothelial growth factor, epidermal growth factor receptor or vascular endothelial growth factor receptor 2, have been developed and are widely used to treat cancer patients. However, since the therapeutic options are still limited in terms of therapeutic efficacy and types of cancers that can be treated, efforts are being made to identify and evaluate novel RTK-mediated signaling pathways as targets for more efficacious cancer treatment. The hepatocyte growth factor/c-Met signaling pathway has come into the spotlight as a promising target for development of potent cancer therapeutic agents. Multiple antibody-based therapeutics targeting hepatocyte growth factor or c-Met are currently in preclinical or clinical development. This review focuses on the development of inhibitors of the hepatocyte growth factor/c-Met signaling pathway for cancer treatment, including critical issues in clinical development and future perspectives for antibody-based therapeutics.

An injectable liquid crystal system for sustained delivery of entecavir.

Liquid crystal (LC) technology has attracted much interest for new injectable sustained-release (SR) formulations. In this study, an injectable liquid crystal-forming system (LCFS) including entecavir was prepared for the treatment of hepatitis B. In particular, an anchoring effect was introduced because LCFSs are relatively hydrophobic while entecavir is a slightly charged drug. The physicochemical properties of LCFSs were investigated by cryo-transmission electron microscopy (cryo-TEM), polarized optical microscopy, and small-angle X-ray scattering (SAXS), showing typical characteristics of the liquid crystalline phase, which was classified as the hexagonal phase. A pharmacokinetic study in rats showed sustained release of entecavir for 3-5 days with a basic LCFS formulation composed of sorbitan monooleate (SMO), phosphatidyl choline (PC), and tocopherol acetate (TA) as the main LC components. 1,2-Dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), an anionic phospholipid, was added to increase the anchoring effect between the cationic entecavir and the anionic DPPA, which resulted in a 1.5-times increase in half-life in rats. In addition, anchoring was strengthened by optimizing the pH to 2.5-4.5, increasing the half-life in the rat and dog. Also, due to the increasing terminal half-life from rat to dog resulting from species differences, LCFS produced one week delivery of entecavir in rat and two weeks delivery in dog. Therefore, LCFS injection using the anchoring effect for entecavir can potentially be used to deliver the drug over more than 2 weeks or even 1 month for the treatment of hepatitis B.

Spatiotemporal distribution of vasoactive intestinal polypeptide receptor 2 in mouse suprachiasmatic nucleus.

Vasoactive intestinal polypeptide (VIP) signaling is critical for circadian rhythms. For example, the expression of VIP and its main receptor, VPAC2R, is necessary for maintaining synchronous daily rhythms among neurons in the suprachiasmatic nucleus (SCN), a master circadian pacemaker in animals. Where and when VPAC2R protein is expressed in the SCN and other brain areas has not been examined. Using immunohistochemistry, we characterized a new antibody and found that VPAC2R was highly enriched in the SCN and detectable at low levels in many brain areas. Within the SCN, VPAC2R was circadian, peaking in the subjective morning, and abundantly expressed from the rostral to caudal margins with more in the dorsomedial than ventrolateral area. VPAC2R was found in nearly all SCN cells including neurons expressing either VIP or vasopressin (AVP). SCN neurons mainly expressed VPAC2R in their somata and dendrites, not axons. Finally, constant light increased VIP and AVP expression, but not VPAC2R. We conclude that the circadian clock, not the ambient light level, regulates VPAC2R protein localization. These results are consistent with VPAC2R playing a role in VIP signaling at all times of day, broadly throughout the brain and in all SCN cells.