Background: In the central nervous system, astrocytic L-glutamate (L-Glu) transporters maintain extracellular L-Glu below neurotoxic levels, but their function is impaired with neuroinflammation. Microglia become activated with inflammation; however, the correlation between activated microglia and the impairment of L-Glu transporters is unknown.Methods: We used a mixed culture composed of astrocytes, microglia, and neurons. To quantify L-Glu transporter function, we measured the extracellular L-Glu that remained 30 min after an application of L-Glu to the medium (the starting concentration was 100 μM). We determined the optimal conditions of lipopolysaccharide (LPS) treatment to establish an inflammation model without cell death. We examined the predominant subtypes of L-Glu transporters and the changes in the expression levels of these transporters in this inflammation model. We then investigated the role of activated microglia in the changes in L-Glu transporter expression and the underlying mechanisms in this inflammation model.Results: Because LPS (10 ng/mL, 72 h) caused a significant increase in the levels of L-Glu remaining but did not affect cell viability, we adopted this condition for our inflammation model without cell death. GLAST was the predominant L-Glu transporter subtype, and its expression decreased in this inflammation model. As a result of their release of L-Glu, activated microglia were shown to be essential for the significant decrease in L-Glu uptake. The serial application of L-Glu caused a significant decrease in L-Glu uptake and GLAST expression in the astrocyte culture. The hemichannel inhibitor carbenoxolone (CBX) inhibited L-Glu release from activated microglia and ameliorated the decrease in GLAST expression in the inflammation model. In addition, the elevation of the astrocytic intracellular L-Glu itself caused the downregulation of GLAST.Conclusions: Our findings suggest that activated microglia trigger the elevation of extracellular L-Glu through their own release of L-Glu, and astrocyte L-Glu transporters are downregulated as a result of the elevation of astrocytic intracellular L-Glu levels, causing a further increase of extracellular L-Glu. Our data suggest the new hypothesis that activated microglia collude with astrocytes to cause the elevation of extracellular L-Glu in the early stages of neuroinflammation.

A new reward premium system, “Reward Premiums for the Promotion of Innovative Drug Discovery and Resolution of Off-label Use Issue, etc,” unique to Japan, was introduced in 2010 and was used in 2010, 2012, and 2014. If the new rewards system is applied to a drug, the company can recover costs early and reinvest in research and development of unapproved or innovative drugs. However, a detailed analysis of the drugs to which this new system was applied has not been conducted. In this study, a data analysis was performed to determine what factors were strongly associated with receiving a reward. Domestic pharmaceuticals in the top 500 sales in September 2009, 2011, and 2013 were selected for this study. Multivariable or univariate logistic regression was performed. The following factors were significantly related to receiving a reward: a more recent launch, a global promotional company, and orphan drug designation. Cardiovascular drugs were less likely to receive rewards, whereas drugs acting on the central nervous system, anticancer agents, and immunomodulatory agents were significantly more likely to receive rewards. These findings suggest that orphan drugs and drugs with specific Anatomical Therapeutic Chemical (ATC) classifications such as N and L are likely to receive rewards. We can verify that this incentive program works well in Japan, launching truly innovative drugs that meet high medical needs.

A new incentive program, “Premium Rewards for the Promotion of Innovative Drug Discovery and Resolution of Off-Label Use, etc,” was introduced in Japan in 2010 and subsequently applied in 2010, 2012, and 2014. In our previous study, we reported statistical evidence regarding the attributes of drugs for which these premium rewards were used. We found that orphan drugs and drugs in certain therapeutic areas, including neuroscience, oncology, and immunology, were likely to receive rewards. However, that investigation was not conducted to clarify how the rewards system affects pharmaceutical sales or whether drugs that received rewards led to more profits for pharmaceutical companies. The data set for this study was a list of the 500 top-selling pharmaceuticals in Japan from 2010 to 2014, which was obtained from the IMS Japan Pharmaceutical Market database. To clarify the effect of the rewards system on different therapeutic areas, total sales and quantity sold were compared between drugs that received rewards and those that did not. The rewards system positively affected drug sales in specific therapeutic areas, including those with Anatomical Therapeutic Chemical (ATC) classifications neuroscience (N) and oncology (L), as well as the quantity sold of neuroscience-related drugs. Our study suggests that this system has been working according to its original aim of promoting the development of innovative drugs in Japan.