Tumor Accumulation of PIP-Based KRAS Inhibitor KR12 Evaluated by the Use of a Simple, Versatile Chicken Egg Tumor Model.

BACKGROUND: The KRAS inhibitor KR12, based on pyrrole-imidazole polyamide (PIP), has been developed and shown to exhibit efficacy in mouse experiments. Because some PIP species exhibit tumor accumulation capability, we decided to evaluate whether the PIP portion of KR12 exhibits tumor accumulation. We employed the CAM assay that provides a simple method for tumor accumulation evaluation. METHODS: KR12 PIP was synthesized and conjugated to TAMRA to produce a fluorescently labeled reagent (KR12-TAMRA). This reagent was injected into a fertilized chicken egg that has been transplanted with human cancer cells. Distribution of the red fluorescence was examined by cutting out tumor as well as various organs from the embryo. RESULTS: The red fluorescence of KR12-TAMRA was found to overlap with the green fluorescence of the tumor formed with GFP-expressing cancer cells. We also observed nuclear localization of KR12-TAMRA. Treatment of KR12 that contained the alkylating agent CBI in the tumor-bearing chicken egg resulted in tumor growth inhibition. CONCLUSIONS: KR12 contains a PIP that has two key features: tumor accumulation and nuclear localization. KR12 conjugated with CBI exhibits inhibition of tumor growth in the CAM model.

[Efficacy of Saxagliptin for Blood Glucose Control in Hemodialysis Patients with Diabetes].

Studies on the drug saxagliptin (marketed in Japan since 2013) suggest favorable efficacy in hemodialysis patients, but included small sample sizes. Noting that some hemodialysis patients at our medical institution had been switched to saxagliptin 2.5 mg from treatment with other dipeptidyl peptidase-4 inhibitors, we decided to evaluate the effects of switching to saxagliptin on blood glucose control in these patients. The study included 11 patients. Before switching drugs, six of the patients used teneligliptin 20 mg and five used linagliptin 5 mg. Mean glycated albumin (GA) from before to 4 months after switching tended to increase in the previous users of teneligliptin 20 mg (18.4±3.0% to 19.5±2.7%) and tended to decrease in the previous users of linagliptin 5 mg (18.8±3.3% to 17.7±1.4%). Lack of a substantial change in GA when the previous users of teneligliptin 20 mg and linagliptin 5 mg were switched to saxagliptin 2.5 mg indicates that these three agents might have comparable antihyperglycemic profiles when used in patients on hemodialysis. Future research following from this pilot study must evaluate the risk of cardiac failure and incidences of adverse events in a larger population, to investigate the long-term efficacy and safety of switching to saxagliptin.

CSF histamine contents in narcolepsy, idiopathic hypersomnia and obstructive sleep apnea syndrome.

STUDY OBJECTIVE: To (1) replicate our prior result of low cerebrospinal fluid (CSF) histamine levels in human narcolepsy in a different sample population and to (2) evaluate if histamine contents are altered in other types of hypersomnia with and without hypocretin deficiency. DESIGN: Cross sectional studies. SETTING AND PATIENTS: Sixty-seven narcolepsy subjects, 26 idiopathic hypersomnia (IHS) subjects, 16 obstructive sleep apnea syndrome (OSAS) subjects, and 73 neurological controls were included. All patients were Japanese. Diagnoses were made according to ICSD-2. RESULTS: We found significant reductions in CSF histamine levels in hypocretin deficient narcolepsy with cataplexy (mean +/- SEM; 176.0 +/- 25.8 pg/mL), hypocretin non-deficient narcolepsy with cataplexy (97.8 +/- 38.4 pg/mL), hypocretin non-deficient narcolepsy without cataplexy (113.6 +/- 16.4 pg/mL), and idiopathic hypersomnia (161.0 +/- 29.3 pg/ mL); the levels in OSAS (259.3 +/- 46.6 pg/mL) did not statistically differ from those in the controls (333.8 +/- 22.0 pg/mL). Low CSF histamine levels were mostly observed in non-medicated patients; significant reductions in histamine levels were evident in non-medicated patients with hypocretin deficient narcolepsy with cataplexy (112.1 +/- 16.3 pg/ mL) and idiopathic hypersomnia (143.3 +/- 28.8 pg/mL), while the levels in the medicated patients were in the normal range. CONCLUSION: The study confirmed reduced CSF histamine levels in hypocretin-deficient narcolepsy with cataplexy. Similar degrees of reduction were also observed in hypocretin non-deficient narcolepsy and in idiopathic hypersomnia, while those in OSAS (non central nervous system hypersomnia) were not altered. The decrease in histamine in these subjects were more specifically observed in non-medicated subjects, suggesting CSF histamine is a biomarker reflecting the degree of hypersomnia of central origin.

Expression pattern of FOS in orexin neurons during sleep induced by an adenosine A2A receptor agonist.

The present study examined the expression pattern of FOS in the hypothalamic peptide neurons during the sleep-dominant state induced by an adenosine A2A receptor agonist. The control rats, those that received the microdialysis-perfusion of their ventral striatum with artificial cerebrospinal fluid in the dark-active phase, spent 24% of the 90-min period prior to sacrifice in non-rapid eye movement (non-REM) sleep and 2.3% of that in REM sleep. These rats exhibited FOS, a transcription factor, in 21% of their orexin neurons and in 1.0% of their melanin-concentrating hormone (MCH) neurons in the perifornical/lateral hypothalamic areas. However, the rats perfused with 50 microM CGS21680, an adenosine A2A receptor agonist, spent 60% of the 90-min period prior to sacrifice in non-REM sleep and 11% of that in REM sleep. These rats exhibited FOS in 1.7% of their orexin neurons and FOS in 0.5% of their MCH neurons. When the sleep-dominant state was disturbed by mild stimulation and the rats were kept in the sleepy state by treatment with a sleep-inducing dose of CGS21680, the rats exhibited FOS in 13.3% of their orexin neurons, which percentage was about half of that for the control rats. These results suggest that the sleep-promoting process induced by this adenosine A2A receptor agonist was associated with a decline in the activity of orexin neurons. MCH neurons are not likely to change their activities during this sleep-promoting process.

FOS expression in orexin neurons following muscimol perfusion of preoptic area.

Unilateral microdialysis-perfusion of the preoptic area with 50 microM muscimol decreased the sleep period of rats to less than 3% of the baseline value over a 90 min period before death (p = 0.018 by Wilcoxon signed-rank test). These rats showed the expression of FOS in 36% of the orexin neurons located in the perifornical/lateral hypothalamic areas on the side ipsilateral to the perfusion site, but in only 3% of the orexin neurons on the side contralateral to it (p = 0.018 by Wilcoxon signed-rank test). These results suggest that subpopulations of the preoptic neurons give an inhibitory tone to the activities of the orexin neurons in the perifornical/lateral hypothalamic areas.

Inhibition of rostral basal forebrain neurons promotes wakefulness and induces FOS in orexin neurons.

The present study examined whether the activities of the rostral basal forebrain neurons alter the activities of the orexin (also known as hypocretin) neurons in the tuberal part of the hypothalamus in rats. We performed microdialysis perfusion of the ventromedial portion of the rostral basal forebrain with the GABAA receptor agonist muscimol to inhibit focally the neuronal activities in the rostral basal forebrain. Then, we monitored sleep/wake behaviour and investigated the pattern of activities of orexin neurons by examining the expression of FOS as an indicator of cellular activation. Bilateral perfusion with muscimol (5, 15, and 50 micro m) produced a dose-dependent decrease in the amount of sleep. This perfusion with muscimol at 50 micro m produced FOS-like immunoreactivity in 37% of the orexin neurons located in the tuberal part of the hypothalamus, whereas the FOS-like immunoreactivity was sparse in orexin neurons of the sleeping control rats (P = 0.001 by Mann-Whitney U-test). Unilateral perfusion with muscimol (50 micro m) also suppressed sleep. In this case, FOS-like immunoreactivity was seen in 40% of the orexin neurons on the side ipsilateral to the perfusion site but only in 10% of orexin neurons on the contralateral side (P = 0.018 by Wilcoxon signed rank test). These functional data suggested that a sleep-generating element in the ventromedial part of the rostral basal forebrain provides an inhibitory influence on the activities of the orexin neurons in the tuberal part of the hypothalamus.

A role of the C-terminus of aquaporin 4 in its membrane expression in cultured astrocytes.

BACKGROUND: Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, which is localized in the astrocyte plasma membrane. Membrane targeting of AQP4 is essential to perform its function. The mechanism(s) of membrane targeting is not clear in astrocytes. RESULTS: We investigated the role of the C-terminus of AQP4 (short isoform) in its membrane targeting by an expression study of C-terminal mutants of AQP4 in cultured astrocytes. The deletion of 26 C-terminal residues of AQP4 (AQP4[Delta276-301aa]) results in the intracellular localization of the protein. However, smaller deletions than 21 C-terminal residues did not alter its plasma membrane localization. These results suggest that C-terminal residues between Val(276) and Ile(280) play an important role in the expression of AQP4 in the plasma membrane. However, the plasma membrane localization of the AQP4(A(276)AAAA(280)) mutant (alanine substitution of Val(276)-Ile(280) of AQP4) suggests that another signal for membrane targeting exists in the C-terminus of AQP4. The deletion or point mutations of the PDZ binding motif of the AQP4(A(276)AAAA(280)) mutant resulted in the intracellular localization of the proteins. These results suggest that the PDZ binding motif may also be involved in the membrane targeting of AQP4. CONCLUSIONS: We found that the C-terminal sequence of AQP4 contains two important signals for membrane expression of AQP4 in cultured astrocytes. One is a hydrophobic domain and the other is a PDZ binding motif that exists in the C-terminus.