Chikamasa Yamashita

Rhythmic Control of the ARF-MDM2 Pathway by ATF4 Underlies Circadian Accumulation of p53 in Malignant Cells

The sensitivity of cancer cells to chemotherapeutic agents varies according to circadian time. Most chemotherapeutic agents ultimately cause cell death through cell-intrinsic pathways as an indirect consequence of DNA damage. The p53 tumor suppressor gene (TRP53) configures the cell deaths induced by chemotherapeutic agents. In this study, we show that the transcription factor ATF4, a component of the mammalian circadian clock, functions in circadian accumulation of p53 protein in tumor cells. In murine fibroblast tumor cells, ATF4 induced the circadian expression of p19ARF (Cdkn2a). Oscillation of p19ARF interacted in a time-dependent manner with MDM2, a specific ubiquitin ligase of p53, resulting in a rhythmic prevention of its degradation by MDM2. Consequently, the half-life of p53 protein varied in a circadian time-dependent manner without variation in mRNA levels. The p53 protein accumulated during those times when the p19ARF-MDM2 interaction was facilitated. Notably, the ability of the p53 degradation inhibitor nutlin-3 to kill murine fibroblast tumor cells was enhanced when the drug was administered at those times of day during which p53 had accumulated. Taken together, these results suggested that ATF4-mediated regulation of the p19ARF-MDM2 pathway underlies the circadian accumulation of p53 protein in malignant cells. Furthermore, they suggest an explanation for how the sensitivity of cancer cells to chemotherapeutic agents is enhanced at those times of day when p53 protein has accumulated, as a result of circadian processes controlled by ATF4.

Pulmonary administration of integrin-nanoparticles regenerates collapsed alveoli.

Chronic obstructive pulmonary disease (COPD) is an intractable pulmonary disease, causes widespread and irreversible alveoli collapse. In search of a treatment target molecule, which is able to regenerate collapsed alveoli, we sought to identify a factor that induces differentiation in human alveolar epithelial stem cells using all-trans retinoic acid (ATRA), whose alveolar repair capacity has been reported in animal experiments. When human alveolar epithelial stem cells were exposed to ATRA at a concentration of 10μM for over seven days, approximately 20% of the cells differentiated into each of the type-I and type-II alveolar epithelial cells that constitute the alveoli. In a microarray analysis, integrin-α1 and integrin-β3 showed the largest variation in the ATRA-treated group compared with the controls. Furthermore, the effect of the induction of differentiation in human alveolar epithelial stem cells using ATRA was suppressed by approximately one-fourth by siRNA treatments with integrin α1 and integrin β3. These results suggested that integrin α1 and β3 are factors responsible for the induction of differentiation in human alveolar epithelial stem cells. We accordingly investigated whether integrin nanoparticles also had a regenerative effect in vivo. Elastase-induced COPD model mouse was produced, and the alveolar repair effect of pulmonary administration using nanoparticles of integrin protein was evaluated by X-ray CT scanning. Improvement in the CT value in comparison with an untreated group indicated that there was an alveolar repair effect. In this study, it was shown that the differentiation-inducing effect on human alveolar epithelial stem cells by ATRA was induced by increased expression of integrin, and that the induced integrin enhanced phosphorylation signaling of AKT, resulting in inducing differentiations. Furthermore, the study demonstrated that lung administration of nanoparticles with increased solubility and stability of integrin repaired the alveolus of an Elastase-induced COPD model mouse. Those results show that those integrin nanoparticles are effective as novel COPD treatment target compounds.

Pulmonary administration of phosphoinositide 3-kinase inhibitor is a curative treatment for chronic obstructive pulmonary disease by alveolar regeneration

Chronic obstructive pulmonary disease (COPD) is an intractable pulmonary disease, causing widespread and irreversible alveoli collapse. The discovery of a low-molecular-weight compound that induces regeneration of pulmonary alveoli is of utmost urgency to cure intractable pulmonary diseases such as COPD. However, a practically useful compound for regenerating pulmonary alveoli is yet to be reported. Previously, we have elucidated that Akt phosphorylation is involved in a differentiation-inducing molecular mechanism of human alveolar epithelial stem cells, which play a role in regenerating pulmonary alveoli. In the present study, we directed our attention to phosphoinositide 3-kinase (PI3K)-Akt signaling and examined whether PI3K inhibitors display the pulmonary alveolus regeneration. Three PI3K inhibitors with different PI3K subtype specificities (Wortmannin, AS605240, PIK-75 hydrochloride) were tested for the differentiation-inducing effect on human alveolar epithelial stem cells, and Wortmannin demonstrated the most potent differentiation-inducing activity. We evaluated Akt phosphorylation in pulmonary tissues of an elastase-induced murine COPD model and found that Akt phosphorylation in the pulmonary tissue was enhanced in the murine COPD model compared with normal mice. Then, the alveolus-repairing effect of pulmonary administration of Wortmannin to murine COPD model was evaluated using X-ray CT analysis and hematoxylin-eosin staining. As a result, alveolar damages were repaired in the Wortmannin-administered group to a similar level of normal mice. Furthermore, pulmonary administration of Wortmannin induced a significant recovery of the respiratory function, compared to the control group. These results indicate that Wortmannin is capable of inducing differentiation of human alveolar epithelial stem cells and represents a promising drug candidate for curative treatment of pulmonary alveolar destruction in COPD.

Pulmonary administration of Am80 regenerates collapsed alveoli.

Chronic obstructive pulmonary disease (COPD) is an intractable pulmonary disease, which causes widespread and irreversible alveoli collapse. Nevertheless, there is no effective drug therapy that regenerates lung tissue or prevents the progression of COPD and clinical management of patients remains mostly supportive. The aim of this study was to evaluate whether Am80 is useful as a novel pulmonary emphysema therapeutic drug. In this study, we treated the human alveolar epithelial stem cells with Am80 to clarify the differentiation-inducing mechanism and administrated Am80 transpulmonarily into elastase-induced COPD model mice to evaluate the effect of Am80 on pulmonary emphysema. First, we accordingly investigated whether Am80 had a differentiation-inducing effect on human alveolar epithelial stem cells, Am80 induced differentiation of human alveolar epithelial stem cells to alveolar type I and II cells dose dependently, and the proportion of differentiated into type I and type II alveolar epithelial cells as a result of treatment with 10 μM of Am80 for 7 days was approximately 20%. Second, we attempted to identify the major factor involved in the differentiation-inducing effect of human alveolar epithelial stem cells induced by Am80 using microarray analysis. In a microarray analysis, WNT1, lectin, SLIT, chordin, ck12, ck11, and neurexin3 showed the largest variation in the Am80-treated group compared with the controls. In quantitative polymerase-chain-reaction assay, Am80 resulted in significant reduction in the WNT1 expression ratio whereas increase in the neurexin3 expression ratio. We evaluated the repairs effect for collapsed alveoli by Am80 of pulmonary administration. In untreated and Am80-treated mice the average CT value at 2 days was, respectively, -506 and -439 and there was a significant difference. Likewise, the assessment of the distance between alveolar walls, Lm, confirmed that there was a significant difference between control (68.0±3.8 μm) and Am80-treated group (46.8±1.8 μm). These indicated that treatment with Am80 caused a reversal of lung tissue damage in elastase-induced COPD model mouse. Those results suggested that Am80 were effective as novel COPD therapeutic compounds.

Antidepressant-like effects exerted by the intranasal administration of a glucagon-like peptide-2 derivative containing cell-penetrating peptides and a penetration-accelerating sequence in mice

HIGHLIGHTSPAS‐CPPs‐GLP‐2 (i.n.) exerted antidepressant‐like effects in naïve mice.PAS‐CPPs‐GLP‐2 (i.n.) also exerted antidepressant‐like effects in ACTH‐treated mice.PAS‐CPPs‐GLP‐2 (i.v.) and CPPs‐GLP‐2 (i.n.) did not affect the FST immobility time.FITC‐labeled PAS‐CPPs‐GLP‐2 (i.n.) was distributed through the mouse brain.PAS‐CPPs may enhance the i.n. delivery to the brain compared with CPPs only. ABSTRACT The intracerebroventicular (i.c.v.) administration of glucagon‐like peptide‐2 (GLP‐2) to rodents was shown to have antidepressant‐like effects in imipramine‐resistant depression‐model mice. In order to utilize GLP‐2 as a clinical treatment tool for depression, we herein focused on the intranasal delivery that is non‐invasive approach, because the i.c.v. administration is invasive and impractical. In the present study, we prepared a GLP‐2 derivative containing cell penetrating peptides (CPPs) and a penetration accelerating sequence (PAS) (PAS‐CPPs‐GLP‐2) for the intranasal (i.n.) administration. PAS‐CPPs‐GLP‐2 (i.n.) exhibited antidepressant‐like effects in the forced‐swim test (FST) and tail suspension test (TST) in naïve mice as well as adrenocorticotropic hormone (ACTH) treated‐mice. However, PAS‐CPPs‐GLP‐2 (i.v.) and the GLP‐2 derivative containing CPPs without a PAS (CPPs‐GLP‐2) (i.n.) did not affect the immobility time in the mouse FST. Moreover, fluorescein isothiocyanate (FITC)‐labeled PAS‐CPPs‐GLP‐2 (i.n.), but not FITC‐labeled CPPs‐GLP‐2 (i.n.) was distributed through the mouse brain after the FST session. These results suggest that PAS‐CPPs‐GLP‐2 is effective for i.n. delivery to the brain, and may be useful in the clinical treatment of major depression.

Pulmonary administration of 1,25-dihydroxyvitamin D3 to the lungs induces alveolar regeneration in a mouse model of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disease with several causes, including smoking, and no curative therapeutic agent is available, particularly for destructive alveolar lesions. In this study, we investigated the differentiation-inducing effect on undifferentiated lung cells (Calu-6) and the alveolar regenerative effect of the active vitamin 1,25-dihydroxy vitamin D3 (VD3) with the ultimate goal of developing a novel curative drug for COPD. First, the differentiation-inducing effect of VD3 on Calu-6 cells was evaluated. Treatment with VD3 increased the proportions of type I alveolar epithelial (AT-I) and type II alveolar epithelial (AT-II) cells constituting alveoli in a concentration- and treatment time-dependent manner, demonstrating the potent differentiation-inducing activity of VD3 on Calu-6 cells. We thus administered VD3 topically to the mice lung using a previously developed intrapulmonary administration via self-inhalation method. To evaluate the alveolus-repairing effect of VD3, we administered VD3 intrapulmonarily to elastase-induced COPD model mice and computed the mean distance between the alveolar walls as an index of the extent of alveolar injury. Results showed significant decreases in the alveolar wall distance in groups of mice that received 0.01, 0.1, and 1μg/kg of intrapulmonary VD3, revealing excellent alveolus-regenerating effect of VD3. Furthermore, we evaluated the effect of VD3 on improving respiratory function using a respiratory function analyzer. Lung elasticity and respiratory competence [forced expiratory volume (FEV) 1 s %] are reduced in COPD, reflecting advanced emphysematous changes. In elastase-induced COPD model mice, although lung elasticity and respiratory competence were reduced, VD3 administered intrapulmonarily twice weekly for 2weeks recovered tissue elastance and forced expiratory volume in 0.05s to the forced vital capacity, which are indicators of lung elasticity and respiratory competence, respectively, to levels comparable to those in normal mice. These results revealed the potent activity of VD3 in inducing differentiation of the Calu-6 cells and the effect of topical administration of VD3 to the lungs to induce lung regeneration at histological and functional levels, demonstrating the potential of VD3 as a curative agent for alveolar destruction in COPD.

Pulmonary Administration of GW0742, a High-Affinity Peroxisome Proliferator-Activated Receptor Agonist, Repairs Collapsed Alveoli in an Elastase-Induced Mouse Model of Emphysema

Pulmonary emphysema is a disease in which lung alveoli are irreversibly damaged, thus compromising lung function. Our previous study revealed that all-trans-retinoic acid (ATRA) induces the differentiation of human lung alveolar epithelial type 2 progenitor cells and repairs the alveoli of emphysema model mice. ATRA also reportedly has the ability to activate peroxisome proliferator-activated receptor (PPAR) β/δ. A selective PPARβ/δ ligand has been reported to induce the differentiation of human keratinocytes during wound repair. Here, we demonstrate that treatment using a high-affinity PPARβ/δ agonist, GW0742, reverses the lung tissue damage induced by elastase in emphysema-model mice and improves respiratory function. Mice treated with elastase, which collapsed their alveoli, were then treated with either 10% dimethyl sulfoxide (DMSO) in saline (control group) or GW0742 (1.0 mg/kg twice a week) by pulmonary administration. Treatment with GW0742 for 2 weeks increased the in vivo expression of surfactant proteins A and D, which are known alveolar type II epithelial cell markers. GW0742 treatment also shortened the average distance between alveolar walls in the lungs of emphysema model mice, compared with a control group treated with 10% DMSO in saline. Treatment with GW0742 for 3 weeks also improved tissue elastance (cm H2O/mL), as well as the ratio of the forced expiratory volume in the first 0.05 s to the forced vital capacity (FEV 0.05/FVC). In each of these experiments, GW0742 treatment reversed the damage caused by elastase. In conclusion, PPARβ/δ agonists are potential therapeutic agents for pulmonary emphysema.

Dosing-time-dependent effect of rivaroxaban on coagulation activity in rats

The anticoagulant effect of rivaroxaban, a direct inhibitor of activated factor X (FX), might be influenced by its dosing time because the activity of the coagulofibrinolytic system exhibits daily rhythmicity. In rats, FX activity follows a 24-h rhythm with a peak in the middle of the light phase and a trough at the beginning of the dark phase. Consistent with these findings, a single dose of rivaroxaban had a stronger inhibitory effect on FX activity after dosing at the beginning of the light phase than after dosing at the beginning of the dark phase. A similar chronopharmacological effect was seen in a quantitative model of venous stasis thrombosis. In comparison, the dosing time had minimal influence on the pharmacokinetics of rivaroxaban. These data indicate that the anticoagulant effect of rivaroxaban is influenced by the dosing time. Further studies should confirm this finding in a clinical setting.

LARETH-25 and β-CD improve central transitivity and central pharmacological effect of the GLP-2 peptide.

Depression is a common mental disorder. More than 350 million people of all ages suffer from depression worldwide. Although a number of antidepressants are available, >20% of patients with major depressive disorder suffer from treatment-resistant depression. Therefore, development of novel therapeutics to overcome this condition is required. We reported that intracerebroventricular administration of glucagon-like peptide-2 (GLP-2) exerts antidepressant-like effects treated with or without adrenocorticotropic hormone. In the present study, we developed a nasal formulation of GLP-2 containing 5% polyoxyethylene (25) lauryl ether and 1% β-cyclodextrin that enhanced the resistance of GLP-2 to inactivation by dipeptidyl peptidase-4. Intranasal administration of this formulation (60μg/kg) increased the delivery of GLP-2 to the brain and had antidepressant-like effects on rats. These results suggest the potential of the GLP-2 nasal formulation for use as a novel antidepressant.

Intranasal administration of neuromedin U derivatives containing cell-penetrating peptides and a penetration-accelerating sequence induced memory improvements in mice

HighlightsIntranasally administered neuromedin U derivatives prevented LPS‐induced amnesia.Intranasally administered neuromedin U derivatives ameliorated LPS‐induced amnesia.Derivatives contained two different sequences exhibited similar anti‐amnesic effects. ABSTRACT Neuromedin U (NMU) is a neuropeptide that is expressed and secreted in the brain and gut. We previously demonstrated that the intracerebroventricular (i.c.v.) administration of NMU inhibited inflammation‐mediated memory impairment in mice. In order to utilize NMU as a clinical treatment tool for inflammation‐mediated amnesia, we herein focused on non‐invasive intranasal delivery because the i.c.v. administration route is invasive and impractical. In the present study, we prepared two NMU derivatives containing cell‐penetrating peptides (CPPs), octaarginine (R8), and each penetration‐accelerating sequence, namely FFLIPKG (PASR8‐NMU) and FFFFG (F4R8‐NMU), for intranasal (i.n.) administration. In the Y‐maze test, the i.c.v. administration of lipopolysaccharide (LPS) (10 &mgr;g/mouse) significantly decreased spontaneous alternation behavior, and this was prevented by the prior administration of PASR8‐NMU or F4R8‐NMU (5.6 &mgr;g/mouse, i.n.). Moreover, the administration of PASR8‐NMU or F4R8‐NMU (5.6 &mgr;g/mouse, i.n.) just before the Y‐maze test also improved LPS‐induced memory impairment. Indocyanine green (ICG)‐labeled PASR8‐NMU (i.n.) was significantly observed in the hippocampus and paraventricular hypothalamic nucleus 30 min after its i.n. administration. PASR8‐NMU, but not F4R8‐NMU guaranteed the stability of the administration liquid for 24 h. These results suggest that PASR8‐NMU is effective for i.n. delivery to the brain, and may be useful in the clinical treatment of inflammation‐mediated amnesia.