GLP-1 derivatives with functional sequences transit and migrate through trigeminal neurons.

Patients with dementia are increasing with the aging of the population, and dementia has become a disease with high unmet medical needs. Glucagon-like peptide-1 (GLP-1), a neuropeptide, has been reported to improve learning and memory following intracerebroventricular administration. We focused on intranasal administration, which can deliver drugs noninvasively and efficiently to the brain. Although much of the human nasal mucosa is occupied by respiratory epithelium, many capillaries are present in the paracellular route of respiratory epithelium. Therefore, to incorporate GLP-1 into cells, we created a GLP-1 derivative by adding cell-penetrating peptides (CPP) and penetration accelerating sequences (PAS) to GLP-1. We investigated in vitro and in vivo function of PAS-CPP-GLP-1 to enable the translocation of GLP-1 directly from nose to brain. PAS-CPP-GLP-1 enhanced cellular uptake by macropinocytosis with CPP, efficiently escaped from the endosomes due to PAS, and exited the cells. PAS-CPP-GLP-1 also transited trigeminal nerve cells through axon transport and migrated to the adjacent trigeminal nerve cell. Moreover, PAS-CPP-GLP-1 showed significant improvement in learning memory in mice within 20 min of intranasal administration. These results suggested CPP and PAS may be important for the efficient transfer of GLP-1 to the site of action in the brain following intranasal administration.

Characterization of a spontaneous mouse model of mild, accelerated aging via ECM degradation in emphysematous lungs.

Emphysema limits airflow and causes irreversible progression of chronic obstructive pulmonary disease (COPD). Strain differences must be considered when selecting mouse models of COPD, owing to disease complexity. We previously reported that a novel C57BL/6JJcl substrain, the Mayumi-Emphysema (ME) mouse, exhibits spontaneous emphysema; however, the other characteristics remain unknown. We aimed to characterize the lungs of ME mice and determine their experimental availability as a model. ME mice had a lower body weight than the control C57BL/6JJcl mice, with a median survival time of ~80 weeks. ME mice developed diffused emphysema with respiratory dysfunction from 8 to 26 weeks of age, but did not develop bronchial wall thickening. Proteomic analyses revealed five extracellular matrix-related clusters in downregulated lung proteins in ME mice. Moreover, EFEMP2/fibulin-4, an essential extracellular matrix protein, was the most downregulated protein in the lungs of ME mice. Murine and human EFEMP2 were detected in the pulmonary artery. Furthermore, patients with mild COPD showed decreased EFEMP2 levels in the pulmonary artery when compared to those without COPD. The ME mouse is a model of mild, accelerated aging with low-inflammatory emphysema and respiratory dysfunction that progresses with age and pulmonary EFEMP2 decrease, similar to that observed in patients with mild COPD.

Intracellular Drug Delivery Process of Am80-Encapsulated Lipid Nanoparticles Aiming for Alveolar Regeneration.

Chronic obstructive pulmonary disease (COPD) results in obstructive ventilatory impairment caused by emphysema, and current treatment is limited to symptomatic therapy or lung transplantation. Therefore, the development of new treatments to repair alveolar destruction is especially urgent. Our previous study revealed that 1.0 mg/kg of synthetic retinoid Am80 had a repair effect on collapsed alveoli in a mouse model of elastase-induced emphysema. From these results, however, the clinical dose calculated in accordance with FDA guidance is estimated to be 5.0 mg/60 kg, and it is desirable to further reduce the dose to allow the formulation of a powder inhaler for clinical application. To efficiently deliver Am80 to the retinoic acid receptor in the cell nucleus, which is the site of action, we focused on SS-cleavable proton-activated lipid-like material O-Phentyl-P4C2COATSOME®SS-OP, hereinafter referred to as "SS-OP"). In this study, we investigated the cellular uptake and intracellular drug delivery process of Am80-encapsulated SS-OP nanoparticles to elucidate the mechanism of Am80 by nanoparticulation. Am80-encapsulated SS-OP nanoparticles were taken up into the cells via ApoE, and then Am80 was efficiently delivered into the nucleus via RARα. These results indicated the usefulness of SS-OP nanoparticles as drug delivery system carriers of Am80 for COPD treatment.

Involvement of trigeminal axons in nose-to-brain delivery of glucagon-like peptide-2 derivative.

In our previous study, we created a glucagon-like peptide-2 (GLP-2) derivative with the functional sequence PAS-CPP to achieve efficient uptake by the respiratory epithelium and trigeminal nerve. By using octaarginine for cell penetrating peptides (CPP) and FFLIPKG, a reverse sequence of a part of the cathepsin D sequence for the penetration accelerating sequence (PAS), we found that the derivative was taken up by the cells through macropinocytosis and efficiently escaped from the endosomes and exited the cells. Moreover, it showed drug effects by intranasal (in.) administration at the same dose as intracerebroventricular (icv.) administration, which is direct drug administration into the brain. The purpose of this study was to elucidate the cause of the drug effect of in. administered PAS-CPP-GLP-2 at the same dose as that by icv. administration. The present results suggested that although icv. administered PAS-CPP-GLP-2 entered the cerebrospinal fluid, it barely penetrated the perivascular space of the brain, and therefore, only a small amount of the administered dose may have reached the site of action in the brain. In contrast, it was qualitatively suggested that in. administered PAS-CPP-GLP-2 migrates from the trigeminal nerve to the central nervous system via the principal sensory trigeminal nucleus and then through the trigeminal lemniscus. The present results show that nose-to-brain delivery by trigeminal axons, which is assumed to be a transcellular pathway, may be possible. As the drug can be delivered into the nerve, it is expected to be applied not only as a central delivery route but also for the treatment of neurological diseases.

A Mouse Model for Tuberculosis Combined With Inhalable Imiquimod-PLGA Nanocomposite Particles Based on Macrophage Phenotype.

BACKGROUND/AIM: In vivo models of tuberculosis are effective tools for developing new drugs. The objective of this study was to prepare in vivo models for tuberculosis by utilizing nanocomposite particles (NCPs) containing imiquimod-loaded poly(lactic-co-glycolic acid) nanoparticles. MATERIALS AND METHODS: NCPs were prepared from dichloromethane with imiquimod and poly(lactic-co-glycolic acid) using a spray dryer. Mice were treated with NCPs in the lungs by inhalation, and then infection with Mycobacterium bovis bacille Calmette-Guerin was performed (treatment groups). The concentrations of the pro-inflammatory cytokines, tumor necrosis factor-α and interferon-γ were measured in bronchoalveolar lavage fluid using an enzyme-linked immunosorbent assay. RESULTS: When animals were treated with NCPs, the concentrations of tumor necrosis factor-α and interferon-γ in bronchoalveolar lavage fluid were significantly higher than in animals not treated with NCPs. In addition, high bacterial counts and circular granuloma were observed. CONCLUSION: NCPs prepared in this study enhanced the level of inflammation in the lungs and support the preparation of in vivo models of tuberculosis.

Intracerebroventricular administration of oxytocin and intranasal administration of the oxytocin derivative improve ß-amyloid peptide (25-35)-induced memory impairment in mice.

AIM: We previously reported that oxytocin, a peptide hormone, can reverse the ß-amyloid peptide (25-35) (Aß25-35 )-induced impairments of the murine hippocampal synaptic plasticity. In this study, we examined the effects of oxytocin on the Aß25-35 -induced impairment of cognitive behavior in murine in order to investigate the potential of oxytocin as a clinical treatment tool for Alzheimer's disease (AD). METHODS: The Y-maze and Morris water maze (MWM) tests were performed. Since the intracerebroventricular (ICV) administration is both invasive and impractical, we further utilized intranasal (IN) delivery to the brain. For this purpose, we prepared an oxytocin derivative containing cell-penetrating peptides and a penetration accelerating sequence, which was subsequently used in our IN administration experiments. RESULTS: We herein showed that the ICV administration of oxytocin in mice exerted memory-improving effects on the Aß25-35 -induced amnesia in both the Y-maze and MWM tests. The IN administration of the oxytocin derivative exhibited memory-improving effects in the Y-maze test. Moreover, we acquired evidence that the fluorescein isothiocyanate-labeled oxytocin derivative was distributed throughout the mouse brain following its IN administration. CONCLUSION: Our results suggest that the oxytocin derivative is effective for its IN delivery to the brain and may be particularly useful in the clinical treatment of cognitive impairment, such as that characterizing AD.

Radiolabeling Method for Lyophilizate for Dry Powder Inhalation Formulations.

Human lung deposition data is non-mandatory for drug approval but very useful for the development of orally inhaled drug products. Lung deposition of inhaled drugs can be quantified by radionuclide imaging, for which one of the first considerations is the method used to radiolabel formulations. In this study, we report the development of a radiolabeling method for lyophilizate for dry powder inhalation (LDPI) formulations. TechneCoatTM is one method that can radiolabel drug particles without using solvents. In this method, particles are radiolabeled with a dispersion of 99mTc-labeled nanoparticles called TechnegasTM. Because a LDPI formulation is not comprised of particles but is a lyophilized cake aerosolized by air impact, the TechneCoat method cannot be used for the radiolabeling of LDPI formulations. We therefore modified the TechneCoat apparatus so that LDPI formulations were not aerosolized by the Technegas flow. Radiolabeling using a modified TechneCoat apparatus was validated with model LDPI formulations of interferon alpha (IFN). IFN of 99mTc-unlabeled, IFN of 99mTc-labeled, and 99mTc of 99mTc-labeled LDPI formulations showed similar behavior, and differences from IFN of 99mTc-unlabeled LDPI formulations were within ±15% in aerodynamic particle size distribution measurement. Our radiolabeling method for LDPI formulations may be useful for the quantification of drug deposition in human lungs.

Am80-Encapsulated Lipid Nanoparticles, Developed with the Aim of Achieving Alveolar Regeneration, Have an Improvement Effect on Pulmonary Emphysema.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitis and emphysema, and current drug treatments target its symptoms. Thus, the development of a therapeutic drug to repair alveolar destruction is urgently needed. Our previous research revealed that the synthetic retinoic acid Am80 (1.0 mg/kg) showed a repairing effect on collapsed alveoli in a mouse model of elastase-induced emphysema. However, a further reduction in the dose is desirable to facilitate the development of a powder inhalation formulation for clinical application. We, therefore, focused on SS-OP to deliver Am80 efficiently. As a result, 0.01 mg/kg of Am80-encapsulated SS-OP nanoparticles repaired collapsed alveoli and improved the respiratory function in the mouse model of elastase induced emphysema. The results suggested that, with the use of SS-OP, the Am80 dose could be reduced. This could contribute to the development of a powder inhalation system as a curative medicine for COPD.

Induction of mucosal immunity by pulmonary administration of a cell-targeting nanoparticle.

We previously found that a nanoparticle constructed with an antigen, benzalkonium chloride (BK) and γ-polyglutamic acid (γ-PGA) showed high Th1 and Th2-type immune induction after subcutaneous administration. For prophylaxis of respiratory infections, however, mucosal immunity should be induced. In this study, we investigated the effect of pulmonary administration of a nanoparticle comprising ovalbumin (OVA) as a model antigen, BK, and γ-PGA on induction of mucosal immunity in the lungs and serum. The complex was strongly taken up by RAW264.7 and DC2.4cells. After pulmonary administration, lung retention was longer for the OVA/BK/γ-PGA complex than for OVA alone. OVA-specific serum immunoglobulin (Ig)G was highly induced by the complex. High IgG and IgA levels were also induced in the bronchoalveolar lavage fluid, and in vivo toxicities were not observed. In conclusion, we effectively and safely induced mucosal immunity by pulmonary administration of an OVA/BK/γ-PGA complex.

Usefulness of cell-penetrating peptides and penetration accelerating sequence for nose-to-brain delivery of glucagon-like peptide-2.

Neuropeptides are expected as therapeutic drug candidates for central nervous system (CNS) disorders. Intracerebroventricular (i.c.v.) administration of glucagon-like peptide-2 (GLP-2) has an antidepressant-like effect not only in depression model mice but also in treatment-resistant depression model mice. However, because i.c.v. administration is very invasive, research is progressing on brain delivery using intranasal administration as a non-invasive method. After intranasal administration of the drug, there are two routes to the brain. That of direct delivery from the paracellular route of olfactory epithelium to the brain via the olfactory bulb has been studied, and that of systemic absorption via the paracellular route of respiratory epithelium has been put to practical use. The high degree of vascularization and permeability of the nasal mucosa enables drug delivery via the paracellular route that leads to systemic delivery. Therefore, suppressing systemic absorption may increase drug delivery to brain, so we focused on the transcellular route. We created a GLP-2 derivative by adding cell-penetrating peptides (CPP) and penetration accelerating sequences (PAS), which are reported to provide efficient intracellular uptake, to GLP-2. However, to deliver GLP-2 by the transcellular route, GLP-2 must not only be taken up into cells but also move out of the cells. We investigated in vitro and in vivo function of PAS-CPP-GLP-2 to enable the translocation of GLP-2 directly from the nose to the brain. Derivatization of PAS-CPP-GLP-2 prevented its degradation. In the evaluation of intracellular dynamics, PAS-CPP-GLP-2 enhanced cellular uptake by macropinocytosis with CPP and promoted escape from endosomal vesicles by PAS. This study also showed that PAS-CPP-GLP-2 can move out of cells. Furthermore, only this PAS-CPP-GLP-2 showed an antidepression-like effect within 20 min of intranasal administration. Intranasal administered PAS-CPP-GLP-2 surprisingly showed the effect at the same dose with i.c.v. administration, but intravenous administered PAS-CPP-GLP-2 did not show the effect. These results suggested that PAS-CPP-GLP-2 can be efficiently delivered from the nose to the CNS and show a pharmacological effect, demonstrating the usefulness of PAS and CPP for nose-to-brain delivery of GLP-2.

Optimization of Very Low-Dose Formulation of Vitamin D3 with Lyophilizate for Dry Powder Inhalation System by Simple Method Based on Time-of-Flight Theory.

It has been previously reported that active vitamin D3 (VD3) is a candidate drug that can repair alveolar damage in chronic obstructive pulmonary disease at a very low dose. We herein report the optimization of a very low-dose formulation of VD3 for dry powder inhalation by a simple method based on time-of-flight (TOF) theory. As the preparation content of VD3 is very low, aerodynamic particle size distribution cannot be measured by pharmacopeial methods that require quantification of the main drug. Thus, a simple method based on TOF theory, which can measure aerodynamic particle size distribution without quantification, was used. The optimized formulation for an inhalation system using a lyophilized cake contained phenylalanine as the excipient (VD3 1 µg/vial + phenylalanine 0.3 mg/vial) and showed high performance with fine particle fraction ≤ 3 µm = 47.2 ± 4.4%. The difference between the results of pharmacopeial methods and simple method was examined using the formulation containing 10 µg/vial of VD3 and was within 5.0%. The preparation is expected to efficiently deliver VD3 to the lungs. Our simple method can optimize dry powder inhalation formulations more easily and rapidly even when the content of the main drug in a preparation is very low.

Systemic Delivery of hGhrelin Derivative by Lyophilizate for Dry Powder Inhalation System in Monkeys.

Ghrelin is the peptide that increases the hunger sensation and food intake and is expected to be clinically applied for treatment of diseases such as cachexia and anorexia nervosa. In the clinical application of ghrelin, injections are problematic in that they are invasive and inconvenient. Thus, we aimed to develop a formulation that can eliminate the need for injections and can be applied clinically. We prepared formulations of an hGhrelin derivative, in which the octanoyl group essential for expression of activity is modified to avoid rapid des-acylation, using lyophilizate for a dry powder inhalation (LDPI) system. The formulation of hGhrelin derivative was optimized by the addition of phenylalanine, of which the fine particle fraction of 5 µm or less was 41.7 ± 3.8%. We also performed pharmacokinetic/pharmacodynamic tests in monkeys using the optimum formulation that can be applied clinically. The absolute bioavailability of inhaled hGhrelin derivative with respect to that intravenously injected was 16.9 ± 2.6%. An increase in growth hormone was shown as an effect of the inhaled hGhrelin derivative similar to intravenous injection. The LDPI formulation can deliver the hGhrelin derivative systemically, and it is expected to be applied clinically as a substitute for injections.

Simple Method to Measure the Aerodynamic Size Distribution of Porous Particles Generated on Lyophilizate for Dry Powder Inhalation.

Recently, statistical techniques such as design of experiments are being applied for efficient optimization of oral formulations. To use these statistical techniques for inhalation formulations, efficient methods for rapid determination of the aerodynamic particle size distribution of many samples are needed. Therefore, we aimed to develop a simple method to measure aerodynamic particle size distribution that closely agrees with the results of inhalation characteristic tests. We added attachments for dispersion to the aerodynamic particle sizer (APS) so that formulations could be dispersed under the same condition as for multi-stage liquid impinger (MSLI) measurement. Then, we examined the correlation between MSLI and APS using lyophilizate for dry powder inhalation formulations that generate porous particles just on inhalation. It is difficult to obtain the accurate aerodynamic particle size distribution of porous particles by APS because the particle density is difficult to estimate accurately. However, there was a significant correlation between MSLI and APS when the particle density settings for APS measurement was calculated by a conversion factor based on the result of MSLI. The APS with dispersion attachments and this conversion factor can measure a number of samples in a short time, thereby enabling more efficient optimization of dry powder inhalers.

Expression of clock gene Dbp in omental and mesenteric adipose tissue in patients with type 2 diabetes.

INTRODUCTION: We previously reported in ob/ob mice, one of animal models of human type 2 diabetes mellitus (DM2), that (i) acetylation of histone H3 lysine 9 (H3K9) at the promoter region of clock gene Dbp and DBP mRNA expression are reduced in epididymal adipose tissue, (ii) binding of DBP to the promoter region of peroxisome proliferator-activated receptor (Ppar)-γ and mRNA expression of PPAR-γ1sv were decreased in preadipocytes and (iii) adiponectin secretion was decreased, leading to the impaired insulin sensitivity. RESEARCH DESIGN AND METHODS: The present study was undertaken to evaluate whether such the changes in visceral adipose tissue were detected in patients with DM2. We obtained omental and mesenteric adipose tissue during surgery of lymph node dissection for gastric and colorectal cancers, and investigated these variables in adipose tissue (omental from gastric cancer; 13 non-DM, 12 DM2: mesenteric from colorectal cancer; 12 non-DM, 11 DM2). RESULTS: Acetylation of histone H3K9 at the promoter region of Dbp and DBP mRNA expression in omental, but not in mesenteric adipose tissue were significantly lower in DM2 than in patients without DM. PPAR-γ mRNA expression in omental adipose tissue was also lower in patients with DM2, but not in mesenteric adipose tissue. CONCLUSIONS: The changes in DBP-PPAR-γ axis observed in mice with diabetes were also detected in patients with DM2. Because adiponectin secretion is reported to be enhanced through the PPAR-γ-related mechanism, this study supports the hypothesis that omental adipose tissue is involved in the mechanism of DM2.

Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues.

The present study investigated a pulmonary delivery system of plasmid DNA (pDNA) and its application to melanoma DNA vaccines. pCMV-Luc, pEGFP-C1, and pZsGreen were used as a model pDNA to evaluate transfection efficacy after inhalation in mice. Naked pDNA and a ternary complex, consisting of pDNA, dendrigraft poly-l-lysine (DGL), and γ-polyglutamic acid (γ-PGA), both showed strong gene expression in the lungs after inhalation. The transgene expression was detected in alveolar macrophage-rich sites by observation using multi-color deep imaging. On the basis of these results, we used pUb-M, which expresses melanoma-related antigens (ubiquitinated murine melanoma gp100 and tyrosinase-related protein 2 (TRP2) peptide epitopes), as DNA vaccine for melanoma. The inhalation of naked pUb-M and its ternary complex significantly inhibited the metastasis of B16-F10 cells, a melanoma cell line, in mice. The levels of the inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, which enhance Th1 responses, were higher with the pUb-M ternary complex than with naked pUb-M and pEGFP-C1 ternary complex as control. In conclusion, we clarified that the inhalation of naked pDNA as well as its ternary complex are a useful technique for cancer vaccination.

The effects of 1α,25-dihydroxyvitamin D3 on alveolar repair and bone mass in adiponectin-deficient mice.

Chronic obstructive pulmonary disease (COPD) is a major cause of death worldwide. However, no drugs can regenerate lung tissue in COPD patients, and differentiation-inducing drugs that can effectively treat damaged alveoli are needed. In addition, the presence of systemic comorbidities is also considered problematic. Our previous study revealed that a retinoic acid derivative improved emphysema in elastase-induced COPD model mice at a dose of 1.0 mg/kg, whereas 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) showed an emphysema-improving effect in the same model at 0.1 µg/kg. Elastase-induced COPD model mice do not exhibit a systemic disease state, so evaluation in a model that better reflects the human disease state is considered necessary. To solve this problem, we focused on the adiponectin-deficient mouse and examined the effects of 1,25(OH)2D3 on alveolar regeneration. Fifty-week-old adiponectin-deficient mice were treated with 1,25(OH)2D3 (0.1 µg/kg) twice a week, for 30 weeks. The effects of pulmonary administration on alveolar repair were evaluated according to the distance between alveolar walls (Lm values) and computed tomography (CT) parameters. Bone density was evaluated based on CT. The administration of 1,25(OH)2D3 was confirmed to show a significant therapeutic effect. The Lm values in the control and 1,25(OH)2D3-treated groups were 98 ± 4 µm and 63 ± 1 µm, respectively. However, on CT, the average CT value and % of low attenuation area showed no significant change. In adiponectin-deficient mice, the reduction of bone density (cortical, spongy, and total bone), which is a systemic symptom of COPD, was significantly suppressed by 1,25(OH)2D3 at 80 weeks of age. The present study suggests that 1,25(OH)2D3 could be a potential candidate drug that may provide a radical cure for the lung disease and comorbidities of COPD patients. This work can lead to the development drugs that may provide a radical cure for COPD.

A novel diagnostic system to evaluate epidermal growth factor receptor impact as a prognostic and therapeutic indicator for lung adenocarcinoma.

Many driver pathways for cancer cell proliferation have been reported. Driver pathway activation is often evaluated based on a single hotspot mutation such as EGFR L858R. However, because of complex intratumoral networks, the impact of a driver pathway cannot be predicted based on only a single gene mutation. Here, we developed a novel diagnostic system named the "EGFR impact score" which is based on multiplex mRNA expression profiles, which can predict the impact of the EGFR pathway in lung cancer cells and the effect of EGFR-tyrosine kinase inhibitors on malignancy. The EGFR impact score indicated robust predictive power for the prognosis of early-stage lung cancer because this score can evaluate the impact of the EGFR pathway on the tumor and genomic instability. Additionally, the molecular features of the poor prognostic group resembled those of biomarkers associated with immune checkpoint inhibitors. The EGFR impact score is a novel prognostic and therapeutic indicator for lung adenocarcinoma.

Efficient optimization of high-dose formulation of novel lyophilizates for dry powder inhalation by the combination of response surface methodology and time-of-flight measurement.

Inhalation of proteins/peptides has recently received attention as various biopharmaceuticals have emerged on the market. Novel lyophilisates for dry powder inhalation (LDPIs), which are aerosolized by air impact, have been reported and LDPIs are considered an attractive option for the pulmonary administration of biopharmaceuticals. However, desirable disintegration and aerosolization properties have been unavailable in high-dose formulations, which has been a critical issue. This study aimed to investigate high-dose LDPIs and their optimization. In the present study, lysozyme (Lysoz) was used as a stable model protein and formulated with various amino acids. Furthermore, response surface methodology (RSM) and time-of-flight measurement were applied for efficient optimization. Superior disintegration and aerosolization properties were confirmed in the LDPIs with phenylalanine (Phe) and leucine (Leu). RSM results revealed that 0.5 mg/vial of Phe and 1.0 mg/vial of Leu are the optimal quantities for high-dose formulation. Based on these optimum quantities, high-dose LDPI formulations were prepared and the maximum formulable quantity of Lysoz with acceptable inhalation performance was confirmed to be 3.0 mg/vial. The results suggest that LDPI can cover the milligram-order pulmonary administration of proteins/peptides. LDPIs are expected to have biopharmaceutical applications.

Method for Pulmonary Administration Using Negative Pressure Generated by Inspiration in Mice.

When developing inhaled medicines for respiratory diseases, such as chronic obstructive pulmonary disease, drugs need to be administered by pulmonary delivery to animals in non-clinical tests. Common methods require application of pressure during administration, and it may cause lung injury, so we focused on the inhalation of liquid medicines by mice themselves. This study aimed to evaluate a negative pressure method of pulmonary administration in mice by self-inhalation. First, to confirm the accuracy of delivery of liquid medicines into lungs and the potential for lung injury, Institute of Cancer Research (ICR) mice received methylene blue tetrahydrate or saline by the negative pressure method. We assessed drug distribution and usefulness of this method by administering porcine pancreatic elastase and all-trans-retinoic acid (ATRA) to mice. Consequently, we confirmed good distribution of the dye and no injury such as disruption of blood flow or destruction of alveoli in lungs of mice. Following production of the murine emphysema model, the mean linear intercept (Lm) was calculated as 78 ± 4 µm. Moreover, a significant therapeutic effect of administration of the ATRA was confirmed. These results suggest that this negative pressure method of administration may be useful for pulmonary administration in non-clinical tests.

Induction of Dbp by a histone deacetylase inhibitor is involved in amelioration of insulin sensitivity via adipocyte differentiation in ob/ob mice.

We previously reported that a histone deacetylase inhibitor (HDACi) increases D-site binding protein (Dbp) mRNA expression in adipose tissue and subsequently improved insulin sensitivity of obese (ob/ob) mice. However, the potential mechanism of this phenomenon was unclear. Thus, the aim of this study was to clarify the molecular mechanism involved in enhanced Dbp mRNA expression and improvement of insulin sensitivity in mice. Ob/ob mice were treated with HDACi every second day for 3 weeks. At the end of treatment, an insulin tolerance test was performed and epididymal adipose tissue obtained for fractionation into adipocytes and preadipocytes. HDACi improved insulin sensitivity in ob/ob mice and significantly increased Dbp mRNA in epididymal adipose tissue. Further, epididymal adipocytes of ob/ob mice showed a tendency towards a larger size distribution, while HDACi increased the proportion of smaller sized cells in fractionated preadipocytes. Dbp knocked-down 3T3-L1 cells down-regulated peroxisome proliferator-activated receptor-γ (PPAR-γ1) protein expression during adipogenesis, which suppressed adipocyte differentiation. These data indicate that DBP promotes adipocyte differentiation via direct up-regulation of PPAR-γ1 production in preadipocytes. In fractionated preadipocytes of ob/ob mice, DBP binding to the promoter region of the Ppar-γ gene and splicing variant of Ppar-γ (Ppar-γ1sv) mRNA expression were suppressed. HDACi significantly increased DBP binding to the Ppar-γ gene and Ppar-γ1sv transcription. Altogether, this indicates a modification in genetic regulation downstream from the circadian clock that can ameliorate an environmental function of adipose tissue, leading to improved insulin sensitivity in ob/ob mice.