Journal of Controlled Release 213 (2015) 112–119

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Pulmonary administration of phosphoinositide 3-kinase inhibitor is a curative treatment for chronic obstructive pulmonary disease by alveolar regeneration Michiko Horiguchi ⁎, Yuki Oiso, Hitomi Sakai, Tomoki Motomura, Chikamasa Yamashita ⁎ Department of Pharmaceutics and Drug Delivery, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

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Article history: Received 6 February 2015 Received in revised form 11 June 2015 Accepted 2 July 2015 Available online 6 July 2015 Keywords: Chronic obstructive pulmonary disease (COPD) Phosphoinositide 3-kinase (PI3K) Akt Wortmannin Alveolar regeneration

a b s t r a c t 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. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Emphysema and chronic bronchitis that cause respiratory failure due to destruction of alveolar structures are collectively referred to as chronic obstructive pulmonary disease (COPD), which is currently the fourth leading cause of death worldwide [1]. The number of patients with COPD reportedly exceeds 200 million in the world including the potential ones on the basis of epidemiological surveys by WHO (Global Alliance Against Chronic Respiratory Diseases (GARD)). However, no therapeutic drug is available for curative treatment of COPD, and the development of a therapeutic agent to repair alveolar destruction is of particular urgency. The targets of human lung alveolar remodeling are resident stem and progenitor cells in the lung that function in tissue repair and homeostasis. The adult lung consists of the following four major biologically distinct components: the trachea, bronchi, bronchioles, and alveoli. ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Horiguchi), [email protected] (C. Yamashita).

http://dx.doi.org/10.1016/j.jconrel.2015.07.004 0168-3659/© 2015 Elsevier B.V. All rights reserved.

Each component is biologically distinct and has its own stem and progenitor population [2–5]. Alveoli are terminal structures of distal airways specialized for gas exchange. The gaseous alveolar surface is lined by alveolar type I cells (AT-I) and alveolar type II cells (AT-II) [6]. Recent studies have characterized resident alveolar stem cells in human [7]. However, potent regenerative compounds have not been identified for human alveolar stem cells. We identified a differentiation-inducing agent for human alveolar epithelial stem cells responsible for the regeneration of pulmonary alveoli, and further studied its differentiation-inducing molecular mechanism. Based on this study, for first time, we reported that suppression of the phosphorylation of Ser/Thr kinase Akt leads to differentiation of human alveolar epithelial stem cells into AT-I and AT-II, which constitute pulmonary alveoli [8]. In this study, we focus on phosphoinositide 3-kinase (PI3K)-Akt signaling pathway. PI3K is categorized into class I, class II, and class III according to its primary structure and substrate specificity, with class I being involved in cellular survival and differentiation [9]. Class I PI3K is further divided into four subunits, α and β subunits involved in cellular survival, δ and γ subunits reported to be involved in inflammation [10,11]. While PI3K-δ and PI3K-γ have been reported

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to suppress inflammation in COPD [12,13], their differentiation-inducing effect on alveolar epithelial stem cells has yet to be elucidated. Thus, in this study, we focused on phosphoinositide 3-kinase (PI3K), which phosphorylates Akt, and examined whether PI3K inhibitors induced the differentiation of human alveolar epithelial stem cells. We further examined the effectiveness of the PI3K inhibitors on the repair of pulmonary alveoli and improvement of respiratory function in a murine COPD model. Pulmonary administration is a drug delivery system (DDS) that is superior to other methods of drug administration in delivering a drug to the lung. Previously, the efficacy of PI3K inhibitors has been studied primarily in oral administration, and no study has been reported on their pharmacological effects, including the representative antitumor effect, through pulmonary administration. This study is the first to report the effect of PI3K inhibitors on the lung when they are delivered by pulmonary administration. We demonstrated the alveolus-repairing effect of PI3K inhibitors in mice using a pulmonary administration method of breath-actuated inhalation, and found the possibility of an additional indication of PI3K inhibitors for COPD. 2. Materials and methods 2.1. Animals and cells Male ICR mice were purchased from Sankyo Labo Service Corporation (Tokyo, Japan). Animals were housed in a temperature-controlled (24 ± 1 °C) facility maintained on a light (12 h):dark (12 h) cycle with standard food available ad libitum. All animal procedures followed the guidelines established by the Animal Care and Use Committee of the Tokyo University of Science. Six-week-old male mice were anesthetized with isoflurane, and a solution of porcine pancreatic elastase (Elastin Products Company, Owensville, MO) (4.05 or 7.5 U/50 μl of saline) was administered intratracheally. We administered pulmonary administration of 0.2 mg/kg Wortmannin to elastase-induced COPD model mouse twice a week and evaluated the results using X-ray CT scanning or hematoxylin and eosin staining. Mice were sacrificed after 4 weeks. Human alveolar epithelial stem cells were provided by Dr. Hiroshi Kubo (Tohoku University, Sendai, Japan) [7] and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) and 20% ReproFF2 Medium (ReproCELL, Yokohama, Japan) in a humidified 5% CO2 atmosphere maintained at 37 °C. Experiments using human cells were conducted in accordance with the guidelines of the Research Ethics Committee of the Tokyo University of Science and Tohoku University. 2.2. Induction of differentiation to AT-I or AT-II cells To induce the differentiation of human alveolar epithelial cells to AT-I or AT-II cells, a culture system described previously was adopted with some modifications [14]. In brief, cells (5 × 105) at passages 3–6 were plated on cell culture inserts (BD Biosciences, Franklin Lakes, NJ) that had been coated with a mixture of 60% Matrigel (BD Biosciences, Franklin Lakes, NJ) and 40% rat tail collagen I (BD Biosciences, Franklin Lakes, NJ) in 5% FBS/E-MEM. After 4 h, media were changed to DMEM containing 5% FBS with or without Wortmannin, AS605240, PIK-75 Hydrochloride (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The media were changed every other day until analysis on day 7. The cultured cells were fixed with 4% paraformaldehyde in phosphate buffer for 15 min at room temperature. Samples were blocked using Blocker BSA (Thermo Fisher, Waltham, MA) for 30min at room temperature. Cells were then incubated with the following primary antibodies overnight at 4 °C: goat anti-human CD90 (Thy-1) polyclonal antibody, goat anti-human aquaporin-5 (AQP-5) polyclonal antibody, and goat anti-human SP-A monoclonal antibody (Santa Cruz Biotechnology, Billerica, MA). Alexa Fluor 488-conjugated anti-goat IgG (each

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at 1:500, Molecular Probes, Carlsbad, CA) were used as secondary antibodies. After mounting the samples and staining nuclei using ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) (Molecular Probes, Carlsbad, CA), samples were observed using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) and an A1Rsi confocal laser scanning microscope system (Nikon, Tokyo, Japan). By using image analysis software, ImageJ (NIH), the number of positive cells in immunostaining images at 20 × magnification was counted. Then the ratios of CD90, AQP-5, and SP-A positive cells were determined against the number of DAPI positive cells, i.e., the nuclear marker. Ten views were taken of each sample, and CD90, AQP-5, and SP-A positive cell numbers were measured for each sample, for a total of 500 DAPI positive cells. 2.3. Evaluation of the safety of Wortmannin Treatment dose-dependent cytotoxicity of Wortmannin on human alveolar epithelial stem cells was evaluated. Wortmannin was dissolved in DMSO, and the resulting solution was diluted with DMEM to give Wortmannin solutions at final concentrations of 1, 10, and 100 μM containing DMSO at a final concentration of 0.1%. Human alveolar epithelial stem cells were seeded at 1 × 104 cells/cm2, ATP derived from metabolically active cells was detected every 4 h for 24 h with the CellTiter-Glo™ Luminescent Cell Viability Assay kit (Promega, WI, USA), and the chemiluminescence signals were quantified with an ARVO (PerkinElmer, MA, USA). The proportion of viable cells was evaluated over time, using the cell viability at time 0 as 100%. To evaluate body weight changes depending on the Wortmannin dose, Wortmannin was administered twice a week via the pulmonary route to elastase-induced COPD model mice at doses of 0.05, 0.1, 0.2, and 0.4 mg/kg, and the body weight was measured 1, 2, and 3 weeks after starting the administration. 2.4. Immunohistochemical analysis Six-week-old male mice were anesthetized with isoflurane, and a solution of porcine pancreatic elastase (Elastin Products Company, Owensville, MO) (4.05 or 7.5 U/50 μl of saline) was administered intratracheally. After 4 weeks, the lung was fixed with 4% (w/v) paraformaldehyde in phosphate buffer, pH 6.9, and thereafter treated with Blocker BSA (Thermo Fisher, Waltham, MA) for 30 min at room temperature. Sliced lung tissues were incubated with the antibodies against p-Akt and Akt (1:200, Cell Signaling Technology, Inc., Boston, MA) at 4 °C for 12 h. After washing with cold PBS, cells were incubated with Alexa Fluor 546-conjugated anti-rabbit IgG (1:500, Santa Cruz Biotechnology, Billerica, MA) for 3 h. After mounting the samples and staining nuclei using ProLong Gold antifade reagent with DAPI (4′,6diamidino-2-phenylindole) (Molecular Probes, Carlsbad, CA), samples were observed using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) and an A1Rsi confocal laser scanning microscope system (Nikon, Tokyo, Japan). 2.5. Oral, intraperitoneal, and pulmonary administration methods Oral administration was conducted in the usual manner. Specifically, the tip of a gastric tube for oral administration (No. KN-348, for mouse, Natsume Seisakusho, Japan) was placed in the oral cavity of a retained mouse and inserted along the maxillary. Once the tip entered the stomach, the inner cylinder was pushed to inject the drug solution. Intraperitoneal administration was conducted in the usual manner. Specifically, while the mouse was retained, an injection needle (NN-2613S, 26G, Terumo Corporation, Japan) was inserted subcutaneously to an approximate depth of 5 mm in the lower abdomen slightly left to the midline. The needle was then let stand upright and proceed to the peritoneal cavity, in which the drug solution was injected.

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Pulmonary administration was carried out with a gastric tube for oral administration (No. KN-348, for the mouse, Natsume Seisakusho, Japan) equivalent in diameter to the mouse airway. Using the Mouse Intubation Platform-Model MIP (Penn-Century, PA, USA) as a mouse retainer for pulmonary administration, front teeth of the mouse were retained in the retaining position approximately at 90° to facilitate the tracheal access of the tube. The airway was confirmed with the Small Animal Laryngoscope-Model LS-2 (Penn-Century, PA, USA) as a tracheal endoscope for the mouse, the tube was inserted to the airway, and the drug solution was administered in synchronization with the air intake of the mouse.

2.6. Evaluation of effects of differences in the administration method on drug distribution and pharmacological effect The drugs were administered by oral, intraperitoneal, and pulmonary methods to 5 and 6-week-old male ICR mice (Sankyo Labo Service Corporation, Japan). To evaluate effects of differences in the administration methods on drug distribution, 50 μl of a 0.9 μg/μl solution of indocyanine green (ICG) in saline was respectively administered to the mice using each administration method, and the distribution image of the drug administered was photographed 10 min after the administration with the in vivo photo imager Clairvivo OPT plus (Shimadzu, Japan). Effects of differences in the administration methods on pharmacological effects were verified by evaluating the extent of lung damage after administrating elastase, an enzyme that induces pulmonary damages by different methods. Elastase (Elastin Products Company, MO, USA) was administered to 6-week-old male ICR mice as a 50-μl solution containing 7.5 U in saline. After 2 weeks, the mice were analyzed with the animal X-ray CT scanner Latheta LCT-200 (Hitachi ALOKA, Japan), and low attenuation area (LAA) from − 871 to − 610 HU, which represents the area of lung damages, was computed using a standard analysis software pre-installed in the CT scanner. Moreover, 3D images of the damaged areas of the lung were created with the 3D image processing software Amira version 5.4.0 (Maxnet, Japan).

2.7. Pulmonary physiology and histology Six-week-old male mice were anesthetized with isoflurane, and a solution of porcine pancreatic elastase (Elastin Products Company, Owensville, MO) (4.05 U/50 μl of saline) was administered intratracheally. We administered pulmonary administration of 0.2 mg/kg Wortmannin to elastase-induced COPD model mouse twice a week and evaluated the results using hematoxylin and eosin staining. Mice were sacrificed after 4 weeks. Tissue sections were stained with hematoxylin and eosin. The mean linear intercept (Lm), an indicator of air space size, was calculated for each mouse from 5 randomly selected fields (n = 6 mice × 5 selected fields).

2.8. Computed tomography Mice were anesthetized with isoflurane and were placed in the chamber of a Latheta LCT-200 computed tomography (CT) scanner for small animals (Aloka, Tokyo, Japan). The CT scanner was calibrated according to the manufacturer's recommendations. CT scans were performed at 192-μm intervals (100 slices), and image acquisition was respiratory-gated. Images captured between the apex and base of the lung used for quantitative assessment with Latheta software v.3.2. ROI was sated − 200 to − 1000 for analyzing of average CT level. CT scans were performed in accordance with the guidelines established by the X-ray Care and Use Committee of the Tokyo University of Science.

2.9. Assessment of lung function Lung function in mice was evaluated with the flexiVent system (flexiVent™ SCIREQ©, Montreal, QC, Canada). The flexiVent™ is a device to evaluate the pulmonary function in an animal based on the pneumodynamics. The flexiVent™ system equips a precision piston pump and a pressure control exercise computer, and is capable of measuring the pneumodynamics accurately and reproducibly. Using flexiVent™, it is possible to evaluate the respiratory capacity per unit time, which is similar to the forced expiratory volume in 1 s/forced vital capacity ratio clinically used as an index of the elasticity and respiratory function of the lung, as well as measurement of airway resistance and compliance. In COPD, the elasticity of lung tissues is reduced due to emphysematous changes of the lung, and the breathing capacity per unit time declines. The effectiveness of PI3K inhibitors for COPD is evaluated by measuring the elasticity and respiratory capacity of the lung using flexiVent™. The associated protocols adhered to the manufacturer's instructions. We administered pulmonary administration of 0.2 mg/kg Wortmannin to elastase-induced COPD model mouse twice a week and evaluated the results using flexiVent system. Ventilator rates of 200/min, tidal volume of 10 ml/kg with positive end-expiratory pressures of 3 cm H2O and a pressure limit of 30 cm H2O were used. 2.10. Statistics For each parameter measured, the values from individual samples were averaged, and the standard error (S.E.) was calculated. Data were compared using the unpaired t-test. Differences among groups were assessed for significance using analysis of variance (ANOVA) and post-hoc Bonferroni comparisons. A 5% probability was considered significant. 3. Results First, we tested three PI3K inhibitors (AS605240, PIK-75 hydrochloride, Wortmannin) to evaluate their differentiation-inducing effect on human alveolar epithelial stem cells. AS605240 and PIK-75 hydrochloride are inhibitors highly specific to PI3K-γ and PI3K-α [15,16], respectively, whereas Wortmannin is a PI3K inhibitor with low substrate specificity. The differentiation-inducing effects of the three PI3K inhibitors on human alveolar epithelial stem cells were evaluated by counting cells positive for CD90 as a specific marker of human alveolar epithelial stem cells [7,17], aquaporin-5 (AQP-5) as an AT-I-specific marker, and surfactant protein-A (SP-A) as an AT-II-specific marker. The results indicated that all inhibitors induced differentiation into AT-I and A-II in an inhibitor concentration- and treatment time-dependent manner (Fig. 1). When cultured with 10 μM Wortmannin for 6 days, 48.8% and 32.2% of the cells were differentiated into AT-I and AT-II, respectively (Fig. 1B); culture with 10 μM AS605240 for 6 days resulted in differentiation into AT-I and AT-II with percentages of 16.8% and 16.6%, respectively (Fig. 1D); and culture with 10 μM PIK-75 hydrochloride for 6 days induced the differentiation into AT-I and AT-II by 12.8% and 14.0%, respectively (Fig. 1F). From these results, Wortmannin demonstrated to have the most potent differentiation-inducing activity on human alveolar epithelial stem cells (Fig. 1G). Moreover, the differentiationinduction effect of Wortmannin was characterized by a higher proportion of AT-I-differentiated cells than that of AT-II, whereas AS605240 and PIK-75 hydrochloride induced differentiation to AT-I and AT-II nearly equally (Fig. 1G). In evaluation of cytotoxicity of Wortmannin on human alveolar epithelial stem cells, treatment of the cells with 1 μM or 10 μM Wortmannin showed no significant cytotoxicity compared to the control group (Fig. 2). On the other hand, significant cytotoxicity was observed when the cells were treated with 100 μM Wortmannin (Fig. 2). In evaluation of the dose-dependent effects of Wortmannin on the body weight

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Fig. 1. PI3K-inhibitor induces alveolar epithelial stem cell differentiation. Representative morphologic appearances of cells cultured on a mixture of Matrigel and rat tail collagen with representative CD90 in alveolar epithelial stem cells, AQP-5 in AT-I cells, SP-A in AT-II cells, and DAPI nucleic acid stain. Dose-dependent increase in the differentiation profile of AT-I and AT-II cells cultured from human alveolar epithelial stem cells following 7 days of treatment with (A) Wortmannin, (C) AS605240, (E) PIK-75 hydrochloride. Each value represents the mean ± S.E. (n = 4–6). Temporal differentiation profile of AT-I and AT-II cells cultured from human alveolar epithelial stem cells after treatment with 10 μM of (B) Wortmannin, (D) AS605240, (F) PIK-75 Hydrochloride. Each value represents the mean ± S.E. (n = 4–6). Statistical analyses demonstrated that Wortmannin had significantly higher AT-I and AT-II differentiationinducing activities than AS605240 and PIK-75 hydrochloride (G).

decrease in elastase-induced COPD model mice, no significant body weight changes were observed after pulmonary administration of Wortmannin at any doses tested between 0.05 and 0.2 mg/kg (Fig. 5). In the lung tissue of patients with COPD, severity-dependent increase of Akt phosphorylation has been reported (13). Therefore, we measured the level of phosphorylated Akt (p-Akt) and the expression level of Akt to determine whether the PI3K-Akt signaling is activated in an elastase-induced murine COPD model, which is the representative COPD model used in this study. After pulmonary administration of 4.05 U of elastase twice a week for 4 weeks, the lung tissue was excised and subjected to immunostaining using anti-p-Akt (Fig. 3A) and anti-Akt (Fig. 3B) antibodies, and 4′,6-diamidino-2-phenylindole

(DAPI) for nuclear labeling. The result showed that Akt phosphorylation was elevated in the murine COPD model (Fig. 3). The results from evaluation of the effects of differences in the administration methods on drug distribution indicated that the orally administered drug was distributed in the oral cavity and around the stomach (Fig. 4A-a), and the intraperitoneally administered drug was distributed over a wide range of the abdomen (Fig. 4B-a). In contrast, the drug administered via the pulmonary route was localized in the oral cavity and lung (Fig. 4C-a). The effects of differences in the administration methods on the pharmacological activity were evaluated. Elastase, an enzyme that induces pulmonary damages, was administered orally and intraperitoneally, and X-ray CT analysis of the damaged lung areas

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Fig. 2. Evaluation of cytotoxicity of Wortmannin. The proportion of viable cells over time in human alveolar epithelial stem cells exposed to 1, 10, and 100 μM Wortmannin is shown. The proportion of viable cells was computed using the cell viability at the start of the test (time 0) as 100%.

(shown in red) 2 weeks later revealed nearly no damages in the lung (Fig. 4A-b and 4B-b). In the case of pulmonary elastase administration, the lung was damaged extensively (Fig. 4C-b), and LAA%, which represents the affected area in the lung, was significantly greater in the pulmonary administration group compared to that in the oral and intraperitoneal administration groups (Fig. 4D). From these results, pulmonary administration was demonstrated to be very effective for evaluation of the drug effect in the lung. Therefore, this murine COPD model was used to evaluate the repair effect of Wortmannin on pulmonary alveoli. Murine COPD model prepared by pulmonary administration of 4.05 U or 7.5 U of elastase twice a week underwent pulmonary administration of 0, 0.05, 0.1, 0.2, or 0.4 mg/kg of Wortmannin twice a week, followed by lung imaging

Fig. 3. Quantification of phosphorylated Akt in elastase-induced COPD model mouse. Sixweek-old male mice were anesthetized with isoflurane, and a solution of porcine pancreatic elastase (4.05/50 μl of saline) was administered intratracheally. After 4 weeks, the lung was fixed with 4% (w/v) paraformaldehyde in phosphate buffer for 30 min at room temperature. Slice lung tissues were incubated with the antibodies against p-Akt and Akt. Samples were observed using a BZ-9000 fluorescence microscope and an A1Rsi confocal laser scanning microscope system by lens of ×20. Scale bar = 50 μm.

Fig. 4. Evaluation of pulmonary delivery of the drug administered by different methods. The distribution and pulmonary effect of the drugs following (A) oral administration, (B) intraperitoneal administration, and (C) pulmonary administration were assessed. For distribution after administration of an indocyanine green solution, in vivo imaging pictures after (A-a) oral administration, (B-a) intraperitoneal administration, and (C-a) pulmonary administration are shown. The scale bar is 20 mm in length. For distribution of LAA (red) in the lung, which represents a damaged lung area, 2 weeks after (A-b) oral administration, (B-b) intraperitoneal administration, and (C-b) pulmonary administration of elastase, 3D image pictures created with 3D analysis software Amira using LAA computed by the X-ray CT scanner are shown. (D) Proportions of LAA, which represents a damaged lung area, calculated 2 weeks after oral administration, intraperitoneal administration, and pulmonary administration are shown.

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with an X-ray CT scanner for small animals three weeks after the initial Wortmannin administration. Four weeks after the initial Wortmannin administration, the lung tissue was excised and stained with hematoxylin–eosin. As a result, the linear intercept of the alveolar airspace, which is indicative of the severity of the alveolar damages, significantly decreased in the 0.2 and 0.4 mg/kg Wortmannin-treated groups compared to the control group. However, a decrease in weight was also observed in the 0.4 mg/kg Wortmannin-treated group; and thus, the Wortmannin dose was set to 0.2 mg/kg (Fig. 5). The linear intercept of the alveolar airspace was 72.8 μm in the control group and that of the 0.2 mg/kg Wortmannin-treated group was 38.7 μm, demonstrating recovery to a similar level of that observed in normal mice, which was 37.3 μm (Fig. 6). Similarly, in the evaluation of the X-ray CT lung images, average CT values of the Wortmannin-treated group increased to − 405.4 HU, which was significantly higher than that of the control group (− 488.3 HU), demonstrating the recovery to a similar level of that observed in normal mice (−378.1 HU) (Fig. 7A). Furthermore, in the histogram based on the X-ray CT analysis, the Wortmannin-treated group showed a decrease in the low-absorption region, indicating a recovery to a similar level of that observed in normal mice (Fig. 7B). Then, we evaluated whether Wortmannin recovered the respiratory functions of murine COPD model using a high-performance respiratory function analysis system (flexiVentTM). As a result, tissue elastance, which indicates the elasticity and surface tension of the lung, significantly increased in the Wortmannin-treated group (14.3 cm H2O/mL) compared with that of the control group (7.0 cm H2O/mL), indicating recovery of the contraction/expansion function of the lung (Fig. 8A). Furthermore, the ratio of forced expiratory volume in 0.05 s (FEV0.05) against forced vital capacity (FVC), which is a respiratory function index, increased to 73.3% in the Wortmannin-treated group, which was significantly higher than that of the control group (46.3%), demonstrating the recovery of respiratory function (Fig. 8B).

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Fig. 6. Wortmannin treatment abrogates elastase-induced COPD model mouse. Lung sections and average of airspace from 0.2 mg/kg Wortmannin and control treated at 3 weeks after the administration of Wortmannin were stained with hematoxylin and eosin. Scale bar = 50 μm. Data represent the mean ± S.E. (n = 6 mice × 5 selected fields), *P b 0.05 for between-group comparisons.

evaluated with systemic administration rather than local administration to the lung, and evaluation with local pulmonary delivery systems, like this study, may become increasingly more important in the future.

4. Discussion To the best of our knowledge, our results are the first to demonstrate a high potential of PI3K inhibitors as a novel class of therapeutic agents for regenerating pulmonary alveoli by inducing the differentiation of human alveolar epithelial stem cells. The results from evaluation of the distribution and pulmonary pharmacological effect of the drugs administered via pulmonary, oral, and intraperitoneal routes demonstrated the effectiveness of pulmonary administration as a means for local drug delivery to the lung, and proved pulmonary administration to be very effective as a DDS strategy for lung diseases. Drug effects for pulmonary diseases have been

Fig. 5. Effects of Wortmannin on the body weight decrease. Wortmannin was administered twice a week via the pulmonary route to elastase-induced COPD model mice at 0.05, 0.1, 0.2, and 0.4 mg/kg, and the mean body weights measured 1, 2, and 3 weeks after starting the administration are shown.

Fig. 7. Wortmannin treatment abrogates elastase-induced COPD model mouse. (A) Representative CT images from 0.2 mg/kg Wortmannin and control treated (saline containing 5% EtOH) lungs after 2 weeks in a mouse model of elastase-induced COPD. Bars represent the mean ± S.E. *P b 0.05 for between-group comparisons. (B) Representative histogram from 0.2 mg/kg Wortmannin and control treated (saline containing 5% EtOH) lungs after 2 weeks in a mouse model of elastase-induced COPD. Bars represent the mean ± S.E. (n = 6).

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pulmonary alveoli regenerating effect on human alveolar epithelial stem cells has been demonstrated, and screening for a therapeutic agent against COPD needs to be performed. The high alveolusregenerating effect of Wortmannin revealed in this study could potentially serve for the development of innovative therapeutic agents for COPD, which has been considered incurable. 5. Conclusions PI3K inhibitors were found to induce the differentiation of human alveolar epithelial stem cells, and Wortmannin, which was the most potent differentiation inducer, was shown to indeed repair pulmonary alveoli and recover respiratory function in murine COPD model. Wortmannin was novel curative treatment of pulmonary alveolar destruction in COPD. Fig. 8. Effect of Wortmannin on airway function in COPD model mouse. (A) Those tissue elastances were determined after 2 weeks 0.2 mg/kg Wortmannin treatment in elastase-induce COPD mouse model. (B) The FEV0.05/FVC was determined after 2 weeks 0.2 mg/kg Wortmannin treatment in elastase-induce COPD mouse model. Bars represent the mean ± S.E. (n = 6). *P b 0.05 for between-group comparisons.

It was beyond our expectation that Wortmannin characterized by low PI3K subtype specificity, would show the most potent differentiationinducing activity. When cultured with 10 μM Wortmannin for 6 days, 48.8% and 32.2% of the cells were differentiated into AT-I and AT-II, respectively (Fig. 1B); culture with 10 μM AS605240 for 6 days resulted in differentiation into AT-I and AT-II with percentages of 16.8% and 16.6%, respectively (Fig. 1D); and culture with 10 μM PIK-75 hydrochloride for 6 days induced the differentiation into AT-I and AT-II by 12.8% and 14.0%, respectively (Fig. 1F). From these results, Wortmannin demonstrated to have the most potent differentiation-inducing activity on human alveolar epithelial stem cells. Given that Wortmannin acts on not only PI3K class I, but also class II and class III, as well as PI4K, MLCK, and PLD [18–21], it is required to elucidate how these target molecules contribute to differentiation induction of human alveolar epithelial stem cells in the future. Wortmannin did not show cytotoxicity on human alveolar epithelial stem cells up to 10 μM, a concentration at which the differentiationinducing effect was observed, and did not induce body weight changes in elastase-induced COPD model mice at a dose of 0.2 mg/kg, at which the alveolus-repairing effect was observed. These results suggested the safety of Wortmannin at a dose showing the alveolus-repairing effect. However, cytotoxicity was observed when human alveolar epithelial stem cells were treated with the high concentration of Wortmannin (100 μM), and therefore, Wortmannin may have toxicity at a high dose. In this study, local delivery of the drug to the lung by pulmonary administration not only enhanced the alveolus-repairing effect but also potentially helped reduce the systemic toxicity. The Global Initiative for Chronic Obstructive Lung Disease (GOLD) provided guidelines on the diagnosis and management of stable COPD. The COPD grades of GOLD are associated with a postbronchodilator forced expiratory volume (FEV) to forced vital capacity (FVC) ratio ≤ 0.70, indicating that airway obstruction is only partially reversible. These guidelines define four grades of COPD that stratify severity based on spirometry measurement of FEV in 1 s (FEV1) as a percentage of that predicted. In the case of mouse, FEV in 0.05 s (FEV0.05) was a measure of lung function. We evaluated whether Wortmannin recovered the respiratory functions of murine COPD model. The ratio of forced expiratory volume in 0.05 s (FEV0.05) against forced vital capacity (FVC), which is a respiratory function index, increased to 73.3% in the Wortmannin-treated group, which was significantly higher than that of the control group (46.3%), demonstrating the recovery of respiratory function (Fig. 8B). These results have shown that Wortmannin is an excellent curative treatment of COPD. Currently, various PI3K inhibitors are being developed as antitumor agents [22]. However, neither their differentiation-inducing effect nor

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