Heterogenous Intrapulmonary Distribution of Aerosolized Model Compounds in Mice with Bleomycin-Induced Pulmonary Fibrosis.

Background: A distinctive pathological feature of idiopathic pulmonary fibrosis (IPF) is the aberrant accumulation of extracellular matrix components in the alveoli in abnormal remodeling and reconstruction following scarring of the alveolar structure. The current antifibrotic agents used for IPF therapy frequently result in systemic side effects because these agents are distributed, through the blood, to many different tissues after oral administration. In contrast to oral administration, the intrapulmonary administration of aerosolized drugs is believed to be an efficient method for their direct delivery to the focus sites in the lungs. However, how fibrotic lesions alter the distribution of aerosolized drugs following intrapulmonary administration remains largely unknown. In this study, we evaluate the intrapulmonary distribution characteristics of aerosolized model compounds in mice with bleomycin-induced pulmonary fibrosis through imaging the organs and alveoli. Methods: Aerosolized model compounds were administered to mice with bleomycin-induced pulmonary fibrosis using a Liquid MicroSprayer®. The intrapulmonary distribution characteristics of aerosolized model compounds were evaluated through several imaging techniques, including noninvasive lung imaging using X-ray computed tomography, ex vivo imaging using zoom fluorescence microscopy, frozen tissue section observation, and three-dimensional imaging with tissue-clearing treatment using confocal laser microscopy. Results: In fibrotic lungs, the aerosolized model compounds were heterogeneously distributed. In observations of frozen tissue sections, model compounds were observed only in the fibrotic foci near airless spaces called honeycombs. In three-dimensional imaging of cleared tissue from fibrotic lungs, the area of the model compound in the alveolar space was smaller than in healthy lungs. Conclusion: The intrapulmonary deposition of extracellular matrix associated with pulmonary fibrosis limits the intrapulmonary distribution of aerosolized drugs. The development of delivery systems for antifibrotic agents to improve the distribution characteristics in fibrotic foci is necessary for effective IPF therapy.

Development of a Compensated Förster Resonance Energy Transfer Imaging for Improved Assessment of the Intrapulmonary Distribution of Polymeric Nanoparticles.

Inhalation-based drug delivery systems have gained attention as potential therapeutic options for various respiratory diseases. Among these systems, nanoparticles are being explored as drug carriers because of their ability to deliver therapeutic agents directly to the lungs. It is essential to accurately evaluate the intrapulmonary behavior of nanoparticles to optimize drug delivery and achieve selective targeting of lung lesions. Prior research used the Förster resonance energy transfer (FRET) phenomenon to study the in vivo behavior of nanoparticles as drug carriers. In this study, image reconstruction involving bleed-through compensation was used to quantitatively assess the behavior of FRET nanoparticles in the lungs. When the nanoparticles for FRET fluorescence imaging, which employed 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) as the donor and as 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine iodide (DiR) the acceptor, were administered to mouse lungs, whole-body in vivo imaging could not compensate for the influence of respiration and heartbeat. However, ex vivo imaging of excised lungs enabled the quantitative evaluation of the time-concentration profiles and distribution of nanoparticles within the lungs. This imaging technique is particularly useful for the development of inhalable nanoparticles that specifically target the lesions and exhibit controlled-release capabilities within the lungs.

Development of visualization and analysis methods for evaluating intratumoral nanoparticle kinetics for tumor-targeted drug delivery using Förster resonance energy transfer in vivo live imaging and tissue clearing techniques.

In this study, the imaging methods for evaluating the kinetics of nanoparticles as drug delivery systems in tumor tissues were improved in BxPC3 tumor-bearing mice. First, Förster resonance energy transfer (FRET) live imaging was selected to quantitatively evaluate nanoparticle kinetics in the tumor tissue of mice. Briefly, and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine iodide (as an acceptor)-and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (as a donor)-coloaded nanoparticles were administered intravenously to the mice, and imaging was performed using a fluorescence in vivo imager. The fluorescence intensities of images were acquired in the FRET, donor, and acceptor channels, and the nanoparticle kinetics in the tumor region was quantified by compensating for bleed-through. Second, in the cleared tumor tissue of mice, the difference in evaluation properties between the two- and three-dimensional visualization of the nanoparticles was examined. In brief, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-loaded nanoparticles were intravenously administered to the mice after fluorescently labeled tomato lectin treatment to visualize tumor vessels. Excised tumor tissue was cleared and observed using laser-scanning confocal microscopy, and three-dimensional images were reconstructed. The three-dimensional minimum distances traveled by DiI from the tumor vessels were calculated using information about the two-dimensional distance and the slicing position using the Pythagoras theorem. These imaging techniques should facilitate the development of drug delivery systems for cancer.

Improvement of the pharmacokinetics and antifibrotic effects of nintedanib by intrapulmonary administration of a nintedanib-hydroxypropyl-γ-cyclodextrin inclusion complex in mice with bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic lung disease that is characterized by progressive abnormal reprogramming following injury of the pulmonary structure. In this study, we prepared a nintedanib (antifibrotic agent) and cyclodextrin (CyD) inclusion complex to improve the pharmacokinetics and antifibrotic effects of nintedanib following intrapulmonary administration. Hydroxypropyl-γ-CyD (HP-γ-CyD) enhanced the solubility of nintedanib without cytotoxic effects on WI-38 cells (lung fibroblasts) and NCI-H441 cells (alveolar epithelium model). Compared with nintedanib ethanesulfonate salt, the nintedanib-HP-γ-CyD inclusion complex exhibited prolonged distribution in the lungs following intrapulmonary administration in mice with bleomycin-induced pulmonary fibrosis. In addition, compared with nintedanib ethanesulfonate salt, the nintedanib-HP-γ-CyD inclusion complex exhibited higher stability in the bronchoalveolar lavage fluid and lower permeability in NCI-H441 cell monolayers. These results suggested that the inclusion complexation of nintedanib into HP-γ-CyD improved its pharmacokinetics following intrapulmonary administration by increasing its stability in the lungs and reducing its permeability through the alveolar cell membrane. Intrapulmonary administration of the nintedanib-HP-γ-CyD inclusion complex significantly reduced the intrapulmonary hydroxyproline content and limited pathological fibrotic changes. Overall, this study indicates that antifibrotic agent-CyD inclusion complexation intended for intrapulmonary administration can be used to prolong distribution in the lungs and lead to the expansion of idiopathic pulmonary fibrosis therapy.

Aerosolized Liposomal Formulation Prevents Enhanced Drug Transfer from Fibrotic Lungs to the Systemic Circulation.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive respiratory disease characterized by the destruction of the alveolar structure. In pulmonary fibrosis, aerosolized drugs are easily transferred to the systemic circulation via leakage through the injured alveolar epithelium. Therefore, pulmonary drug delivery systems for sustained distribution in fibrotic lungs are needed. OBJECTIVE: We evaluated the intrapulmonary pharmacokinetics of aerosolized liposomes as pulmonary drug delivery systems in mice with bleomycin-induced pulmonary fibrosis. METHODS: The aerosolized liposomal formulations and solutions of model compounds, including indocyanine green and 6-carboxyfluorescein (6-CF), were intrapulmonary administered to mice with bleomycin-induced pulmonary fibrosis. In vivo imaging for indocyanine green and 6-CF measurements in lung tissues and plasma were performed. Additionally, in vitro permeation experiments using NCI-H441 cell monolayers as a model of alveolar epithelial cells were performed. RESULTS: The fluorescence signals of indocyanine green following the administration of liposomal formulations were observed longer in the lungs than those in solution-treated mice. Compared with the solution, the 6-CF concentrations in lung tissues after the administration of liposomal formulations were determined higher, whereas those in the plasma were lower. 6-CF permeability was significantly increased by transforming growth factor-ß1 in NCI-H441 cell monolayers treated with the solution but unchanged in the presence of the liposomal formulation. CONCLUSION: The aerosolized liposomal formulation can prevent enhanced drug transfer from fibrotic lungs into the systemic circulation via the injured alveolar epithelium. This system may be useful for the sustained distribution of anti-fibrotic agents in fibrotic lungs and the optimization of IPF therapy.

Evaluation of tissue-clearing techniques for intraorgan imaging of distribution of polymeric nanoparticles as drug carriers.

OBJECTIVE: The development of drug delivery systems using nanocarriers requires intraorgan imaging techniques for evaluating the distribution of nanocarriers. In this study, we evaluated the tissue-clearing techniques for the imaging of polymeric nanoparticles, a nanocarrier, in the liver used as a model of pigment-rich organ in mice. SIGNIFICANCE: The intraorgan imaging method of polymeric nanoparticles was examined without sectioning of organ samples for evaluating the delivery efficiency in preclinical studies. METHODS: DiI-loaded polymeric nanoparticles and fluorescence-tagged tomato lectin for fluorescence labeling of liver general structures were intravenously administered to mice. Tissue-clearing treatment of the mouse liver was performed using ClearT2, ScaleSQ(0), clearing agent comprising fructose, urea, and glycerol for imaging (FUnGI), clear unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and modified CUBIC techniques. Intraorgan fluorescence imaging in the liver was performed by confocal laser microscopy. RESULTS: ClearT2 treatment exhibited insufficient clearing capability in the mouse liver. Although CUBIC treatment exhibited the best clearing capability, the CUBIC caused DiI leakage. ScaleSQ(0), FUnGI, and modified CUBIC treatments exhibited better clearing capability than ClearT2 technique while preserving the DiI. In the fluorescence imaging, the CUBIC and modified CUBIC exhibited deeper visualization than with the ScaleSQ(0) and FUnGI; however, the CUBIC led to a change in DiI distribution. The modified CUBIC enabled the deepest visualization while preserving the distribution of DiI. CONCLUSION: The intraorgan imaging method was established using modified CUBIC technique by the intravenous administration of fluorescence-tagged tomato lectin for evaluating the distribution of polymeric nanoparticles in mouse pigment-rich organs.

Prolonged distribution of aerosolized PEGylated liposomes in the lungs of mice with bleomycin-induced pulmonary fibrosis.

OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic lung disease characterized by abnormal remodeling of the lung parenchyma with subsequent scarring of the alveolar structure. In this study, we examined the distribution characteristics of aerosolized polyethylene glycol (PEG)ylated liposomes in the lungs of mice with bleomycin-induced pulmonary fibrosis. SIGNIFICANCE: The present study details the utility of aerosolized PEGylated liposomes for improving intrapulmonary pharmacokinetics in fibrotic lungs. METHODS: Aerosolized PEGylated liposomes were administered to fibrotic mouse lungs using a MicroSprayer. Intrapulmonary pharmacokinetics was evaluated via in vivo imaging, measurement of liposome concentrations in bronchoalveolar lavage fluid (BALF) and alveolar macrophages (AMs), and observation of lung tissue sections. In addition, in vitro accumulation experiments using WI-38, A549, and RAW264.7 cells were performed. RESULTS: The decrease of the fluorescence intensity of the PEGylated liposomes was slower than that of the non-modified liposomes. Compared with the non-modified liposomes, the PEGylated liposomes were determined higher in BALF, whereas those in the AMs were lower. Both PEGylated and non-modified liposomes were widely dispersed in fibrotic regions in tissue sections. No difference in accumulation in WI-38 and A549 cells was noted between PEGylated and non-modified liposomes, whereas the PEGylated liposomes exhibited lower intracellular accumulation than non-modified liposomes in RAW264.7 cells. CONCLUSION: Aerosolized drug delivery systems using PEGylated liposomes exhibited prolonged distribution in both healthy and fibrotic mouse lungs. PEGylated liposomes were determined to be efficient drug delivery systems for anti-fibrotic agents targeting lung fibroblasts and alveolar epithelial cells for optimizing the treatment of IPF.

Multiscale Live Imaging Using Förster Resonance Energy Transfer (FRET) for Evaluating the Biological Behavior of Nanoparticles as Drug Carriers.

To develop targeted drug delivery systems using nanoparticles for treating various diseases, the evaluation of nanoparticle behavior in biological environments is necessary. In the present study, the biological behavior of polymeric nanoparticles was directly traced in living mice and cells. The dissociation of nanoparticles was detected by Förster resonance energy transfer (FRET) imaging. DiR and DiD were encapsulated in the nanoparticles for near-infrared FRET imaging, and they were traced using in vivo FRET imaging and intravital FRET imaging at the whole-body and tissue scales, respectively. In vivo FRET imaging revealed that the nanoparticles dissociated over time following intravenous administration. Intravital FRET imaging revealed that the nanoparticles dissociated in the liver and blood vessels following intravenous administration. DiI and DiO were encapsulated in nanoparticles for FRET imaging using confocal microscopy, and they were traced using in vitro FRET imaging in HepG2 cells. In vitro FRET imaging revealed that the nanoparticles dissociated and released fluorescent dyes that distributed in the cell membrane. Finally, live imaging was performed using FRET at the whole-body, tissue, and cellular scales. This method is suitable for obtaining information regarding the biological kinetic properties of nanoparticles and their use in targeted drug delivery.

Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix.

Idiopathic pulmonary fibrosis is a progressive, chronic lung disease characterized by the accumulation of extracellular matrix proteins, including collagen and elastin. Imaging of extracellular matrix in fibrotic lungs is important for evaluating its pathological condition as well as the distribution of drugs to pulmonary focus sites and their therapeutic effects. In this study, we compared techniques of staining the extracellular matrix with optical tissue-clearing treatment for developing three-dimensional imaging methods for focus sites in pulmonary fibrosis. Mouse models of pulmonary fibrosis were prepared via the intrapulmonary administration of bleomycin. Fluorescent-labeled tomato lectin, collagen I antibody, and Col-F, which is a fluorescent probe for collagen and elastin, were used to compare the imaging of fibrotic foci in intact fibrotic lungs. These lung samples were cleared using the ClearT2 tissue-clearing technique. The cleared lungs were two dimensionally observed using laser-scanning confocal microscopy, and the images were compared with those of the lung tissue sections. Moreover, three-dimensional images were reconstructed from serial two-dimensional images. Fluorescent-labeled tomato lectin did not enable the visualization of fibrotic foci in cleared fibrotic lungs. Although collagen I in fibrotic lungs could be visualized via immunofluorescence staining, collagen I was clearly visible only until 40 µm from the lung surface. Col-F staining facilitated the visualization of collagen and elastin to a depth of 120 µm in cleared lung tissues. Furthermore, we visualized the three-dimensional extracellular matrix in cleared fibrotic lungs using Col-F, and the images provided better visualization than immunofluorescence staining. These results suggest that ClearT2 tissue-clearing treatment combined with Col-F staining represents a simple and rapid technique for imaging fibrotic foci in intact fibrotic lungs. This study provides important information for imaging various organs with extracellular matrix-related diseases.

Intratumoral delivery and therapeutic efficacy of nanoparticle-encapsulated anti-tumor siRNA following intrapulmonary administration for potential treatment of lung cancer.

This study evaluated the delivery efficiency and antitumor effects of the intrapulmonary administration of antitumor small interfering ribonucleic acid (siRNA)-containing nanoparticles to mice with metastatic lung tumor. Fluorescence-labeled, siRNA-containing nanoparticles were administered using Liquid MicroSprayer® to mice with metastatic lung tumors induced by the murine melanoma cell line B16F10. Fluorescent signals in the whole lung and in the tumor region following the intrapulmonary administration of siRNA-containing nanoparticles were stronger than those following intravenous administration. The intrapulmonary administration of nanoparticles containing a mixture of siRNA against MDM2, c-Myc, and vascular endothelial growth factor (VEGF) significantly improved survival and prolonged the survival of mice with metastatic lung tumor. In addition, after the intrapulmonary or intravenous administration of the mixture, the activity levels of interleukin-6 and -12, markers of systemic toxicity, were similar to those of nontreatment. These results indicate that the antitumor siRNA-containing nanoparticles were delivered efficiently and specifically to tumor cells, effectively silencing the oncogenes in the lung metastasis without any significant systemic toxicity.

Evaluation of various tissue-clearing techniques for the three-dimensional visualization of liposome distribution in mouse lungs at the alveolar scale.

PURPOSE: To develop a three-dimensional visualization method for evaluating the distribution of pulmonary drug delivery systems and compare four tissue-clearing techniques (ClearT2, CUBIC, ScaleS, and SeeDB2) using intrapulmonary liposomes as drug carriers. METHODS: Rhodamine B-labeled liposomes were administered intrapulmonarily to mice using a MicroSprayer, and then fluorescent-labeled tomato lectin was administered intravenously to visualize the general lung structure. Tissue-clearing treatment of the mouse lungs was performed using the standard protocols of the ClearT2, CUBIC, ScaleS, and SeeDB2 techniques. Lung clearing was clarified using laser-scanning confocal microscopy, and three-dimensional images were reconstructed. RESULTS: Fluorescent-labeled tomato lectin was preserved using ClearT2 and SeeDB2 but not using CUBIC and ScaleS. In addition, the liposomes were stable in ClearT2 reagent, but they were mostly degraded in other reagents by surface-active agents. ClearT2 treatment enabled the three-dimensional visualization of intrapulmonary rhodamine B-labeled liposomes at the alveolar scale. CONCLUSIONS: These results suggest that the ClearT2 tissue-clearing technique was appropriate for the three-dimensional visualization of intrapulmonary liposomes at the alveolar scale. This study provides important information for selecting and optimizing suitable optical tissue-clearing techniques in lungs for evaluating the distribution of pulmonary drug delivery systems.

Tissue-Clearing Techniques Enable Three-Dimensional Visualization of Aerosolized Model Compound and Lung Structure at the Alveolar Scale.

In this study, we examined the usefulness of a tissue-clearing technique for the evaluation of the lung distribution of aerosolized drugs. An aerosol formulation of TexasRed dextran (70 kDa), a model compound of drug carrier for aerosolized drugs, was administered intrapulmonarily to mice using a MicroSprayer, and then DyLight 488-conjugated tomato lectin was administered intravenously to visualize general lung structure via the fluorescent labeling of alveolar and bronchial epithelial cells. Tissue clearing followed by laser scanning confocal microscopy enabled the three-dimensional visualization of intrapulmonary TexasRed dextran and the evaluation of its distribution at the alveolar scale without the preparation of thin tissue sections. These findings suggest that tissue-clearing techniques are useful for the evaluation of intrapulmonary distribution and development of pulmonary drug delivery systems.

Aerosolized liposomes with dipalmitoyl phosphatidylcholine enhance pulmonary absorption of encapsulated insulin compared with co-administered insulin.

OBJECTIVE: We have previously shown that aerosolized liposomes with dipalmitoyl phosphatidylcholine (DPPC) enhance the pulmonary absorption of encapsulated insulin. In this study, we aimed to compare insulin encapsulated into the liposomes versus co-administration of empty liposomes and unencapsulated free insulin, where the DPCC liposomes would serve as absorption enhancer. SIGNIFICANCE: The present study provides the useful information for development of noninvasive treatment of diabetes. METHODS: Co-administration of empty DPPC liposomes and unencapsulated free insulin was investigated in vivo to assess the potential enhancement in protein pulmonary absorption. Co-administration was compared to DPPC liposomes encapsulating insulin, and free insulin. RESULTS: DPPC liposomes enhanced the pulmonary absorption of unencapsulated free insulin; however, the enhancing effect was lower than that of the DPPC liposomes encapsulating insulin. The mechanism of the pulmonary absorption of unencapsulated free insulin by DPPC liposomes involved the opening of epithelial cell space in alveolar mucosa, and not mucosal cell damage, similar to that of the DPPC liposomes encapsulating insulin. In an in vitro stability test, insulin in the alveolar mucus layer that covers epithelial cells was stable. These findings suggest that, although unencapsulated free insulin spreads throughout the alveolar mucus layer, the concentration of insulin released near the absorption surface is increased by the encapsulation of insulin into DPPC liposomes and the absorption efficiency is also increased. CONCLUSION: We revealed that the encapsulation of insulin into DPPC liposomes is more effective for pulmonary insulin absorption than co-administration of DPPC liposomes and unencapsulated free insulin.

Evaluation of permeability alteration and epithelial-mesenchymal transition induced by transforming growth factor-ß1 in A549, NCI-H441, and Calu-3 cells: Development of an in vitro model of respiratory epithelial cells in idiopathic pulmonary fibrosis.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease, which is accompanied by changes in lung structure. With regard to treatment, aerosolized drugs administered intrapulmonarily are rapidly distributed into the plasma and do not remain in the lungs due to damage to the alveolar epithelium that occurs from pulmonary fibrosis. In this study, we sought to develop an in vitro model of respiratory epithelial cells in IPF for the evaluation of the intrapulmonary distribution of aerosolized drugs. We investigated transforming growth factor (TGF)-ß1-induced epithelial-mesenchymal transition (EMT) and permeability alteration in A549, NCI-H441, and Calu-3 cell monolayers. METHODS: After TGF-ß1 treatment of A549, NCI-H441, and Calu-3 cells, EMT markers including E-cadherin and vimentin and tight junction proteins including claudins-1, -3, and -5 were stained using immunofluorescence methods and detected using immunoblotting methods. Transport experiments were performed using TGF-ß1-treated cell monolayers and fluorescein isothiocyanate dextrans (FD; 4.4, 10, and 70kDa). In addition, TGF-ß1-induced apoptosis and necrosis were evaluated by flow cytometry using Annexin V and ethidium homodimer III, respectively. RESULTS: In NCI-H441 cells, incomplete EMT, destruction of claudins-1 and -3, and enhancement of FD permeability were caused by TGF-ß1 treatment. In A549 cells, complete EMT occurred but was not adequate for transport experiments because of low transepithelial electrical resistance. Whereas in Calu-3 cells, no changes were observed. TGF-ß1-induced apoptosis and necrosis were not observed in any of the cell lines. DISCUSSION: Incomplete EMT and permeability enhancement were observed in the alveolar epithelium of IPF. Therefore, our results indicate that TGF-ß1-treated NCI-H441 cell monolayers may serve as a useful in vitro model of respiratory epithelial cells for IPF.

Sustained distribution of aerosolized PEGylated liposomes in epithelial lining fluids on alveolar surfaces.

The distribution characteristics of aerosolized PEGylated liposomes in alveolar epithelial lining fluid (ELF) were examined in rats, and the ensuing mechanisms were investigated in the in vitro uptake and protein adsorption experiments. Nonmodified or PEGylated liposomes (particle size 100 nm) were aerosolized into rat lungs. PEGylated liposomes were distributed more sustainably in ELFs than nonmodified liposomes. Furthermore, the uptake of PEGylated liposomes by alveolar macrophages (AMs) was less than that of nonmodified liposomes. In further in vitro uptake experiments, nonmodified and PEGylated liposomes were opsonized with rat ELF components and then added to NR8383 cells as cultured rat AMs. The uptake of opsonized PEGylated liposomes by NR8383 cells was lower than that of opsonized nonmodified liposomes. Moreover, the protein absorption levels in opsonized PEGylated liposomes were lower than those in opsonized nonmodified liposomes. These findings suggest that sustained distributions of aerosolized PEGylated liposomes in ELFs reflect evasion of liposomal opsonization with surfactant proteins and consequent reductions in uptake by AMs. These data indicate the potential of PEGylated liposomes as aerosol-based drug delivery system that target ELF for the treatment of respiratory diseases.

Alteration in Intrapulmonary Pharmacokinetics of Aerosolized Model Compounds Due to Disruption of the Alveolar Epithelial Barriers Following Bleomycin-Induced Pulmonary Fibrosis in Rats.

Idiopathic pulmonary fibrosis is a lethal lung disease that is characterized by the accumulation of extracellular matrix and a change in lung structure. In this study, intrapulmonary pharmacokinetics of aerosolized model compounds were evaluated using rats with bleomycin-induced pulmonary fibrosis. Aerosol formulations of indocyanine green, 6-carboxyfluorescein (6-CF), and fluorescein isothiocyanate dextrans (FD; 4.4, 10, 70, and 250 kDa) were administered to rat lungs using a MicroSprayer. Indocyanine green fluorescence signals were significantly weaker in fibrotic lungs than in control lungs and 6-CF and FD concentrations in the plasma of pulmonary fibrotic animals were markedly higher than in the plasma of control animals. Moreover, disrupted epithelial tight junctions, including claudins-1, -3, and -5, were observed in pulmonary fibrotic lesions using immunofluorescence microscopy. In addition, destruction of tight junctions on model alveolar epithelial cells (NCI-H441) by transforming growth factor-ß1 treatment enhanced the permeability of 6-CF and FDs through NCI-H441 cell monolayers. These results indicate that aerosolized drugs are easily distributed into the plasma after leakage through damaged tight junctions of alveolar epithelium. Therefore, the development of delivery systems for anti-fibrotic agents to improve intrapulmonary pharmacokinetics may be necessary for effective idiopathic pulmonary fibrosis therapy.

Ingestibility and Formulation Quality of Lansoprazole Orally Disintegrating Tablets.

Objectives. We evaluated the ingestibility and formulation quality of one branded (formulation A) and five generic (formulations B, C, D, E, and F) lansoprazole orally disintegrating (OD) tablets. Methods. Ingestibility, including the oral disintegrating time, taste, mouth feeling, and palatability, was examined by sensory testing in healthy subjects. Formulation qualities, including salivary stability, gastric acid resistance, and intestinal dissolution behavior, were examined. Results and Discussion. The oral disintegration time of formulation F (52 s) was significantly longer than that of other formulations (32-37 s). More than 90% of subjects did not experience bitterness with formulations A, E, and F, whereas 50% of subjects felt rough and powdery sensations with formulations B, C, and D. More than 80% of subjects suggested that formulations A, E, and F had good palatability. Ingestibility was different between formulations. OD tablets consist of enteric granules containing lansoprazole, which is unstable in gastric acid. Enteric granules of each formulation were stable in artificial saliva and gastric juice. No differences were observed in dissolution behaviors among the formulations, indicating that the formulation quality of the formulations was almost equivalent. Conclusions. This study provides useful information for selecting branded or generic lansoprazole OD tablets for individualized treatments.

Efficient delivery to human lung fibroblasts (WI-38) of pirfenidone incorporated into liposomes modified with truncated basic fibroblast growth factor and its inhibitory effect on collagen synthesis in idiopathic pulmonary fibrosis.

In the present in vitro study, we assessed the delivery of pirfenidone incorporated into liposomes modified with truncated basic fibroblast growth factor (tbFGF) to lung fibroblasts and investigated the anti-fibrotic effect of the drug. The tbFGF peptide, KRTGQYKLC, was used to modify the surface of liposomes (tbFGF-liposomes). We used the thin-layer evaporation method, followed by sonication, to prepare tbFGF-liposomes containing pirfenidone. The cellular accumulation of tbFGF-liposomes was 1.7-fold greater than that of non-modified liposomes in WI-38 cells used as a model of lung fibroblasts. Confocal laser scanning microscopy showed that tbFGF-liposomes were widely localized in WI-38 cells. The inhibitory effects of pirfenidone incorporated into tbFGF-liposomes on transforming growth factor-ß1 (TGF-ß1)-induced collagen synthesis in WI-38 cells were evaluated by measuring the level of intracellular hydroxyproline, a major component of the protein collagen. Pirfenidone incorporated into tbFGF-liposomes at concentrations of 10, 30, and 100 µM significantly decreased the TGF-ß1-induced hydroxyproline content in WI-38 cells. The anti-fibrotic effect of pirfenidone incorporated into tbFGF-liposomes was enhanced compared with that of pirfenidone solution. These results indicate that tbFGF-liposomes are a useful drug delivery system of anti-fibrotic drugs to lung fibroblasts for the treatment of idiopathic pulmonary fibrosis.

Involvement of intestinal permeability in the oral absorption of clarithromycin and telithromycin.

The involvement of intestinal permeability in the oral absorption of clarithromycin (CAM), a macrolide antibiotic, and telithromycin (TEL), a ketolide antibiotic, in the presence of efflux transporters was examined. In order independently to examine the intestinal and hepatic availability, CAM and TEL (10 mg/kg) were administered orally, intraportally and intravenously to rats. The intestinal and hepatic availability was calculated from the area under the plasma concentration-time curve (AUC) after administration of CAM and TEL via different routes. The intestinal availabilities of CAM and TEL were lower than their hepatic availabilities. The intestinal availability after oral administration of CAM and TEL increased by 1.3- and 1.6-fold, respectively, after concomitant oral administration of verapamil as a P-glycoprotein (P-gp) inhibitor. Further, an in vitro transport experiment was performed using Caco-2 cell monolayers as a model of intestinal epithelial cells. The apical-to-basolateral transport of CAM and TEL through the Caco-2 cell monolayers was lower than their basolateral-to-apical transport. Verapamil and bromosulfophthalein as a multidrug resistance-associated proteins (MRPs) inhibitor significantly increased the apical-to-basolateral transport of CAM and TEL. Thus, the results suggest that oral absorption of CAM and TEL is dependent on intestinal permeability that may be limited by P-gp and MRPs on the intestinal epithelial cells.

Subcellular distribution of azithromycin and clarithromycin in rat alveolar macrophages (NR8383) in vitro.

Azithromycin (AZM), a 15-membered ring macrolide antimicrobial agent, has an antibacterial spectrum that includes intracellular parasitic pathogens that survive or intracellularly multiply in alveolar macrophages (AMs). The subcellular distribution of AZM in AMs was evaluated in vitro in comparison with clarithromycin (CAM). AZM and CAM (50 µM) were applied to the NR8383 cells, used as an in vitro model of AMs, followed by incubation at 37°C or 4°C. The total amount of AZM in cells and subcellular distribution (cell fractionation) was determined after incubation. High level of AZM accumulation was observed in the NR8383 cells at 37°C, and the equilibrium intracellular to extracellular concentration ratio (I/E ratio) was approximately 680, which was remarkably higher than that of CAM (equilibrium I/E ratio=28). The intracellular accumulation of AZM and CAM was temperature dependent. In addition, AZM distributed to the granules fraction including organelles and soluble fraction including cytosol in the NR8383 cells, whereas CAM mainly distributed in soluble fraction. The amount of AZM in the granules fraction was markedly reduced in the presence of ammonium chloride for increase in intracellular pH. These results indicate that AZM is distributed in acidic compartment in AMs. This study suggests that high AZM accumulation in the NR8383 cells is due to the trapping and/or binding in acidic organelles, such as lysosomes.