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The evaluation of subvisible particles, including protein aggregates, in therapeutic protein products has been of great interest for both pharmaceutical manufacturers and regulatory agencies. To date, the flow imaging (FI) method has emerged as a powerful tool instead of light obscuration (LO) due to the fact that (1) protein aggregates contain highly transparent particles and thereby escape detection by LO and (2) FI provides detailed morphological characteristics of subvisible particles. However, the FI method has not yet been standardized nor listed in any compendium. In an attempt to assess the applicability of the standardization of the FI method, we conducted a collaborative study using FI and LO instruments in a Japanese biopharmaceutical consortium. Three types of subvisible particle preparations were shared across 12 laboratories and analyzed for their sizes and counts. The results were compared between the methods (FI and LO), inter-laboratories, and inter-instruments (Micro Flow Imaging and FlowCam). We clarified the marked difference between the detectability of FI and LO when counting highly transparent protein aggregates in the preparations. Although FlowCam provided a relatively higher number of particles compared with MFI, consistent results were obtained using the instrument from the same manufacturer in all 3 samples.

Chronic obstructive pulmonary disease (COPD) is an intractable pulmonary disease that causes widespread and irreversible alveolar collapse. Although COPD occurs worldwide, only symptomatic therapy is currently available. Our objective is the development of therapeutic agents to eradicate COPD. Therefore, we focused on 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carbamoyl] benzoic acid (Am80), which is a derivative of all-trans retinoic acid. We evaluated the effects of Am80 on alveolar repair in a novel COPD model of adiponectin-deficient mice. This mouse model has more symptoms similar to human COPD than the classic elastase-induced emphysema mouse model. Lung volume, computed tomography (CT) values, low-attenuation area ratios, and bone and fat mass were measured by CT. However, the administration of Am80 did not affect these results. To examine the degree of destruction in the alveoli, the mean linear intercept of the alveolar walls was calculated, and assessment of this value confirmed that there was a significant difference between the control (46.3 ± 2.3 μm) and 0.5 mg/kg Am80-treated group (34.4 ± 1.7 µm). All mice survived the treatment, which lasted for more than 6 months, and we did not observe any abnormalities in autopsies performed at 80 weeks of age. These results suggested that Am80 was effective as a novel therapeutic compound for the treatment of COPD.

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.

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

Pulmonary emphysema is a disease in which alveoli in the lung are irreversibly destroyed, compromising lung function. All-trans-retinoic acid (ATRA) reportedly repairs the alveoli of emphysema model mice. In addition, ATRA can activate two nuclear receptors, nuclear retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR) β/δ. Both of these receptors induce differentiation in many different cell types. In this study, PPARβ/δ stimulation is shown to induce differentiation of human alveolar epithelial progenitor cells into alveolar type II epithelial (AT-II) cells. PPARβ/δ activation also stimulates the expression of adipose differentiation-related protein mRNA in human alveolar epithelial progenitor cells. These data suggest that PPARβ/δ selective agonists could differentiate human alveolar epithelial progenitor cells without cytotoxic of ATRA derived from stimulation to RAR.