ブデソニド/グリコピロニウム/ホルモテロールの３剤による吸入合剤はNFκBの不活性化を介してCOPDマウスモデルの肺高血圧への進展を抑制する

概要

Kobe University Repository : Kernel
PDF issue: 2024-05-02

Budesonide/glycopyrronium/formoterol fumarate
triple therapy prevents pulmonary hypertension
in a COPD mouse model via NFκB activation

Suraya, Ratoe
(Degree)
博士（医学）

(Date of Degree)
2023-03-25

(Resource Type)
doctoral thesis

(Report Number)
甲第8599号

(URL)
https://hdl.handle.net/20.500.14094/0100482347
※ 当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。

（課程博士関係）

学位論文の内容要旨

Budesonide/glycopyrronium/formoterol fumarate triple
therapy prevents pulmonary hypertension in a COPD
mouse model via NFκB inactivation

ブ デ ソ ニ ド /グ リ コ ピ ロ ニ ウ ム / ホ ル モ テ ロ ー ル の ３ 剤 に よ る 吸 入 合 剤 は
NFκB の 不 活 性 化 を 介 し て COPD マ ウ ス モ デ ル の 肺 高 血 圧 へ の 進 展 を 抑
制する

神戸大学大学院医学研究科医科学専攻
呼吸器内科学
(指導教員：平田 健一 教授）

RATOE SURAYA

Background and purpose:
Chronic obstructive pulmonary disease (COPD) has been a major health problem,
and one of the main complications that contributes to COPD-related death is the eventual
development of pulmonary hypertension (PH), grouped into group 3 PH by WHO.
Hypoxia-hypoxemia due to obstructed airflow is known to induce vascular remodeling in
COPD-PH; however, unlike other classes of PH where specific drugs are available, few,
drugs have been shown to successfully treat COPD-PH. Recently, a novel triple inhalation
drug (inhaled corticosteroids/ICS, long-acting beta agonists/LABA, and long-acting
muscarinic antagonists/LAMA) was shown to improve lung function in COPD patients.
This triple therapy includes budesonide (B), glycopyrronium (G), and formoterol (F),
which are the most widely used inhalation drugs and have been established as staples of
COPD therapy. The clinical effects of this triple therapy have also been well established.
Furthermore, the individual classes of drugs that constitute BGF therapy have been
associated with positive effects in pulmonary vasculature cells, suggesting their potential
benefit in treating PH, specifically COPD-PH. This include the cytokine-mediated
inflammatory pathways via NFκB, a pathway controlling vascular tone and the
subsequent remodeling process that is a common link between COPD and PH. In this
study, we hypothesized that BGF combination therapy could act to prevent the
development of pulmonary hypertension in COPD via NFκB-mediated inflammation
pathway deactivation, and we sought to prove this hypothesis accordingly.
Methods:
We utilized one-time intratracheal elastase-induced emphysema mouse model and
performed experiments in three phases illustrating COPD progression: inflammatory
(1 day post-elastase), emphysema (3 weeks post-elastase) and PH (4 weeks post-elastase),
while treatments of BGF combination and controls (vehicle, one-drug treatment with B,
G, or F, and two-drug combinations of BF and GF) were started in prior to elastase
instillation once (inflammatory phase), at day 7 (emphysema) three times weekly, or at
day 14 (PH phase) three times weekly, respectively. Phenotype analyses were performed
in each phase. These include bronchoalveolar lavage fluid (BALF) and tissue cytokine
expression analysis in inflammatory phase, histological morphometric analysis in

emphysema phase, and right ventricular systolic pressure (RVSP) and histological
vascular muscularization analysis in PH phase.
In vitro, A549 lung epithelial cells or isolated mouse lung endothelial cells
(MLEC) were treated with TNFα with/without BGF treatment to analyze NFκB signaling
and cytokine expression changes, while similar TNFα treatment in A549 cells with
additional different combinations of drugs treatment (B, G, F, BF, GF, BGF) were
performed to analyze the effectiveness of each drug combination to prevent cytokine
expression.
Results:
First, in the inflammatory phase of in vivo study, we collected BALF from mice
in all the experimental groups and performed cytological analysis of BALF smears.
Cytology analysis of BALF revealed an increase in the total cell counts after elastase
treatment, while treatment with G, F, BF, GF and BGF significantly decreased the
elastase-induced increase in cell counts. Furthermore, a significant increase in the
neutrophil percentage, with a subsequent decrease in the macrophage percentage were
found after elastase treatment compared to PBS/vehicle treatment, while only GF and
BGF treatments significantly ameliorated the neutrophil percentage increase. Real-time
quantitative PCR analysis of the lung tissue mRNA from all groups revealed that the
elastase-induced increase in the levels of IL-1β and CXCL2 were ameliorated in all of the
treatment groups, including the BGF treatment group.
We then performed an analysis of the 21 days post-elastase emphysema phase.
We first observed the peripheral oxygen saturation levels of the mice at Days 0 (prior to
elastase instillation as baseline), 2, 9, and 16. While similar trends of saturation could be
seen at days 2 and 9, at Day 16 after treatment commenced, the BGF/elastase group
successfully reached an oxygen saturation level similar to that in the PBS/vehicle group.
Further analysis of histological lung sections revealed a highly disrupted alveolar
structure three weeks post-elastase instillation, yet improvement of the lung structure
could be seen in the BGF-treated group, confirmed by semiautomatic mean linear
intercept (MLI) measurements
Lastly, in the PH phase of our in vivo experiments, prior to termination at Day

28, echocardiography examination of the mice was performed, and the results revealed a
significant decrease in the pulmonary artery acceleration time (PAAT) after elastase
treatment, while BGF treatment successfully prevented it. At day 28, invasive
hemodynamic analysis using right heart catheterization to measure RVSP showed the
efficacy of BGF treatment in preventing the increase in the RVSP after elastase treatment.
Consistently, right ventricular hypertrophy measurement using the Fulton index showed
that BGF and BF treatment could prevent the development of right ventricular
hypertrophy. Further, we observed thickened precapillary vessels after elastase treatment
that was not observed in the BGF treatment group. We then confirmed that
muscularization of the precapillary arteries indeed occurred in the PBS/elastase group,
and it was ameliorated after the drug treatments, especially after BGF treatment, after
double immunostaining of the lung sections using von willebrand factor and α-smooth
muscle actin stainings.
We also aimed to prove that BGF could indeed affect this central inflammatory
pathway. We performed in vitro experiments using lung epithelial A549 cells and isolated
mouse lung endothelial cells (MLEC) treated with TNFαor vehicle, and simultaneously
added BGF or vehicle control. Immunoblotting of NFκB p65 showed a marked increase
in the levels of phosphorylated NFκB p65 after TNFα treatment, while the addition of
BGF successfully abolished this phosphorylation in both cells. Additionally, we also
analyzed whether there were differences in the production of cytokines after BGF
treatment. Consistent with the immunoblotting results, TNFα induced increases in the
mRNA levels of cytokines in both epithelial and endothelial cells, and these effects were
successfully abolished by BGF treatment. Lastly, we found that among all drug
combination groups, those containing budesonide (B, BF, BGF) were the ones most
responsible in affecting the cytokine production, shown by the significant reductions in
mRNA expression level of CXCL-8 and IL-6 compared to TNFα-treated A549 cells.
Discussion:
Although PH development is one of the major causes of morbidity and mortality
in COPD patients, therapeutic options that can specifically address COPD-PH conditions
are limited. We present here a novel strategy based on the inhalation of triple therapies

that could help treat this condition while providing molecular evidence about how such
treatments might act on lung cells. It is interesting to see not only that BGF treatment is
confirmed to be effective in both of the COPD phases (acute inflammatory and
emphysematous phases) but also that it can be effective in preventing further development
of PH.
Inflammation is a common mechanism in COPD and subsequent PH
development, and it could also serve as a link between COPD and PH pathogenesis. BGF
treatment is effective in preventing inflammation in epithelial cells, and it can also exert
a direct effect in ameliorating proinflammatory responses in ECs, suggesting a possible
common mechanism underlying the effect of BGF in treating this pathway. Multiple
proinflammatory cytokines are affected by BGF treatment, pointing to a central effect of
BGF in mediating inflammatory reactions. Indeed, we found that BGF exerted its effects
through a similar mechanism involving the NFκB pathway in both A549 cells and
MLECs. The transcription factor NFκB is known to trigger cellular inflammatory
reactions after activation by triggering the mRNA expression of a host of
proinflammatory cytokines Our study showed that BGF can directly ameliorate this
pathway in both cell lines, suggesting that this compound may have a common molecular
mechanism that can ultimately exert similar effects regardless of cell type. We did not
observe any changes, however, to other PH and/or COPD-related pathways that could be
related to the effect of BGF treatment. Further molecular studies are warranted to
elucidate this mechanism.
One aim of our study was to analyze whether there is a preferable combination
that can differentially affect lung cells in different phases. While BGF seemed to exert a
synergistic effect in preventing proinflammatory reactions in all experimental phases that
was not exerted by other drugs, interestingly, dual combination therapy did not exert a
protective effect consistent with that of BGF in vivo, suggesting a synergystic effect
between the three drugs. This could be explained by the interactions and synergism
between steroids and β agonists in exerting anti-inflammatory effects, and between
formoterol and glycopyrronium in affecting different pathways of anti-inflammatory and
bronchodilating effects. Additionally, it is interesting to note that although the dual drug
combinations showed tendencies of improving emphysematous and PH conditions, the

effects were relatively milder and less consistent than those of the BGF combination
treatment. Moreover, the results from these three phases suggest that different drugs can
positively affect COPD and COPD-PH conditions in different manners.
Further, we found that combinations that included budesonide had the strongest
effects on the expression of proinflammatory cytokines, as expected for a potent antiinflammatory agent. Notably, there was a decreasing trend in their expression with the
addition of formoterol and a further reduction with the addition of glycopyrronium. The
mutually beneficial interactions between steroids and β agonist agents could be the cause
of this phenomenon, although the link with glycopyrronium is unknown. Still, the
seemingly positive synergistic effect of the BGF combination warrants further studies in
the future.
In our study, we not only confirmed the efficacy of BGF in COPD but also
showed the additional benefit of BGF for COPD-PH conditions. The usage of the drug
classes LAMA and LABA to specifically treat type 3 PH has not been widely studied in
either basic or clinical settings. Corticosteroids, on the other hand, have been reported to
be potentially effective in treating PH. Nevertheless, there is a notable lack of clinical
data related to the usage of corticosteroids. As such, we hope that the results of our study
will contribute to filling the knowledge gap on this specific topic.
In conclusion, the results of our study contributed to the increasing evidence that
suggests the benefits of BGF treatment in COPD while also suggesting that BGF
treatment could also be effective in preventing the occurrence of COPD-related PH.
Further studies regarding the clinical efficacy of BGF in COPD-PH could potentially
provide a novel way to treat COPD-PH patients in the future.

神 戸大学大学院医学（
系）
研究科（博士課程）

論 文 審 査 の 結 果 の 要 旨
甲第 3
264号

氏

名

受付番号

RATOESURAYA

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