Compositions for managing chronic obstructive pulmonary disease

Abstract

The present invention discloses a composition containing 20% ​​or more by weight of bisdemethoxycurcumin for the regeneration of damaged alveolar cells in emphysematous conditions and for the therapeutic management of chronic obstructive pulmonary disease and acute respiratory distress syndrome. The composition further contains 10-35% by weight of demethoxycurcumin and 10-45% by weight of curcumin. The composition is highly suitable for the treatment of COPD and ARDS caused by viral infections, particularly COVID-19, and for improving lung function in the prognosis.

Description

[Technical Field]

[0001] Cross-reference to related patent applications This application is a PCT application claiming priority to U.S. Provisional Patent Application No. 62,962,343, filed January 17, 2020, and U.S. Provisional Patent Application No. 63,126,920, filed December 17, 2020, the details of which are incorporated herein by reference. The present invention relates to compositions for managing chronic obstructive pulmonary disease. More particularly, the present invention relates to compositions comprising bisdemethoxycurcumin for regenerating alveoli and for managing chronic obstructive pulmonary disease and acute respiratory distress syndrome. [Background technology]

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease that causes airflow obstruction from the lungs. COPD is typically caused by long-term exposure to irritating gases like cigarette smoke, fuel combustion, viral infections, or particulate matter. Emphysema and chronic bronchitis are the two most common conditions that contribute to COPD. Emphysema is a condition in which the alveoli at the end of the smallest airways (bronchioles) in the lungs are destroyed. Emphysema is defined by the American Thoracic Society as "a pulmonary condition characterized by abnormal, persistent enlargement of the air spaces from the terminal bronchioles to the periphery, accompanied by destruction of their walls." Emphysema is a major component of COPD and is characterized by destruction of the alveolar extracellular matrix and a reduction in the alveolar-capillary exchange area, resulting in partially reversible airflow obstruction. This destroys the fragile walls and elastic fibers of the alveoli, causing lung collapse in the small airways and reducing airflow out of the lungs. Signs and symptoms include shortness of breath, especially during physical activity, wheezing, chest tightness, a chronic cough that may produce mucus, frequent respiratory infections, lack of energy, involuntary weight loss (later on), and swelling of the ankles, feet, or lower legs (Thurlbeck et al., Emphysema: Definition, Imaging, and Quantification, Benjamin Felson lecture, AJR 1994;163:1017-1025).

[0003] Evidence demonstrates striking differences in the pathological characteristics of normal and COPD lungs. In healthy individuals, cells respond to mild damage from exogenous ROS by activating repair mechanisms, such as antioxidants, DNA repair, and autophagy. If the damage is severe, cells undergo senescence, thereby preventing oncogenic changes. Stem cell replication plays a key role in tissue regeneration and healing. However, in individuals with COPD, excessive ROS increases damage to lung cells, exacerbated by defective repair mechanisms. The increase in senescent cells further stimulates inflammation, alveolar destruction, and endothelial dysfunction, thereby increasing the risk of oncogenic changes. ROS also induce loss of stem cell replication by inducing a loss of quiescence, leading to stem cell senescence. This leads to a loss of tissue regeneration (Mercado et al., Accelerated aging of the lung in COPD: new concepts, Thorax 2015;70:482-489. doi:10.1136 / thoraxjnl-2014-206084). In addition to emphysematous COPD, non-emphysematous COPD is also common (Occhipinti et al., Emphysematous and Nonemphysematous Gas Trapping in Chronic Obstructive Pulmonary Disease: Quantitative CT Findings and Pulmonary Function, Radiology: 2018, Volume 287: Number 2-683-692).

[0004] Acute respiratory distress syndrome (ARDS) is also observed in the later stages of COPD. ARDS is characterized by dyspnea, severe hypoxemia, decreased lung compliance, and diffuse bilateral pulmonary infiltrates. The pathology of ARDS typically involves three phases: exudative, proliferative, and fibrotic. Type I pneumocytes are irreversibly damaged, with deposition of proteins, fibrin, and cellular debris, creating a hyaline membrane in the denuded air spaces. Damage to type II cells, which produce pulmonary surfactant, also contributes to alveolar collapse. During the proliferative phase, type II cells proliferate, accompanied by regeneration, fibroblastic response, and remodeling of some epithelial cells (Udobi et al., Acute Respiratory Distress Syndrome, Am Fam Physician. 2003 Jan 15;67(2):315-322). Thus, alveolar degeneration is observed in both COPD and ARDS.

[0005] Recently, it has been observed that COPD may be a risk factor for more severe COVID-19 disease (Alqahtani et al. Prevalence, severity, and mortality associated with COPD and smoking in patients with COVID-19: a rapid systematic review and meta-analysis. PLoS One 2020; 15: e0233147). A nationwide analysis of 1,590 COVID-19 patients across China found that COPD had an odds ratio of 2.681 (95% CI 1.424–5.048; p=0.002) for ICU admission, mechanical ventilation, or death, even after adjusting for age and smoking. 62.5% of severe cases (compared to only 15.3% of non-severe cases) had a history of COPD, and 25% of deaths (compared to only 2.8% of survivors) were COPD patients (Leung et al., COVID-19 and COPD, European Respiratory Journal 2020 56: 2002108; DOI: 10.1183 / 13993003.02108-2020). This indicates that COPD is the most prominent cause of COVID-19 pathology.

[0006] Typically, COVID-19 pathogenesis can be divided into three stages: the first stage of the disease is intranasal, the second stage is in the conducting airways, bronchi, and bronchioles, and the third stage of the disease is fatal as the infection spreads to the gas-exchanging parts of the lungs and infects alveolar type II cells. Alveolar invasion is characterized by severe hypoxia and inflammatory inundation. In addition to damage to alveolar type I and type II cells, extensive damage to the endothelium is observed. This results in leakage of fibrinogen and other plasma proteins into the alveoli, which reduces the surface tension and reduces the ability of pulmonary surfactant to adhere to the surface. Thus, alveolar degeneration plays a critical role in the pathology of viral infections such as COVID-19.

[0007] Alveolar regeneration therapy is now considered an effective treatment for the management of COPD and ARDS. The ability of adult mammals to regenerate organs appears to be limited to the liver. However, mammals can continuously replace cells constituting the epidermis, intestinal endoderm, blood, neurons, etc. at the tissue level rather than the whole organ level. Recently, it has been discovered that retinoic acid can induce alveolar regeneration in experimentally injured adult rat lungs. The present invention discloses natural and novel compositions for alveolar regeneration and for the management of COPD and ARDS.

[0008] Currently, there are many natural compounds used to manage COPD. Turmeric compositions containing curcumin have been reported to be effective in improving the symptoms of COPD and ARDS. The following prior art documents disclose the use of turmeric, particularly curcumin, in the management of COPD: 1.Tang et al., Curcumin ameliorates chronic obstructive pulmonary disease by modulating autophagy and endoplasmic reticulum stress through regulation of SIRT1 in a rat model, Journal of International Medical Research, 2019, Vol. 47(10) 4764-4774. 2.Jawed et al., Effect of Turmeric on Mosquito Coil Induced Emphysema in Rat Lungs, JIIMC 2018 Vol. 13, No.3, 141-145. 3.Moghaddam et al., Curcumin inhibits COPD-like airway inflammation and lung cancer progression in mice, Carcinogenesis vol.30 no.11 pp.1949-1956, 2009. 4.Santana et al., Evidences of Herbal Medicine-Derived Natural Products Effects in Inflammatory Lung Diseases, Mediators of Inflammation, Volume 2016, Article ID 2348968, 14 pages, http: / / dx.doi.org / 10.1155 / 2016 / 2348968. 5. Butler and Kaifer, Herbs and Supplements for COPD (Chronic Bronchitis and Emphysema), Healthline, Updated on August 20, 2018, healthline.com / health / copd / herbs-supplements, accessed 5 January 2021.

[0009] Turmeric contains various compounds, the main components of which are curcumin, dimethoxycurcumin, and bisdemethoxycurcumin. The biological properties of curcumin, bisdemethoxycurcumin, and demethoxycurcumin vary depending on the disease, and the effect of bisdemethoxycurcumin in the management of COPD and related functions has not been evaluated. The present invention discloses the biological potential of compositions containing bisdemethoxycurcumin in alveolar regeneration and in the management of symptoms of COPD and ARDS.

[0010] The primary object of the present invention is to disclose a composition comprising 20% ​​by weight or more of bisdemethoxycurcumin for use in regenerating alveolar cells in pulmonary emphysema. Another object of the present invention is to disclose a composition comprising 20% ​​by weight or more of bisdemethoxycurcumin for use in managing the symptoms of chronic obstructive pulmonary disease. Yet another object of the present invention is to disclose a composition comprising 20% ​​by weight or more of bisdemethoxycurcumin for use in preventing the progression of chronic obstructive pulmonary disease to acute respiratory distress syndrome. The present invention fulfills the above objectives and provides related advantages. Summary of the Invention

[0011] In a most preferred embodiment, the present invention discloses a method for regenerating alveolar cells in a mammal having emphysema, comprising administering to the mammal a composition comprising 20% ​​or more by weight of bisdemethoxycurcumin to result in a reduction in the characteristics of emphysema. In another preferred embodiment, the present invention discloses a method for the therapeutic management of chronic obstructive pulmonary disease in a mammal, comprising administering to said mammal a composition comprising 20% ​​or more by weight of bisdemethoxycurcumin to result in a reduction in the characteristics and symptoms of chronic obstructive pulmonary disease. In another preferred embodiment, the present invention discloses a method for preventing progression of chronic obstructive pulmonary disease to acute respiratory distress syndrome in a mammal, comprising administering to the mammal a composition comprising 20% ​​or more by weight of bisdemethoxycurcumin to result in a reduction in the characteristics and symptoms of chronic obstructive pulmonary disease. Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying images, which illustrate, by way of example, the principles of the invention. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0012] [Figure 1A]1 is a graphical representation showing the dose-dependent reduction in HIF-1α levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to normal and COPD control groups. [Figure 1B] 1 is a graphical representation showing the reduction of HIF-1α levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to curcumin and demethoxycurcumin (DMC). [Figure 1C] 1 is a graphical representation showing the dose-dependent reduction in HIF-1α levels in COPD mice treated with a composition comprising bisdemethoxycurcumin at various doses (mg / kg body weight) (BD3 complex) compared to curcumin C3 complex. [Figure 2A] 1 is a graphical representation showing the dose-dependent increase in pulmonary surfactant protein D levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to normal and COPD control groups. [Figure 2B] FIG. 1 is a graphical representation showing the dose-dependent increase in pulmonary surfactant protein D levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to curcumin and demethoxycurcumin (DMC). [Figure 2C] 1 is a graphical representation showing the dose-dependent increase in pulmonary surfactant protein D levels in COPD mice treated with a composition comprising bisdemethoxycurcumin (BD3 complex) at various doses (mg / kg body weight) compared to curcumin C3 complex. [Figure 3A] 1 is a graphical representation showing the dose-dependent increase in periostin levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to normal and COPD controls. [Figure 3B]1 is a graphical representation showing the dose-dependent increase in periostin levels in COPD mice treated with a composition containing bisdemethoxycurcumin (BD3 complex) at various doses (mg / kg body weight) compared to curcumin C3 complex. [Figure 4A] 1 is a graphical representation showing the dose-dependent reduction of 12 / 15-LOX levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to normal and COPD controls. Bar values ​​represent 12LOX, and lines represent 15LOX. [Figure 4B] 1 is a graphical representation showing the dose-dependent reduction in 12 / 15-LOX levels in COPD mice treated with various doses (mg / kg body weight) of a composition containing bisdemethoxycurcumin (BD3 complex) compared to curcumin C3 complex. Bar values ​​represent 12LOX, and lines represent 15LOX. [Figure 5A] 1 is a graphical representation showing the dose-dependent reduction in bradykinin levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to normal and COPD control groups. [Figure 5B] 1 is a graphical representation showing the reduction in bradykinin levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to curcumin and demethoxycurcumin (DMC). [Figure 5C] 1 is a graphical representation showing the dose-dependent reduction in bradykinin levels in COPD mice treated with a composition comprising bisdemethoxycurcumin at various doses (mg / kg body weight) (BD3 complex) compared to curcumin C3 complex. [Figure 6A] FIG. 1 is a graphical representation showing the dose-dependent reduction in IL-17 and IL-23 levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to normal and COPD control groups. [Figure 6B]1 is a graphical representation showing the dose-dependent reduction in IL-17 and IL-23 levels in COPD mice treated with a composition comprising various doses (mg / kg body weight) of bisdemethoxycurcumin (BD3 complex) compared to curcumin C3 complex. [Figure 7A] 1 is a graphical representation showing the dose-dependent reduction in IL-6 levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to normal and COPD control groups. [Figure 7B] 1 is a graphical representation showing the dose-dependent reduction in IL-6 levels in COPD mice treated with a composition comprising bisdemethoxycurcumin (BD3 complex) at various doses (mg / kg body weight) compared to curcumin C3 complex. [Figure 8A] 1 is a graphical representation showing the dose-dependent reduction in CXCL8 levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to normal and COPD control groups. [Figure 8B] 1 is a graphical representation showing the dose-dependent reduction in CXCL8 levels in COPD mice treated with a composition comprising bisdemethoxycurcumin (BD3 complex) at various doses (mg / kg body weight) compared to curcumin C3 complex. [Figure 9A] 1 is a graphical representation showing the dose-dependent reduction in LDH levels in COPD mice treated with various doses (mg / kg body weight) of bisdemethoxycurcumin (BDMC) compared to normal and COPD control groups. [Figure 9B] 1 is a graphical representation showing the dose-dependent reduction in LDH levels in COPD mice treated with a composition comprising bisdemethoxycurcumin (BD3 complex) at various doses (mg / kg body weight) compared to curcumin C3 complex. [Figure 10] 1 shows immunohistochemical staining images of lung tissue sections from normal mice, COPD mice, and mice treated with 25 mg / kg and 100 mg / kg BDMC showing regeneration of alveolar type II cells. DETAILED DESCRIPTION OF THE INVENTION

[0013] In a most preferred embodiment, the present invention discloses a method for regenerating alveolar cells in a mammal with emphysema, comprising administering to the mammal a composition comprising 20% ​​or more by weight of bisdemethoxycurcumin to result in a reduction in characteristics of emphysema. In a related embodiment, the composition further comprises 10-35% by weight of demethoxycurcumin and 10-45% by weight of curcumin. In another related embodiment, the characteristics of emphysema are selected from the group consisting of hypoxia, decreased levels of pulmonary surfactant proteins, increased alveolar-capillary barrier permeability, inflammation, increased neutrophil accumulation and recruitment, increased intraalveolar pressure, increased oxidative stress, increased inflammatory cytokines and chemokines, increased numbers of activated T lymphocytes, and muscle damage. In yet another related embodiment, the emphysema is induced by enzymes, viruses, bacteria, smoking, and particulate irritants. In another related embodiment, the mammal is a human.

[0014] In another preferred embodiment, the present invention discloses a method for the therapeutic management of chronic obstructive pulmonary disease in a mammal, comprising administering to the mammal a composition comprising 20% ​​or more by weight of bisdemethoxycurcumin to result in a reduction in the characteristics and symptoms of chronic obstructive pulmonary disease. In a related embodiment, the composition further comprises 10-35% by weight of demethoxycurcumin and 10-45% by weight of curcumin. In another related embodiment, the chronic obstructive pulmonary disease is emphysematous or non-emphysematous. In another related embodiment, the characteristics of chronic obstructive pulmonary disease are selected from the group consisting of hypoxia, decreased levels of pulmonary surfactant proteins, increased alveolar-capillary barrier permeability, inflammation, increased neutrophil accumulation and recruitment, increased intra-alveolar pressure, increased oxidative stress, increased inflammatory cytokines and chemokines, increased numbers of activated T lymphocytes, and muscle damage. In another related embodiment, the symptoms of chronic obstructive pulmonary disease are selected from the group consisting of shortness of breath, especially during physical activity, wheezing, chest tightness, chronic cough that may produce mucus, respiratory infections, lack of energy, unintentional weight loss, and swelling of the ankles, feet, or legs. In yet another related embodiment, the chronic obstructive pulmonary disease is induced by enzymes, viruses, bacteria, smoking, and particulate irritants. In another related embodiment, the mammal is a human.

[0015] In another preferred embodiment, the present invention discloses a method for preventing progression of chronic obstructive pulmonary disease to acute respiratory distress syndrome in a mammal, comprising administering to the mammal a composition comprising 20% ​​or more by weight of bisdemethoxycurcumin to prevent the onset of acute respiratory distress syndrome by reducing the characteristics and symptoms of chronic obstructive pulmonary disease. In a related embodiment, the composition further comprises 10-35% by weight of demethoxycurcumin and 10-45% by weight of curcumin. In another related embodiment, the chronic obstructive pulmonary disease is emphysematous or non-emphysematous. In another related embodiment, the characteristics of chronic obstructive pulmonary disease are selected from the group consisting of hypoxia, decreased levels of pulmonary surfactant proteins, increased alveolar-capillary barrier permeability, inflammation, increased neutrophil accumulation and recruitment, increased intra-alveolar pressure, increased oxidative stress, increased inflammatory cytokines and chemokines, increased numbers of activated T lymphocytes, and muscle damage. In another related embodiment, the symptoms of chronic obstructive pulmonary disease are selected from the group consisting of shortness of breath, especially during physical activity, wheezing, chest tightness, chronic cough that may produce mucus, respiratory infections, lack of energy, unintentional weight loss, and swelling of the ankles, feet, or legs. In yet another related embodiment, the chronic obstructive pulmonary disease is induced by enzymes, viruses, bacteria, smoking, and particulate irritants. In another related embodiment, the mammal is a human. Preferred embodiments of the present invention are further described in the following illustrative examples. [Example]

[0016] Example 1 Elastase-induced emphysema Wistar rats were divided into experimental groups and, under anesthesia, received intratracheal administration of porcine pancreatic ELT (48.0 U / mg protein; 0, 20, or 160 U dissolved in 100 μl of calcium- and magnesium-free Dulbecco's phosphate-buffered saline). Twenty-one days after intratracheal administration of ELT, various parameters in bronchoalveolar lavage fluid (BALF), concentrations of proinflammatory mediators and biochemical parameters in lung homogenates, and pulmonary function were evaluated (Inoue et al., Extensive analysis of elastase-induced pulmonary emphysema in rats: ALP in the lung, a new biomarker for disease progression? J. Clin. Biochem. Nutr., 46, 168-176, March 2010). The rats were divided into the following groups:

[0017] [Table 1]

[0018] For clarity, the definitions of the compounds administered in the above groups are provided below. BDMC - Bisdemethoxycurcumin (pure compound) Curcumin C3 Complex / C3 Complex - A composition containing 75-81% curcumin, 15-19% demethoxycurcumin, and 2.2-6.5% bisdemethoxycurcumin. BD3 Complex - a composition comprising 20% ​​or more by weight of bisdemethoxycurcumin, 10-35% by weight of demethoxycurcumin, and 10-45% by weight of curcumin (as disclosed in U.S. Provisional Patent Application No. 63126920, which is incorporated herein by reference). Curcumin - Pure Curcumin DMC - Demethoxycurcumin (pure compound) As an illustrative example, the concentrations of BD3 complex used for the experiments are 32% by weight of BDMC, 30% by weight of DMC, and 38% by weight of curcumin. However, those skilled in the art will understand that the effects shown in the following examples are applicable to concentrations of 20% by weight of bisdemethoxycurcumin, 10-35% by weight of demethoxycurcumin, and 10-45% by weight of curcumin.

[0019] BALF Collection - Rats were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg / kg) and exsanguinated via the abdominal aorta. A cannula was inserted into the trachea and secured with sutures. Both lungs were lavaged twice with 10 ml of sterile saline solution dropped into them with a syringe at 37°C. Fluid was collected by gentle aspiration. The collected fluid was cooled and centrifuged at 300 g for 10 minutes, and the supernatant was collected for analysis (Inoue et al., Extensive analysis of elastase-induced pulmonary emphysema in rats: ALP in the lung, a new biomarker for disease progression? J. Clin. Biochem. Nutr., 46, 168-176, March 2010).

[0020] Pulmonary parameters Pulmonary surfactant proteins and periostin were measured in BALF using commercially available ELISA kits. The assay uses a quantitative sandwich enzyme immunoassay technique. SP-D-specific antibodies were precoated on microplates. Standards and samples were pipetted into wells, allowing any SP-D present to bind to the immobilized antibodies. After removing unbound material, biotin-conjugated SP-D-specific antibodies were added to the wells. After washing, avidin-conjugated horseradish peroxidase (HRP) was added to the wells. After washing to remove any unbound avidin-enzyme reagent, a substrate solution was added to the wells, allowing color development proportional to the amount of SP-D bound in the initial step. Color development was stopped, and the color intensity, which is proportional to the amount of SP-D present, was measured.

[0021] Quantification of HIF-1alpha, 12 / 15LOX, IL-6, and CXCL-8 levels in lung tissue supernatants The lungs of the animals were removed and flushed with ice-cold isotonic saline. An extraction buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 mg / ml aprotinin, and 0.05% Tween 20 in phosphate-buffered saline was added to the tissue at 4 ml / g tissue. The tissue was homogenized on ice using a Polytron, and the homogenate was centrifuged at 5000 g for 15 minutes. Aliquots of the supernatant were used for biochemical analysis. The supernatant was stored at -80°C until cytokine analysis (Pandey et al., Multifunctional neuroprotective effect of withanone, a compound from Withania somnifera roots in alleviating cognitive dysfunction. Cytokine 102 (2018) 211-221).

[0022] Serum collection for IL17 / IL23 and LDH-A measurements Blood was collected from the retro-orbital venous plexus of the animals. Samples were allowed to stand for 2 hours and then centrifuged. Serum samples from various groups of animals were prepared for cytokine analysis using commercially available kits based on sandwich and competitive ELISA methods according to the manufacturer's instructions. The concentrations of all cytokines were measured colorimetrically at 450 nm in an ELISA plate reader by interpolation from a standard curve (Pandey et al., Amelioration of Adjuvant-Induced Arthritis by Apocynin. Phytother Res, 2009 Oct;23(10):1462-8).

[0023] Immunohistochemical detection of AT-2 Lung sections were subjected to immunohistochemistry using mouse anti-human thyroid transcription factor 1 (TTF-1) (clone SPT24) (M / s Master diagnostics, (Cat #MAD-000486QD-12) Granada) to demonstrate AT-2 cells.

[0024] material Immunity chemical products 1) Primary antibody: Mouse anti-human thyroid transcription factor 1 (TTF-1) (clone SPT24), M / s Master diagnostics, (Cat #MAD-000486QD-12) Granada, 1:50 dilution. 2) Secondary antibodies: Super Sensitive™ (SS) Polymer-HRP IHC Detection System, M / s Biogenex, USA, anti-mouse and anti-rabbit secondary antibodies 3) Section adhesive: 3-aminopropyltriethoxysilane (APES), obtained from Sigma chemicals, USA. 4) Hydrogen peroxide (H2O2) methanol solution (3%): 3% H2O2 methanol solution was prepared by adding 1 ml of 30% H2O2 to 9 ml of methanol. 5) Antigen retrieval solution: a.1mM EDTA buffer (pH8.4): b. 1 mM EDTA, pH 8.0 c. EDTA 0.37g d. 1L of distilled water The pH was adjusted to 8.4 with 1 N NaOH. All solutions were freshly prepared immediately before use. 6) DAB+ substrate: 3,3-diaminebenzidine tetrahydrochloride substrate was freshly prepared at the time of use by adding 1 mg of 3,3-diaminebenzidine tetrahydrochloride (Santacruz, USA) to 1 ml of 0.01 M PBS with 12 μl of 3% H2O2. 7) 0.01M Phosphate Buffered Saline (pH 7.2): 500 ml of 10x PBS was prepared by adding the following chemicals: 40 g sodium chloride (MW 58.44), 1 g potassium chloride (MW 74.56), 7.2 g disodium hydrogen orthophosphate (MW 141.96), 1 g anhydrous potassium dihydrogen orthophosphate (MW 136.09), and 500 ml distilled water. A 1x wash buffer was prepared using 10x PBS by adding 25 ml of 10x PBS to 225 ml of distilled water. To this, 125 μl of Tween 20 was added and the pH was adjusted to 7.2. 8) Harris Hematoxylin for intranuclear staining: Harris Hematoxylin was used for intranuclear staining. Counterstaining was performed for 45 seconds.

[0025] Preparation of organosilane (APES) treated slides for IHC: This preparation was carried out using the following steps: 1. Slides were placed on a rack, washed thoroughly with soapy water, rinsed with tap water, and finally rinsed with distilled water, and allowed to dry completely. 2. In a dry staining dish, prepare a 2% solution of 3-aminopropyltriethoxysilane (APES) in acetone. Immerse the slides in this APES solution for 5-15 minutes. 3. The slides were rinsed with acetone, followed by two changes of distilled water. The slides were dried at 37°C for 2 hours and then stored at room temperature for further processing later.

[0026] method 1. Tissue sections were placed on 3-aminopropyltriethoxysilane (APES) coated slides and dried at 37°C for 3 hours, then stored at 4°C for further processing. 2. Paraffin tissue sections were deparaffinized using xylene and rehydrated in descending grades of ethanol. 3. Endogenous peroxidase was blocked by covering the entire section with 100 μl of 3% H2O2 in methanol, which was incubated at room temperature for 15 minutes, and then washed with three changes of wash buffer. 4. Heat-induced epitope retrieval (HIER) was performed by immersing tissue sections in a cooker containing EDTA buffer (pH 8.4) and heating for 6 minutes after reaching maximum pressure. Sections were allowed to cool to room temperature for approximately 30 minutes and then washed with three changes of wash buffer. 5. Addition of primary antibody: Ready-to-use mouse anti-human thyroid transcription factor 1 (TTF-1) (clone SPT24) was applied over the sections, followed by incubation in a humidified chamber at room temperature for 1 hour and washing with wash buffer as described above. 6. Addition of secondary antibodies: Anti-mouse and anti-rabbit secondary antibodies (Super Sensitive™ (SS) Polymer-HRP IHC Detection System, M / s Biogenex, USA) were added to the sections and incubated for 30 minutes at room temperature in a humidified chamber. After incubation, the sections were washed with PBS as described above. 7. Addition of DAB + substrate: Freshly prepared 3,3-diaminebenzidine tetrahydrochloride (DAB) containing 3% H2O2 was poured over the sections. This was incubated for 15-20 minutes or until the desired staining intensity was achieved. The sections were then washed again with three changes of distilled water. 8. Nuclear counterstaining was performed with Harris hematoxylin for 45 seconds. Sections were washed in distilled water, dehydrated in ascending grades of ethanol, cleared in xylene, and coverslipped with DPX mounting medium.

[0027] result Hypoxia Hypoxia is a condition in which cells and tissues in the body do not receive enough oxygen. This can occur even when blood flow is normal. Hypoxia can lead to many serious, sometimes life-threatening, complications. Lung cells commonly experience hypoxia during high-altitude travel and fetal development in acute and chronic lung diseases. Chronic hypoxia leads to increased inflammation and alveolar degeneration, resulting in emphysema, one of the causative factors in the development of COPD and ARDS. In COPD, the hypoxia-inducible factor (HIF-1) signaling pathway is activated, and overexpression of HIF-1α and related proteins, such as VEGF, is associated with decreased lung function, reduced quality of life, and COPD progression.

[0028] Recent evidence indicates that hypoxia is a primary pathophysiological feature and a major cause of death in severe COVID-19 patients, and occurs concomitantly with all stages of COVID-19. Protein targets of HIF-1α are involved in severe hypoxia-induced activation of inflammatory cytokine expression and the subsequent inflammatory process and cytokine storm phase of COVID-19.

[0029] In this study, we evaluated the effect of BDMC on reducing HIF-1α levels and compared it with curcumin and DMC. The results showed that BDMC reduced HIF-1α levels in a dose-dependent manner (Figure 1A). BDMC was also significantly more effective than curcumin and DMC (Figure 1B), demonstrating the non-trivial effect of BDMC in reducing hypoxia. A composition containing BDMC, curcumin, and DMC (BD3 complex) was also more effective in reducing hypoxia by reducing elevated HIF-1α levels than the curcumin C3 complex (Figure 1C).

[0030] Pulmonary surfactant proteins Pulmonary surfactant is vital because it coats the alveoli and reduces surface tension. Its primary functions include: 1) reducing surface tension at the air-liquid interface to prevent alveolar collapse at the end of expiration; 2) interacting with and subsequently killing pathogens; and 3) modulating the immune response. Pulmonary surfactant components are primarily synthesized by alveolar type II cells, which synthesize the surfactant proteins SP-A, SP-B, and SP-D, collectively called collectins. These proteins bind to viruses and bacteria and aid in their clearance. Even moderate inflammation and exudation impair gas exchange, making host defense at the alveolar interface crucial. SP-A and SP-D, present in the mucus layer and alveolar surface, are well-positioned to prevent infection of epithelial cells by neutralizing viruses, aggregating them, and enhancing phagocytosis. SP-A and / or SP-D bind to and inhibit the activity of influenza A virus hemagglutinin and neuraminidase. Pulmonary collectins also bind to viral glycoproteins, including HIV, respiratory syncytial virus (RSV), and severe acute respiratory syndrome (SARS) coronavirus. The following documents disclose the importance of pulmonary surfactant proteins and are incorporated herein by reference: 1.Qi L et al. The ability of pandemic influenza virus hemagglutinins to induce lower respiratory pathology is associated with decreased surfactant protein D binding. Virology. 2011;412:426-434. 2.Meschi J, et al. Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication. J Gen Virol. 2005;86:3097-3107. 3.Hickling TP, et al. A recombinant trimeric surfactant protein D carbohydrate recognition domain inhibits respiratory syncytial virus infection in vitro and in vivo. Eur J Immunol. 1999;29:3478-3484. 4.Leth-Larsen R et al. The SARS coronavirus spike glycoprotein is selectively recognized by lung surfactant protein D and activates macrophages. Immunobiology. 2007;212:201-211.

[0031] In this study, we evaluated the effect of BDMC on increasing pulmonary surfactant protein D levels and compared it with curcumin and DMC. The results showed that BDMC increased pulmonary surfactant protein D levels in a dose-dependent manner (Figure 2A). BDMC was also significantly more effective than curcumin and DMC (Figure 2B), demonstrating the non-obvious effect of BDMC. A composition containing BDMC, curcumin, and DMC (BD3 complex) was also more effective at increasing pulmonary surfactant protein D levels than the curcumin C3 complex (Figure 2C).

[0032] Periostin The influx of blood into alveolar air spaces following pulmonary capillary leakage is a critical step in the progression of lung injury. This influx results from increased permeability of the alveolar-capillary barrier. The extracellular matrix (ECM) between the capillaries and the epithelium is involved in preventing this influx, and the ECM structure is constructed by periostin, a matricellular protein localized in the alveolar wall. Binding sites on periostin contribute to the mechanical strength of connective tissue. Periostin strengthens the interactions between nearby molecules and their assembly into extracellular matrix structures. In this study, periostin levels were significantly decreased in COPD patients. BDMC increased periostin levels in a dose-dependent manner (Figure 3A). A composition containing BDMC, curcumin, and DMC (BD3 complex) was also effective in increasing periostin levels compared with the curcumin C3 complex (Figure 3B).

[0033] 12- and 15-lipoxygenases in alveolar injury. Neutrophil accumulation and recruitment to the lung are critical events in the development of lung injury. Neutrophil recruitment to the lung occurs in a cascade-like process of activation, intravascular accumulation, and transendothelial and transepithelial migration. 12 / 15-LOX regulates pulmonary neutrophil recruitment by regulating chemokine / chemokine receptor homeostasis. 12 / 15-LOX also generates lipid mediators with immunomodulatory properties through the enzymatic oxidation of polyunsaturated fatty acids. It has been reported that lipid mediators can promote the resolution of inflammation by controlling and timing 12 / 15-LOX expression. However, its deregulatory activity contributes to tissue damage, cell death, and chronic inflammation. In this study, 12 / 15-LOX levels were significantly elevated in COPD. BDMC dose-dependently reduced 12 / 15-LOX levels (Figure 4A). A composition containing BDMC, curcumin, and DMC (BD3 complex) was also effective in reducing 12 / 15-LOX levels (Figure 4B).

[0034] Bradykinin Bradykinin is a physiologically and pharmacologically active peptide of the kinin group of proteins, consisting of nine amino acids. Bradykinin is a potent endothelium-dependent vasodilator and mild diuretic, and it lowers blood pressure. Bradykinin also causes contraction of nonvascular smooth muscle of the bronchi and intestine, increases vascular permeability, and is involved in pain mechanisms. In this study, bradykinin levels were significantly elevated in COPD, indicating increased vascular permeability, inflammation, and pain. BDMC dose-dependently reduced bradykinin levels (Figure 5A). BDMC was also significantly more effective at reducing elevated bradykinin levels than curcumin and DMC (Figure 5B). A composition containing BDMC, curcumin, and DMC (BD3 complex) was also effective at reducing bradykinin levels (Figure 5C), thereby reducing pain, inflammation, and vascular permeability.

[0035] Immune markers IL-1, IL-23, and IL-6 Elevated numbers of activated T lymphocytes are observed in the bronchial mucosa of COPD patients. It has been reported that T helper type 17 (Th17) cells release interleukin (IL)-17 as their effector cytokine under the control of IL-22 and IL-23. This then plays a role in neutrophil recruitment and tissue remodeling in the patient's bronchi. Overexpression of IL-17 in mouse lung epithelium induces pulmonary inflammation with a COPD-like phenotype, accompanied by CD4 cell recruitment, mucus hypersecretion, and small airway fibrosis. IL-17 also induces the expression of multiple chemokines (including CXCL1 and CXCL8) and matrix metalloproteinase-9.

[0036] There is considerable evidence supporting the presence of neutrophilic inflammation in COPD. Cigarette smoke, oxidative stress, bacteria, and viruses activate neutrophilic inflammation in airway epithelial cells via nuclear factor-κB (NF-κB) signaling. Macrophages also activate and attract Th17 cells, releasing IL-17, which stimulates the release of IL-6 and CXCL8 from epithelial cells. Neutrophils release neutrophil elastase, a potent inducer of mucus secretion. Neutrophils also generate oxidative stress, which further activates inflammation and induces corticosteroid resistance.

[0037] In this study, IL-17 and IL-23 levels were significantly elevated in COPD, indicating increased neutrophilic inflammation. BDMC dose-dependently reduced the levels of these inflammatory markers (Figure 6A). A composition containing BDMC, curcumin, and DMC (BD3 complex) was also effective in reducing IL-17 and IL-23 levels (Figure 6B), thereby reducing inflammation and the influx of cytokines and chemokines. IL-6 levels were significantly elevated in COPD. BDMC and the BD3 complex dose-dependently reduced IL-6 levels (Figures 7A and 7B).

[0038] Elevated inflammatory chemokines in COPD Chemokines released from epithelial cells and macrophages in the lungs recruit inflammatory cells from the circulating blood, leading to the development of COPD. Blocking the inflammatory cell trafficking orchestrated by multiple chemokines with selective antagonists is an effective anti-inflammatory strategy in this disease. In this study, CXCL8 levels were significantly elevated in COPD, indicating increased inflammation and chemokine influx. BDMC dose-dependently reduced these CXCL8 levels (Figure 8A). A composition containing BDMC, curcumin, and DMC (BD3 complex) was also effective in reducing CXCL8 levels (Figure 8B), thereby reducing inflammation.

[0039] Lactate dehydrogenase Lactate dehydrogenase (LDH) is an enzyme that catalyzes the final step in glycolytic metabolism, regenerating NAD+ from reduced NADH by converting pyruvate to lactate. Cytosolic enzymes, including LDH, are released into the extracellular space upon cell lysis or membrane damage. Increased muscle LDH activity has been observed in elderly men with COPD, who are prone to contractile fatigue. In this study, LDH levels were significantly elevated in the COPD group, indicating increased muscle damage and fatigue. BDMC dose-dependently reduced these LDH levels (Figure 9A). A composition containing BDMC, curcumin, and DMC (BD3 complex) was also effective in reducing LDH levels (Figure 9B).

[0040] Immunohistochemistry Immunohistochemical examination was performed according to the protocol to stain and visualize the cells. In the normal control group, the lesioned tissue showed normal histology with alveolar and bronchial epithelium. The tissue was positive for type II alveolar epithelium by intranuclear staining. A uniform distribution of type II cells was observed in alveolar and bronchial lining cells. However, in the COPD group, the lesioned tissue showed diffuse thickening of the alveolar septa, characterized by intense infiltration of mononuclear cells. The alveoli remained distorted. The number of alveolar type II-positive cells was absent or negligible compared with the normal control group. The bronchial epithelium also showed scattered type II-positive cells, indicating increased alveolar degeneration. The lesioned tissue in the 25 mg / kg body weight treatment group showed diffuse thickening of the alveolar septa with mononuclear cell infiltration. Although the alveoli remained distorted throughout the tissue sections, alveolar type II-positive cells were found diffusely throughout the parenchyma. A significant increase in the number of type II-positive cells was observed compared to the disease control group. Furthermore, in mice treated with 100 mg / kg body weight BDMC, the lesion tissue showed diffuse thickening of the alveolar septa accompanied by mononuclear cell infiltration. The alveoli remained distorted throughout the tissue sections. Type II-positive cells were slightly to relatively more numerous compared to the disease control group. In places, the bronchial epithelium showed type II-positive cells, indicating the regeneration of alveoli damaged by emphysema and COPD (Figure 10).

[0041] Overall, these results suggest that BDMC itself and compositions containing BDMC (BD3 complex) reduce tissue macrophage activation, reduce hypoxia, and decrease inflammatory cytokines, chemokines, and bradykinin, thereby restoring the alveolar-capillary barrier and regenerating alveolar type II cells. This composition is highly suitable for the treatment of COPD and ARDS caused by viral infections, particularly COVID-19, and for improving lung function in the prognosis.

[0042] Other modifications and variations of the present invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Thus, while only certain embodiments of the invention have been specifically described herein, it will be apparent that numerous modifications can be made thereto without departing from the spirit and scope of the invention, to be read only in conjunction with the appended claims. Another aspect of the present invention may be as follows. [1] A method for regenerating alveolar cells in a mammal having emphysema, the method comprising administering to the mammal a composition comprising 20% ​​by weight or more of bisdemethoxycurcumin to result in a reduction in the characteristics of emphysema. [2] The method according to [1] above, wherein the composition further contains 10 to 35% by mass of demethoxycurcumin and 10 to 45% by mass of curcumin. [3] The method according to [1], wherein the characteristics of emphysema are selected from the group consisting of hypoxia, decreased levels of pulmonary surfactant proteins, increased alveolar-capillary barrier permeability, inflammation, increased neutrophil accumulation and recruitment, increased intraalveolar pressure, increased oxidative stress, increased inflammatory cytokines and chemokines, increased numbers of activated T lymphocytes, and muscle damage. [4] The method according to [1] above, wherein the emphysema is induced by enzymes, viruses, bacteria, smoking, and particulate irritants. [5] The method according to [1] above, wherein the mammal is a human. [6] A method for the therapeutic management of chronic obstructive pulmonary disease in a mammal, comprising administering to the mammal a composition comprising 20% ​​by weight or more of bisdemethoxycurcumin to result in a reduction in the characteristics and symptoms of chronic obstructive pulmonary disease. [7] The method according to [6], wherein the composition further contains 10 to 35% by mass of demethoxycurcumin and 10 to 45% by mass of curcumin. [8] The method according to [6] above, wherein the chronic obstructive pulmonary disease is emphysematous or non-emphysematous. [9] The method according to [6], wherein the characteristics of chronic obstructive pulmonary disease are selected from the group consisting of hypoxia, decreased levels of pulmonary surfactant proteins, increased alveolar-capillary barrier permeability, inflammation, increased neutrophil accumulation and recruitment, increased intraalveolar pressure, increased oxidative stress, increased inflammatory cytokines and chemokines, increased numbers of activated T lymphocytes, and muscle damage.

[10] The method according to [6], wherein the symptoms of chronic obstructive pulmonary disease are selected from the group consisting of shortness of breath, particularly during physical activity, wheezing, chest tightness, chronic cough that may produce mucus, respiratory infections, lack of energy, unintentional weight loss, and swelling of the ankles, feet, or lower legs.

[11] The method according to [6] above, wherein the chronic obstructive pulmonary disease is induced by enzymes, viruses, bacteria, smoking, and particulate irritants.

[12] The method according to [6] above, wherein the mammal is a human.

[13] A method for preventing progression of chronic obstructive pulmonary disease to acute respiratory distress syndrome in a mammal, comprising the step of administering to the mammal a composition containing 20% ​​by weight or more of bisdemethoxycurcumin to prevent the onset of acute respiratory syndrome by reducing the characteristics and symptoms of chronic obstructive pulmonary disease.

[14] The method according to

[13] above, wherein the composition further contains 10 to 35% by mass of demethoxycurcumin and 10 to 45% by mass of curcumin.

[15] The method according to

[13] above, wherein the chronic obstructive pulmonary disease is emphysematous or non-emphysematous.

[16] The method according to

[13] , wherein the characteristics of chronic obstructive pulmonary disease are selected from the group consisting of hypoxia, decreased levels of pulmonary surfactant proteins, increased alveolar-capillary barrier permeability, inflammation, increased neutrophil accumulation and recruitment, increased intraalveolar pressure, increased oxidative stress, increased inflammatory cytokines and chemokines, increased numbers of activated T lymphocytes, and muscle damage.

[17] The method according to

[13] , wherein the symptoms of chronic obstructive pulmonary disease are selected from the group consisting of shortness of breath, particularly during physical activity, wheezing, chest tightness, chronic cough that may produce mucus, respiratory infections, lack of energy, unintentional weight loss, and swelling of the ankles, feet, or lower legs.

[18] The method according to

[13] above, wherein the chronic obstructive pulmonary disease is induced by enzymes, viruses, bacteria, smoking, and particulate irritants.

[19] The method according to

[13] above, wherein the mammal is a human.

Claims

1. A composition for use in regenerating alveolar cells in a mammal having emphysema, comprising 20% ​​by mass or more of bisdemethoxycurcumin, wherein the composition is administered to the mammal to bring about a reduction in the characteristics of emphysema.

2. 2. The composition of claim 1, wherein the characteristics of emphysema are selected from the group consisting of hypoxia, decreased levels of pulmonary surfactant proteins, increased alveolar-capillary barrier permeability, inflammation, increased neutrophil accumulation and recruitment, increased intra-alveolar pressure, increased oxidative stress, increased inflammatory cytokines and chemokines, increased numbers of activated T lymphocytes, and muscle damage.

3. 10. The composition of claim 1, wherein the emphysema is induced by enzymes, viruses, bacteria, smoking, and particulate irritants.

4. A composition for use in the therapeutic management of chronic obstructive pulmonary disease in a mammal, comprising at least 20% by mass of bisdemethoxycurcumin, wherein the composition is administered to the mammal to bring about a reduction in the characteristics and symptoms of chronic obstructive pulmonary disease.

5. A composition for use in preventing the progression of chronic obstructive pulmonary disease to acute respiratory distress syndrome in a mammal, comprising 20% ​​by mass or more of bisdemethoxycurcumin, wherein the composition is administered to the mammal to prevent the onset of acute respiratory distress syndrome by bringing about a reduction in the features and symptoms of chronic obstructive pulmonary disease.

6. 6. The composition of claim 1, 4, or 5, wherein the composition further comprises 10 to 35% by weight of demethoxycurcumin and 10 to 45% by weight of curcumin.

7. The composition according to claim 4 or 5, wherein the chronic obstructive pulmonary disease is emphysematous or non-emphysematous.

8. 6. The composition of claim 4 or 5, wherein the characteristics of chronic obstructive pulmonary disease are selected from the group consisting of hypoxia, decreased levels of pulmonary surfactant proteins, increased alveolar-capillary barrier permeability, inflammation, increased neutrophil accumulation and recruitment, increased intra-alveolar pressure, increased oxidative stress, increased inflammatory cytokines and chemokines, increased numbers of activated T lymphocytes, and muscle damage.

9. 6. The composition of claim 4 or 5, wherein the symptoms of chronic obstructive pulmonary disease are selected from the group consisting of shortness of breath, especially during physical activity, wheezing, chest tightness, chronic cough that may produce mucus, respiratory infections, lack of energy, unintentional weight loss, and swelling of the ankles, feet, or lower legs.

10. 6. The composition of claim 4 or 5, wherein the chronic obstructive pulmonary disease is induced by enzymes, viruses, bacteria, smoking, and particulate irritants.

11. The composition of claim 1, 4 or 5, wherein the mammal is a human.

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