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Example 3
Analysis of Lung Function Regulatory Effect of Regorafenib in Animal Model with Chronic Obstructive Pulmonary Disease
In addition, the present inventors performed an experiment to determine whether regorafenib is able to restore lost function of damaged lungs in order to confirm whether regorafenib is able to treat chronic obstructive pulmonary disease.
Specifically, 6-week-old C57BL/6 mice were purchased and acclimatized for one week, after which the acclimatized 7-week-old mice (average 25 g) were subjected to nasal administration with saline and PPE [elastase from porcine pancreas] (EPC, EC134) at a concentration of 5 U. Two weeks after administration, the mice were orally administered with 200 μl of regorafenib (SelleckChem, BAY 73-4506) at a concentration of 5 mg/kg for one week. Then, the mice were anesthetized using 300 μl of anesthetic on week 3, after which the lung function of each mouse was measured using a flexiVent (system for measuring the function of lungs) and quantified by subtracting the error value therefrom.
As shown in
Based on the aforementioned results, the present inventors confirmed that regorafenib is capable of inhibiting increased immune response caused by chronic obstructive pulmonary disease, and of improving and restoring damaged and changed lung structure and lung function, whereby regorafenib can be used as a novel therapeutic agent for chronic obstructive pulmonary diseases including emphysema, chronic bronchitis, asthma, and pneumonia.
As is apparent from the above description, regorafenib according to the present invention is capable of inhibiting increased immune response, which is a symptom of chronic obstructive pulmonary disease, and of improving and restoring changed lung structure and damaged lung function, and can be effectively used for the manufacture of a medicament for the prevention, amelioration, or treatment of chronic obstructive pulmonary disease.
Although preferable exemplary embodiments of the present invention have been disclosed in detail above, it will be obvious to those skilled in the art that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive way. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.
Example 1
Analysis of Immune Response Inhibitory Effect Through Treatment with Regorafenib in Animal Model with Chronic Obstructive Pulmonary Disease
The present inventors performed an experiment to determine whether regorafenib has activity capable of treating chronic obstructive pulmonary disease. The initial response induced by PPE (porcine pancreatic elastase) representatively includes immune responses that occur in damaged lung tissue. The activated immune response promotes the proliferation of various immune cells and the secretion of various inflammatory mediators. Specifically, the overall number of immune cells increases with an increase in individual immune cells involved in the immune response. In particular, an increase in the numbers of macrophages, lymphocytes, eosinophils, and neutrophils is observed. The increase in the number of these cells can be confirmed through a bronchoalveolar lavage (BAL) fluid test.
Therefore, in the present invention, mice with chronic obstructive pulmonary disease, particularly emphysema, in which alveolar damage was induced by reducing elastin content using PPE, were prepared and then treated with regorafenib to thus analyze whether chronic obstructive pulmonary disease was ameliorated. Specifically, 6-week-old C57BL/6 mice were purchased and acclimatized for one week. Thereafter, the acclimatized 7-week-old mice (average 25 g) were orally administered with 200 μl of regorafenib (SelleckChem, BAY 73-4506) at a concentration of 5 mg/kg. The next day, that is, on day 1, each mouse's status was observed, followed by nasal administration of PPE [elastase from porcine pancreas] (EPC, EC134) at a concentration of 5 U. Then, each mouse's status was observed on days 2 and 3, followed by oral administration of 200 μl of regorafenib at a concentration of 5 mg/kg. On day 4, the mice were anesthetized using 200 μl of anesthetic (Zoletil to rompun to saline at a ratio of 1:1:8), after which the neck of each mouse was incised to thus expose the airway, which was then cut in half and a catheter was inserted therein. Thereafter, 1 ml of HBSS [Hanks' balanced salt solution] (Sigma Aldrich, H6648) was placed in a syringe, which was then connected to the catheter, after which procedures of injection and then extraction were performed three times to collect the BAL fluid. The BAL fluid thus obtained was centrifuged to collect cells, and the collected cells were suspended in HBSS, treated with a red blood cell lysis buffer (Sigma Aldrich, 11814389001) at 1:1, allowed to react at room temperature for 2 minutes, further added with HBSS, and then centrifuged. In this procedure, red blood cells are removed. The centrifuged cells were diluted and centrifuged using a Cytospin (Hanil Science), and then attached to a slide. The attached cells were stained using a NovaUltr™ Hema-Diff Stain Kit (IHCWORLD, IW-3017) according to the manufacturer's instructions. The stained slide was analyzed and quantified using a microscope.
According to the method described above, a mouse animal model of chronic obstructive pulmonary disease was prepared by administering the mouse lungs with 5 U of PPE, and in order to verify whether the immune response was modulated, one day before administration of PPE and on days 2 and 3 after administration of PPE, regorafenib (5 mg/kg) was administered thereto, and on day 4, bronchoalveolar lavage fluid was obtained and the immune cells therein were stained to thus quantitatively analyze the type of immune cells and the number of individual cells. Here, mice administered only with saline were used as a control group, and 5 mice were placed in each group.
Based on the results of analysis, the number of immune cells increased significantly in the PPE treatment group compared to the control group, whereas in the regorafenib treatment group, the number of immune cells increased due to PPE decreased by about 50% (
Based on these results, the present inventors have found that regorafenib of the present invention is capable of inhibiting or reducing the increase in the initial immune response, which is a symptom of chronic obstructive pulmonary disease.
Example 2
Analysis of Improvement Effect of Changes in Lung Structure Through Treatment with Regorafenib in Animal Model with Chronic Obstructive Pulmonary Disease
One of the pathological indicators of chronic obstructive pulmonary disease is changed alveolar size. Changes in the alveolar size can be analyzed by quantifying the mean linear intercept (Lm) value, representing the average distance between the alveolar walls, in a microscopic field of view. When chronic obstructive pulmonary disease is induced, this value increases, indicating an increase in the alveolar size.
Therefore, the present inventors analyzed whether it is possible to alleviate changes in lung structure by measuring the change in Lm value due to treatment with regorafenib in a mouse animal model with emphysema, a chronic obstructive pulmonary disease, prepared over three weeks through administration of PPE (
Specifically, 6-week-old C57BL/6 mice were acclimatized for one week, after which the acclimatized 7-week-old mice (average 25 g) were subjected to nasal administration with saline and PPE [elastase from porcine pancreas] (EPC, EC134) at a concentration of 5 U. Two weeks after administration, the mice were orally administered with 200 μl of regorafenib (SelleckChem, BAY 73-4506) at a concentration of 5 mg/kg for one week, and on week 3, the lungs were extracted from each mouse, placed in a cassette, immersed in 10% NBF, and stored for one day, after which a parafilm block was prepared using a tissue processor. The prepared block was cut using a sectioning machine, attached to a slide, subjected to H&E staining, and then quantified by measuring the Lm value based on microscopic images.
Based on the results of analysis, as shown in
Therefore, it has been found that regorafenib has activity capable of improving and regulating structural changes in alveoli accompanying chronic obstructive pulmonary disease.