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.
