hereinafter , embodiments of the present invention will be described herein . however , the embodiments do not limit the present invention . ht - 29 human colon cancer cell - derived epithelial cells and u937 human - derived mononuclear cells were cultured at a temperature of 37 ° c . in the presence of 5 % co2 in an rpmi 1640 medium supplemented with 10 % fbs and 1 % penicillin / streptomycin ( ps ). when the cells were grown in the medium at a density of 80 % or more , the cells were subjected to a subculture process with a ratio of 1 : 3 for passage before being used for the experiments . ht - 29 cells were cultured at a concentration of 2 × 105 cells / cm2 per well in a 24 - well plate , and then , a test drug was pre - treated for 1 hour in a medium supplemented only with 1 % fbs and 1 % ps . next , 10 μg / ml bcecf - am was treated therewith to allow a reaction at a temperature of 37 ° c . for 30 minutes . afterwards , the ht - 29 cells , which were previously treated with the test drug , were allowed to react with u937 cells in which bcecf was loaded , or with tnf - α ( 10 ng / ml ) or interleukin ( il )- 6 , at a temperature of 37 ° c . for 3 hours . after the completion of the reaction , the medium of the ht - 29 cells was removed , and then , the ht - 29 cells were washed with pbs twice , thereby removing u937 cells that were not adhered to the ht - 29 cells . next , in terms of cell dissolution , 0 . 1 % triton x - 100 ( 0 . 1 m tris ) was allowed to react with the cells for 30 minutes at room temperature , and then , the the cells were subjected to quantify by measuring fluorescence thereof using a fluostar optima microplate reader ( bmg labtechnologies , germany ) ( carvalho et al ., 1996 ; thapa et al ., 2008 ). the results of analyzing inhibitory activity of the test drug ( 1 μm ) against the adhesion to the tnf - α - induced intestinal epithelial cells ( i . e ., ht - 29 ) and the mononuclear cells ( i . e ., u937 ) are shown in table 2 , and the results of analyzing inhibitory activity of the test drug ( 1 μm ) against the adhesion to the il - 6 - induced intestinal epithelial cells ( i . e ., ht - 29 ) and the mononuclear cells ( i . e ., u937 ) are shown table 4 . in the case mesalazine ( a positive control ), which is an active metabolite of sulfasalazine that is a drug in use for the treatment of inflammatory bowel disease in current clinical , mesalazine showed a inhibition rate of 6 % or less against the adhesion between the tnf - α - induced intestinal epithelial cells ( i . e ., ht - 29 ) and the mononuclear cells ( i . e ., u937 ), and that is , mesalazine hardly exhibited the inhibitory effect ( see table 3 ). however , in the case of 13 types of aminopyridinol compounds , the compounds showed an inhibition rate of 50 % or more at 1 μm concentration , and more particularly , aminopyridinol compound 11 exhibited significantly excellent inhibition rates of 80 % or more . aminopyridinol compounds 7 , 9 , 10 , 12 , 19 and 34 showed inhibition rates between 70 ˜ 79 %, aminopyridinol compounds 30 , 32 , 33 and 35 showed inhibition rates between 60 ˜ 69 %, and aminopyridinol compounds 31 and 38 showed inhibition rates between 50 ˜ 59 %. in addition , aminopyridinol compounds 20 , 23 , 28 , 29 , 36 and 37 showed inhibition rates between 40 ˜ 49 %, aminopyridinol compounds 4 , 6 , 8 , 13 , 25 and 27 showed inhibition rates between 30 ˜ 39 %, aminopyridinol compounds 1 , 2 , 3 , 14 , 16 , 17 , 21 and 22 showed inhibition rates between 20 ˜ 29 %, and aminopyridinol compounds 5 , 15 , 18 , 24 and 26 showed inhibition rates of less than 20 %. accordingly , it was confirmed that the compounds having inhibition rates of less than 20 % mostly exhibited better inhibitory activity than that of mesalazine . regarding the inhibitory effects of mesalazine against adhesion between the il - 6 - induced intestinal epithelial cells ( i . e ., ht - 29 ) and mononuclear cells ( i . e ., u937 ), mesalazine hardly exhibited inhibitory activity when having an inhibitory rate of 5 % or less at 1 mm concentration ( see table 5 ). however , 13 types of aminopyridinol compounds showed inhibition rates of 50 % or more at 1 μm concentration , and more particularly , aminopyridinol compound 11 exhibited significantly excellent inhibition rates of 70 % or more . aminopyridinol compounds 7 , 9 , 10 , 12 , 19 , 23 , 30 and 34 showed inhibition rates between 60 ˜ 69 %, aminopyridinol compounds 32 , 33 , 35 and 38 showed inhibition rates between 50 ˜ 59 %, aminopyridinol compounds 29 and 31 showed inhibition rates between 40 ˜ 49 %. in addition , aminopyridinol compounds 20 , 28 , 36 and 37 showed inhibition rates between 30 ˜ 39 %, aminopyridinol compounds 4 , 6 , 8 , 13 , 16 , 17 , 21 , 25 and 27 showed inhibition rates between 20 ˜ 29 %, and aminopyridinol compounds 1 , 2 , 3 , 5 , 14 , 15 , 18 , 22 , 24 and 26 showed inhibition rates of less than 20 %. accordingly , it was confirmed that the compounds having inhibition rates of less than 20 % mostly exhibited better inhibitory activity than that of mesalazine . the animals used herein was a 7 to 8 week sprague dawley species purchased from orientbio ( korea ). after the purchase , the animals were stabilized with typical solid feed for 7 days before being used for the experiments . the animals were freely supplied with feed and water during the experiments , and stayed in conditions maintaining a cage temperature of about 25 ± 1 ° c . and relative humidity of about 50 ± 10 %. the cage was equipped with an automatic lighting management system to adjust 12 - hour light - dark cycle . regarding experimental groups , 4 groups ( i . e ., a control group , a group administered with tnbs only , a group administration in combination of tnbs + sulfasalazine 300 mg / kg , and a group administration in combination of tnbs + aminopyridinol 1 mg / kg ) were used for the experiment according to the randomized block design , wherein each group consists of 6 rats having an average weight of about 180 ± 10 g . the rats that had been fasting for 24 hours were anesthetized with diethyl ether , and then , a 1 ml syringe connected with a polyethylene catheter was used to slowly inject 5 % tnbs into the lumen of the colon through the anus , wherein 5 % tnbs was diluted with 50 v / v % ethanol . to prevent leakage of 5 % tnbs from the anus , the rats turned upside down and let stand for 60 seconds . regarding the control group , only a vehicle ( 50 v / v % ethanol ) was injected to the rats of the control group in the same manner as in preparation of other experimental groups ( thapa et al ., 2008 ). to examine the effects of the drug , the rats were administered with a drug every day at a certain time from the second day to the fifth day of the tnbs treatment . changes in weight of the rats used in the experiments were observed by using a digital mass meter , beginning from the fasting step , the tnbs administration , and the drug administration . after the large intestine of the rat was extracted , a 5 to 6 cm - long tissue therefrom was cut from the anus to a size of 1 cm , and then , subjected to measurement of the tissue weight . regarding compounds exhibiting excellent inhibitory activity in in vitro adhesion test , inhibitory activity of the compounds against in vivo enteritis was measured , and the results are shown in table 6 . in an animal model with the rats having a weight ranging from about 180 to about 190 g , having colitis , and causing intestinal inflammation by using 5 % tnbs , changes in the weight were regularly observed for 5 days at a certain time , based on the weight of the rats that were not treated with tnbs yet . consequently , it was confirmed that the control group treated with the vehicle continued to increase the weight of the rate , and that the group treated only with tnbs continued to decrease the weight of the rate , but was slightly recovered on the fifth day . however , the recovered weight of the tnbs - treated group was still significantly smaller than the weight of the rats in the normal group . the weight of the rats in the positive group that was treated with 300 mg / kg of sulfasalazine was slowly recovered , but was still smaller than the vehicle - treated control group . however , the rats in the sulfasalazine - treated group showed a significant increase in the weight thereof , compared to the rats in the group treated only with tnbs , thereby eventually showing excellent weight recovery of about 50 %. even if 1 mg / kg of aminopyridinol compounds 9 , 12 , and 23 were administered to the rats , significant weight recovery rates of the intestine tissue ( 51 % to 83 %) was resulted . the compounds listed in the state of having excellent weight recovery rates are as follows : aminopyridinol compounds 9 & gt ; 23 & gt ; 12 . the large intestine that was extracted after the completion of the drug administration for 5 days was observed with the naked eye . consequently , it was observed that the large intestine of the rats in the tnbs - treated group had swelling and redness , compared to that of the rats in the control group , and in addition , showed appendiceal sweeling , congestion , and synechia of the intestine tissue . in the positive group treated with 300 mg / kg of sulfasalazine , it was confirmed that symptoms observed with the naked eyes and synechia between other organs or inflamed large intestine was significantly inhibited . in the case of the group administered with the aminopyridinol compounds , the symptoms above were significantly improved , compared to the group treated with 300 mg / kg of sulfasalazine . after the large intestine of the rat was extracted , a 5 to 6 cm - long tissue therefrom was cut from the anus , and then , subjected to measurement of the tissue weight . consequently , it was confirmed that the group treated only with tnbs had a significant increase in the weight of the intestine having swelling , compared to the vehicle - treated control group . in the positive control group treated with 300 mg / kg of sulfasalazine , it was confirmed that the weight recovery rate of the intestine tissue was about 70 %. in the case of the group administered with the aminopyridinol compounds , regardless of the administration of 1 mg / kg of the compounds , the weight recovery rate of the intestine tissue was about 74 % to about 97 %. the compounds listed in the state of having excellent recovery rate of the intestine tissue are as follows : aminopyridinol compounds 9 & gt ; 23 & gt ; 12 . male balb / c mice were orally administered once with compound 01 suspended in a 0 . 5 % methylcellose solution , in a dosage of each of 0 . 5 g / kg , 1 g / kg , and 2 g / kg , and then , for 7 days , the survival rate and body weight of the mice were measured . after the administration , the death , clinical symptoms , and body weight change of the mice were analyzed , and a hematological examination and a blood biochemical examination were performed . then , the autopsy of the mice was performed , and abnormality of abdominal cavity organs and thoracic cavity organs was examined . examination results show that all the animals did not have distinguishing clinical symptoms or did not die , and even body weight change , blood examination , blood biochemical examination , and autopsy results showed no toxicity change . as described above , the compounds according to the present invention did not show any toxicity change in mice up to the dosage of 2 g / kg , and accordingly , since the compounds have an oral administration lethal dose ( ld50 ) of 2 g / kg or more , the compounds are considered as a safe material . hereinafter , preparation examples for the preparation of a composition including compound 1 will be described . however , the preparation examples do not limit the present invention and are presented herein for illustrative purpose only . 20 mg of compound 1 , 100 mg of lactose , and 10 mg of talc were mixed , and a sealed bag was filled with the mixture to prepare a powder formulation . 20 mg of compound 1 , 100 mg of corn starch , 100 mg of lactose , and 2 mg of magnesium stearate were mixed , and then , the mixture was compressed according to a conventional tablet preparation method to form a tablet formulation . 10 mg of compound 1 , 100 mg of corn starch , 100 mg of lactose , and 2 mg of magnesium stearate were mixed , and then , the components were mixed according to a conventional capsule preparation method . a gelatin capsule was filled with the result to prepare a capsule formulation . 10 mg of compound 1 , an appropriate amount of injectable sterilized distilled water , and an appropriate amount of a ph controller were mixed , and then , according to a conventional injectable preparation method , an injectable liquid formulation was prepared including the dosage described above per 1 ample ( 2 ml ). 10 mg of compound 1 , 250 mg of peg - 4000 , 650 mg of peg - 400 , 10 mg of white vaseline , 1 . 44 mg of methyl p - hydroxybenzoate , 0 . 18 mg of propyl p - hydroxybenzoate , and the balance of purified water were mixed , and then , an ointment formulation was prepared according to a conventional ointment preparation method . it should be understood that the exemplary embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation , and do not limit the scope of the present invention . accordingly , the substantial scope of the present invention is defined by the following claims and equivalents thereto .
provided is a method of treating inflammatory bowel disease comprising administering a pharmaceutical composition comprising a 6 - aminopyridin - 3 - ol compound or a pharmaceutically acceptable salt thereof as an active ingredient to a subject , which inhibits colitis in a model of inflammatory bowel disease , and accordingly , they may be suitable for use as a drug for the prevention or treatment of inflammatory bowel disease .