Patent Application: US-201113085017-A

Abstract:
a pharmaceutical composition for improving a cardiac function is provided . the pharmaceutical composition comprises an effective amount of an active component being one of a kmup - 3 compound and a salt thereof .

Description:
male rats ( 250 - 300 g ) are provided by the national laboratory animal breeding and research center ( taipei , taiwan ) and housed under constant temperature and controlled illumination . food and water are available ad libitum . mi is induced by ligation of the left anterior descending ( lad ) coronary artery according to liang et al ., 2006 . briefly , under general anaesthesia with pentobarbital sodium ( 30 mg · kg − 1 , i . p . ), the heart is exposed via a small left thoracotomy . the lad is ligated with 6 . 0 silk at 2 mm from the origin , and the wound is closed with primary suture . after the operation , the animals are observed until fully conscious . sham animals undergo similar thoracotomy and pericardiotomy , except that the ligature around the coronary artery is not tied . all the animals are administered with s . c . doses of analgesia ( ketoprofen ; 3 mg · kg − 1 ) and antibiotics ( gentamicin ; 0 . 7 mg · kg − 1 ) for 2 days . this study was approved by the animal care and use committee at kaohsiung medical university . surviving rats are fitted with alzet osmotic minipumps ( model 2ml4 , durect corporation , cupertino , calif ., usa ) immediately after recovery . at this time , most of the rats are still anaesthetized from the previously administered pentobarbital sodium ; a further bolus dose of pentobarbital sodium ( 15 mg · kg − 1 ) is given if necessary . these alzet osmotic minipumps has a 2 ml capacity and a mean pumping rate of 2 . 5 ml · h − 1 within 4 weeks . in the treatment group , the minimumps are filled with hydrochloride of kmup - 3 ( kmup - 3 · hcl ; 0 . 3 mg · kg − 1 · day − 1 ). in the sham and mi groups , the minipumps are filled with saline . echocardiography is performed in all animals at the time point 4 weeks post - surgery . each rat is anaesthetized with pentobarbital sodium ( 30 mg · kg − 1 , i . p .). the anterior chest wall is shaved and acoustic coupling gel was applied . an echocardiography system ( hewlett - packard sonos 1500 , 5 - mhz probe , andover , mass ., usa ) is used through m - mode longitudinal and transverse parasternal views to measure lv end - systolic dimension ( lvesd ) and lvedd . lv fractional shortening ( fs ) is analysed from the lv dimensions using the following formula ( louhelainen et al ., 2007 ): after echocardiography , a pe - 50 catheter is inserted through the right carotid artery to measure the lv systolic pressure ( lvsp ), lv end - diastolic pressure ( lvedp ) and maximum rates of pressure development ( lv + dp / dt ) and relaxation ( lv − dp / dt ). at the end of the recording , the inferior vena cava and pulmonary veins are opened to avoid fluid overload . the hearts are then excised and the atria and right ventricles are dissected out . sections of the lv are embedded in mounting medium and the remaining tissues are transferred in the liquid nitrogen for further evaluation . throughout the whole procedure , a further bolus dose of pentobarbital sodium ( 15 mg · kg − 1 ) is given if required . histological analyses are performed to all of the heart . rats are randomly chosen to be analyzed for area of risk after coronary artery ligature by evan blue staining as described by harada et al . ( 2005 ). infarct size is determined by 2 , 3 -, 5 - triphenyltetrazolium chloride ( ttc ) staining , and is expressed as ratio of the surface area of the infarct wall to the entire surface area of the lv ( vandegriff et al ., 2008 ). the heart section is stained with masson &# 39 ; s trichrome to assess fibrosis as previously described ( lin et al ., 2009 ; yeh et al ., 2010 ). the infarct area is visible as white and non - infarct as red after being stained with ttc . peri - infarct area is defined as the region of myocardium extending 1 mm from the infarct scar . ten sections in each heart are analyzed for the percentage of fibrosis . the mean fibrotic change after mi is compared between study groups and the masson &# 39 ; s trichrome staining of the sham group as control . the total lv and infarct area and fibrosis region are measured by an investigator without knowledge of the treatment group using us nih imagej , 1 . 42q . human cardiac fibroblasts ( hcfs ; catalogue number : 306 - 05f ) are purchased from cell applications inc . ( san diego , calif ., usa ). the cells are cultured as monolayer in dulbecco &# 39 ; s modified eagle &# 39 ; s medium containing 10 % fetal bovine serum at 37 ° c . in 95 % humidity and 5 % co 2 . cells are harvested at passage 3 - 6 for experiment . before treatment , hcfs are washed twice with serum - free medium and switched to serum - free medium for 24 h . to stimulate mmp - 9 expression , the cultures are treated with 10 ng · ml − 1 transforming growth factor - b ( tgf - β ) for 24 h . hcfs are pretreated with kmup - 3 ( 10 mmol · l − 1 ) or in combination with l - name ( 100 mmol · l − 1 ) for 1 h before addition of 10 ng · ml − 1 tgf - β for 24 h . each experiment is repeated three times . the hearts are sonicated in 50 mmol · l − 1 tris for 10 s two times and centrifuged at 13 , 000 rpm at 4 ° c . for 30 min . the protein concentration of the supernatants is determined by using bovine serum albumin as the standard . the cell extracts are boiled in ratio of 4 : 1 with sample buffer ( 100 mmol · l − 1 tris , ph 6 . 8 , 20 % glycerol , 4 % sds , and 0 . 2 % bromophenol blue ). electrophoresis is performed using 10 % sds - polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes ( millipore corporation , billerica , mass ., usa ). the membranes are blocked with tris - buffered saline ( 20 mmol · l − 1 tris and 137 mmol · l − 1 nacl , ph 7 . 6 ) containing 0 . 1 % tween - 20 ( ttbs ) and 5 % nonfat milk at room temperature for 1 h , washed with ttbs , and then incubated overnight at 4 ° c . with mmp - 9 , timp - 1 ( millipore , temecula , calif ., usa ), or enos ( bd transduction laboratories , franklin lakes , n . j ., usa ) primary antibody . the membranes are washed in ttbs before being incubated with horseradish peroxidase - conjugated antibody against mouse or rabbit igg ( santa cruz biotechnology , santa cruz , calif ., usa ) for 1 h . the membranes are then washed in ttbs again and developed with the enhanced chemiluminescence for the detection of the specific antigen . the intensity of the bands is quantified by densitometry . early surgery - related death assigned to severe thoracic bleeding within 6 h after surgery was similar in all groups ( 9 %) and these rats were therefore excluded from final analysis . none of the animals died during the post - mi treatment period . as indicated in table 1 , mi induces significant cardiac hypertrophy with an increased heart weight to body weight ratio . kmup - 3 treatment prevents the cardiac remodelling process . the lv + dp / dt decreased in the mi group , while administration of kmup - 3 ( 0 . 3 mg · kg − 1 · day − 1 ) for 4 weeks improved cardiac systolic function . the lvsp and lv − dp / dt also tends to be decreased in the mi group but none of these differences reached statistical significance . a small increase in heart rate occurrs after kmup - 3 treatment , but this is not statistically significant . as fig2 ( b )-( c ) show , the lvedd and lvesd of mi rats are increased while being compared with the sham - operated group . the decreased systolic function is also demonstrated by the decrease in fs in the mi group ( fig2 ( a )). the cardiac remodeling and dysfunction may be prevented by the administration of kmup - 3 for 4 weeks . the area of risk after coronary artery ligature is determined by evans blue staining , and there is no difference between treatment groups ( fig3 a ). infarction size , determined by ttc staining , decreases significantly after kmup - 3 treatment ( 47 . 4 ± 3 . 7 % vs . 33 . 6 ± 1 . 7 %, respectively , p & lt ; 0 . 05 ) ( fig3 b ). kmup - 3 attenuates cardiac fibrosis in both the central - infarct and peri - infarct areas as determined by masson &# 39 ; s trichrome staining . the anti - fibrotic effect of kmup - 3 is also found in the non - infarcted area , as the percentage of fibrosis is comparable with that of the sham - operated hearts . mmp - 9 expression increases significantly in mi rats , compared with sham - operated rats . the administration of kmup - 3 decreases mmp - 9 expression in mi rats ( fig5 a ). it is also noted that the expression of the mmp - 9 inhibitor timp - 1 increases significantly after treatment with kmup - 3 ( fig5 b ). a small decrease in enos expression is noted in mi rats . after treatment with kmup - 3 , the enos expression increases significantly than rats in the mi group ( fig5 c ). to investigate the mechanism of enos dependent cardiac protection in vitro , hcfs are stimulated with tgf - β ( 10 ng · ml − 1 ) and the expression of mmp - 9 and timp - 1 are measured . mmp - 9 expression increases significantly following tgf - b stimulation ( fig6 a ) and the administration of kmup - 3 ( 10 mmol · l − 1 ) attenuates the expression of mmp - 9 . pretreatment with the enos inhibitor n ω - nitro - l - arginine methyl ester ( l - name ; 100 mmol · l − 1 ) reverses the inhibition of mmp - 9 expression ( fig6 a ). at the same time , timp - 1 expression is significantly enhanced by the treatment with kmup - 3 ( fig6 b ). kmup - 3 ( 8 . 3 g ) is dissolved in a mixture of ethanol ( 10 ml ) and 1n hcl ( 60 ml ) the solution is reacted at 50 ° c . for 20 mins , the ethanol ( or methanol ) is added thereinto under room temperature and the solution is incubated over night for crystallization and filtrated to obtain kmup - 3hcl salt ( 6 . 4 g ). kmup - 3 ( 8 . 3 g ) is dissolved in a mixture of ethanol ( 10 ml ) and citric acid ( 4 g ) and reacted at 50 ° c . for 20 min , the ethanol ( or methanol ) is added thereinto under room temperature and the solution is incubated over night for crystallization and filtrated to obtain kmup - 3 citric acid salt ( 10 . 5 g ). kmup - 3 ( 8 . 3 g ) is dissolved in a mixture of ethanol ( 10 ml ) and nicotinic acid ( 2 . 4 g ) and reacted at 50 ° c . for 20 min , the ethanol ( or methanol ) is added thereinto under room temperature and the solution is incubated over night for crystallization and filtrated to obtain kmup - 3 nicotinic acid salt ( 8 . 3 g ). the following components are weighted respectively and filled into a tabletting machine after mixing for preparing tablets . 1 . creemers e e , davis j n , parkhurst a m , leenders p , dowdy k b , hapke e , et al . ( 2003 ). deficiency of timp - 1 exacerbates lv remodeling after mi in mice . am j physiol heart circ physiol 284 : h364 - 371 . 2 . halapas a , zacharoulis a , theocharis s , karavidas a , korres d , papadopoulos k , et al . ( 2008 ). serum levels of the osteoprotegerin , receptor activator of nuclear factor kappa - b ligand , metalloproteinase - 1 ( mmp - 1 ) and tissue inhibitors of mmp - 1 levels are increased in men 6 months after acute mi . clin chem lab med 46 : 510 - 516 . 3 . harada m , qin y , takano h , minamino t , zou y , toko h et al . ( 2005 ). g - csf prevents cardiac remodeling after myocardial infarction by activating the jak - stat pathway in cardiomyocytes . nat med 11 : 305 - 311 . 4 . lee t m , lin m s , chang n c ( 2008 ). effect of atp - sensitive potassium channel agonists on ventricular remodeling in healed rat infarcts . j am coll cardiol 51 : 1309 - 1318 . 5 . li g h , shi y , chen y , sun m , sader s , maekawa y , et al . 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