Patent Application: US-201113179111-A

Abstract:
the theophylline derivative disclosed in the present invention is characterized by having the pharmaceutical functions of osteoporosis . the theophylline derivative protects against bone resorption and inflammatory mediator infiltration .

Description:
the present invention will now be described more specifically with reference to the following embodiments . it is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for purposes of illustration and description only ; it is not intended to be exhaustive or to be limited to the precise form disclosed . 10 % fbs , 20 ml glutamine , 20 ml antibiotics , 3 g nahco 3 and dmem powder were mixed together in 2000 ml ddh 2 o and was adjusted to ph 7 . 2 . after cells were defrosted , they were added in to 10 cm cell culture plates with suitable amount of the culture medium , an then were put in a incubator with 37 ° c ., 5 % co 2 / air to wait for the attachment of the cells . on the next day , the culture medium was replaced to remove dmso . after the old medium was removed , the cells were flushed down by the new culture medium and were counted by trypan blue and then were separated into other plates . cells were plated onto 96 - well plates at a density of 10 3 cells per well . after 24 hours incubation , the different concentrations of the different drugs were administrated into the plates . at 24 hours and 48 hours , 10 μl / well mtt reagent ( 0 . 05 g / 10 ml pbs ) was added to the medium and the reaction was allowed to proceed for 4 hours . subsequently , the medium was removed , and 100 μl / well isopropanol was added to solved formazan . the plates were shaken for 15 minutes and stood for 15 minutes , and then the absorbance was measured at wavelength of 540 nm and 630 nm . the ratio of the absorbance of treated cells relative to that of the control cells was calculated and expressed as a percentage of cell survival . the brdu assay was followed the instructions of cell proliferation elisa brdu kit . 10 3 cells were plated per well in 96 - well plates . after 24 hours incubation , the 10 ng / ml rankl and the different concentrations of kmup - 1 , theophylline and sildenafil were added into the plates . at 24 hours , 48 hours and 72 hours , 10 μl / well brdu labeling reagent was added to the medium and the reaction was allowed to proceed for 4 hours in dark . subsequently , the medium was removed , and 200 μl / well fixdenat solution was added and the reaction was allowed to proceed for 30 minutes at room temperature . subsequently , the fixdenat solution was removed , and 100 μl / well anti - brdu pod was added and the reaction was allowed to proceed for 90 minutes at room temperature . after washing in wash buffer for three times , 100 μl / well substrate reaction was added and incubated for 30 minutes at room temperature . antibodies or staining kit against tartrate resistant acid phosphatase ( trap stain ) 10 3 cells were plated per well in 96 - well plates . after 24 hours incubation , the 10 ng / ml rankl and the different concentrations of kmup - 1 were added into the plates , and the drug and the medium were renewed every 2 days , and the reaction was allowed to proceed for 4 days , and then the stain was conducted on the fifth day . after three washes in 37 ° c . pbs , 50 μl / well fluid was left . subsequently , fixative solution 200 μl / well was added into the plates , and the reaction was allowed to proceed for 10 minutes in room temperature , and then the plates were washed by ddh 2 o for three times . 50 μl / well fluid was left , and then substrate 200 μl / well was added to react for 15 minutes in room temperature . after the cells were stained in red , the cells were washed by ddh 2 o . at last , the cells were observed and taken pictures by an optical microscopy ( 40 × and 200 ×), and autopano pro v1 . 4 . 2 , kolor , paris , was used to proceed the pictures taken in 40 ×. then , the number of the osteoclasts with at least three nuclei was counted and compared with the control group . 1 . fixative solution : citrate solution 25 ml , acetone 65 ml , 37 % formaldehyde 8 ml . stored in dark in 4 ° c . 2 . citrate solution : 18 mm citric acid , 9 mm sodium citrate , and 12 mm nacl were solved in 100 ml water , ph 3 . 6 , and then 0 . 001 % triton x - 100 was added at last . fast red violet lb were solved in 1 m sodium tartrate acetone ( 0 . 1 m , ph 5 ) and 0 . 1 m , 19 ml sodium acetate . 10 3 cells were plated per well in 96 - well plates . after 24 hours incubation , the 10 ng / ml rankl and the different concentrations of kmup - 1 were added into the plates , and the drug and the medium were renewed every 2 days , and the reaction was allowed to proceed for 4 days , and then the trap activity was examined on the fifth day . after three washes in ice - cold pbs , 50 μl / well lysis buffer ( with 0 . 2 % tritonx - 100 ) was added , and the reaction was allowed to proceed for 10 minutes . a ) 5 μl / well supernatant was added to an other 96 well plate with 150 μl / well substrate ( 0 . 1 m 4 - npp and 0 . 2 m sodium tartrate in 0 . 1 m sodium acetate , ph 5 ), and the reaction was allowed to proceed for 30 minutes in 37 ° c . in an incubator . when the reaction fluid became yellow , 0 . 1 m , 100 μl / well naoh was added to terminate the reaction , and od 405 was examined in 37 ° c . b ) 3 μl / well supernatant was examined by bio - red dc protein assay to detect the protein concentration . 10 5 cells were plated per well in 24 - well plates . after 24 hours incubation , the 10 ng / ml rankl and the different concentrations of kmup - 1 were added into the plates . after 24 hours reaction , the reaction fluid was centrifuged ( 13 , 000 rpm , 4 ° c ., 20 minutes ), and then the supernatant was tasted by elisa kit ( r & amp ; d system and ebioscience ™) to detect the amount of tnf - α , il - 1β , il - 6 , and il - 10 . the experimental process is followed with the instructions of the kit . nuclear proteins were extracted according to the instructions of ne - per kit . 2 . 5 × 10 6 cells were plated per well in 24 - well plates . after 24 hours incubation , the different concentrations of kmup - 1 were added into the plates . after 24 hours reaction , the 10 ng / ml rankl was added into the plates , and the cells were incubated for a specific time . after two washes in pbs , the cells were collected by scraping in 1 ml of ice - cold pbs and were put in centrifuge tubes . after centrifugation ( 5 , 000 rpm , 4 ° c ., 5 minutes ), the supernatant was discarded , and the cells were added by 100 μl ceri and were shaken for 15 seconds and then were put on ice for 10 minutes . subsequently , the cells were added by 5 . 5 μl cerii and shaken for 5 seconds and then put on ice for 1 minute . after that , the cells were shaken again for 5 seconds and centrifuged ( 10 , 000 rpm , 4 ° c ., 5 minutes ). cytoplasm and nuclei could be separated by this process , and the supernatant was cytoplasmic proteins , and the pallet was nuclei . the supernatant was stored in an eppendorf in − 80 ° c . the nuclear pallet was added by 500 μl pbs and centrifuged ( 10 , 000 rpm , 4 ° c ., 5 minutes ), and the supernatant was discarded . the pallet could be washed by this process and prevented contamination . the process that the pallet was added by 50 μl ner , shaken 15 seconds and put on ice for 10 minutes was repeated for four times . after centrifugation ( 13 , 000 rpm , 4 ° c ., 10 minutes ), the supernatant was nuclear proteins . the supernatant ( nuclear proteins ) was stored in an eppendorf in − 80 ° c . a ) 10 6 cells were plated in 6 cm culture dishes . after 24 hours incubation , the different concentrations of kmup - 1 were added into the plates . after 24 hours reaction , the 10 ng / ml rankl was added into the culture dishes , and the cells were incubated for a specific time . b ) after two washes in ice - cold pbs and removing the pbs , the cells were added by 25 μl lysis buffer , and the reaction was allowed to proceed for 10 minutes . the proteins were collected by scraping and were centrifuged ( 13 , 000 rpm , 4 ° c ., 30 minutes ). the supernatant was stored in an eppendorf in − 80 ° c . c ) concentrations of the proteins were examined by bio - red dc protein assay . after knowing the concentrations , the proteins were diluted to specific concentrations by adding ddh 2 o . one forth volume of the diluted proteins of sample buffer was added into the diluted proteins and was put in boiled water for 5 min and then was chilled on ice for 5 minutes . after short time centrifugation , the diluted proteins could be injected into wells of a gel . the gel was run at 100 v for 10 minutes until the proteins were driven to the intersection between a loading gel and a running gel . then , the voltage was swopped to 200 v for 40 minutes . d ) a pvdf membrane was immersed in methanol for 2 minutes . after two washes in ddh 2 o , the membrane was immersed in transfer buffer for 15 minutes . a sds - page was put on the membrane , and wet paper was flanked them . the proteins were transferred to the pvdf membrane using a semi - dry transfer at 20 v for 30 minutes . e ) the band of the pvdf membrane was trimmed and was added by suitable amount of blocking buffer ( 5 % skim milk in washing buffer ) and then was shaken for 1 hour in room temperature to block non specific bindings . then , the band was covered by a diluted 1 ° antibody overnight in 4 ° c . after six washes in the washing buffer for 5 minutes , the band was covered by a 2 ° antibody for 1 hour in room temperature . after six washes in the washing buffer for 5 minutes , the band was probed by ecl . a ) 2 . 5 × 10 6 cells were plated in 6 cm culture dishes . after 24 hours incubation , the 10 ng / ml rankl and the different concentrations of kmup - 1 were added into the culture dishes . after 24 hours reaction , the reaction fluid was centrifuged ( 13 , 000 rpm , 4 ° c ., 20 minutes ), and then the supernatant was stored in − 80 ° c . b ) 10 % gel preparation . 10 mg gelatin was added by 1 ml ddh 2 o and then was heated in water bath until the solution was clear . c ) concentrations of the proteins were examined by bio - red dc protein assay . after knowing the concentrations , the proteins were diluted to specific concentrations by adding ddh 2 o . one forth volume of the diluted proteins of sample buffer was added into the diluted proteins . after shaking , the diluted proteins could be added into wells of the gel . subsequently , 1 × running buffer was added with the gel , and then the gel was run at 100 v for 10 minutes until the proteins were driven to the intersection between a loading gel and a running gel . then , the voltage was swopped to 200 v for 60 minutes . d ) after electrophoresis , the loading gel was discarded , and the left gel was washed by tritonx - 100 buffer for 30 minutes in room temperature twice . subsequently , the left gel was washed by tris - hcl buffer for 20 minutes in room temperature twice , then , the left gel was added by developing buffer in 37 ° c . water bath overnight . e ) after the developing buffer was discarded , the stain and destain processes were conducted in a hood . the left gel was added by r - 250 stain solution ( commassie blue ) and was shaken gently for 40 - 60 minutes in room temperature . after the stain solution was retrieved , the left gel was added by destain solution and was shaken quickly . the destain solution was renewed every 5 - 10 minutes until a white band was appeared . f ) the washed gel was put on a transparent slid and was taken photo by a camera . 200 μl / well , 10 ng / ml rankl and different the different concentrations of kmup - 1 were added into the 16 - well slides of bd biocoat ™ osteologic ™ bone cell culture system . subsequently , 100 μl medium with 1 , 000 cells were added into each well , and the total volume was 300 μl / well . the slides were incubated for 5 days , and the medium and the drugs were renewed every 2 days . after 5 days , the medium was removed , and the cells were washed by ddh 2 o 3 times . subsequently , the cells were covered by bleach solution for staining 5 minutes and then were washed by ddh 2 o for 5 times . after drying , the cells were took a photo by an optical microscopy ( 40 × and 200 ×), and autopano pro v1 . 4 . 2 , kolor , paris , was used to proceed the pictures taken in 40 ×. 2 . 5 × 10 6 cells were plated in 6 cm culture dishes . after 24 hours incubation , the ng / ml rankl and the different concentrations of kmup - 1 were added into the culture dishes , and the reaction was allowed to proceed for 24 hours . the following processes were conducted in a laminar flow with nuclease - free tools . the culture medium was discarded . after 2 - 3 washes in ice - cold pbs , the cells were collected by scraping in 175 μl rna lysis buffer ( a new spatula was used for each plate ). the scraped cell fluid was put in 1 . 5 ml eppendorfs and were added by 350 μl rna dilution buffer and were mixed well . the eppendorfs were put in 70 ° c . water bath for 3 minutes ( do not exceed 3 minutes ), and were centrifuged ( 14 , 000 rpm , 4 ° c ., 10 minutes ). the supernatant was put in a new eppendorf . the supernatant was mixed with 200 μl , 95 % ethanol , and the fluid was transfer into a spin column . then , the spin column was centrifuged ( 14 , 000 rpm , 4 ° c ., 1 minute ), and the fluid in a lower column was discarded . 50 μl dnase buffer was spread in a membrane of the spin column , and the reaction was allowed to proceed for 15 minutes . subsequently , the spin column was added by 200 μl dnase stop solution and was centrifuged ( 14 , 000 rpm , 4 ° c ., 1 minute ). then , 600 μl rna wash solution was added into the spin column , and the spin column was centrifuged ( 14 , 000 rpm , 4 ° c ., 1 minute ). after that , 250 μl rna wash solution was added into the spin column , and the spin column was centrifuged ( 14 , 000 rpm , 4 ° c ., 2 minutes ). the lower column was discarded , and the upper spin column was put in an eppendorf . 100 μl nuclease - free buffer was added into the spin column , and the spin column was centrifuged ( 14 , 000 rpm , 4 ° c ., 1 minute ). 2 μl dissolved rna was conducted a concentration test , and the left rna was stored in − 80 ° c . after 1 μl rna was put in a pcr machine for 10 minutes in 70 ° c ., steam of the rna sample was centrifuged down , and the rna sample was chilled on ice . the rna sample was added by solution a , and subsequently , the rna sample with solution were put in the pcr machine ( 42 ° c . for 60 minutes , 95 ° c . for 5 minutes ). after the reaction , cdna concentration was examined and then added 80 μl water each tube . the rna sample was stored in 4 ° c . preparation of solution a : 25 mm , 4 μl mgcl , 2 μl reverse transcription 10 × buffer , 10 mm , 2 μl dvtp mixture , 0 . 5 μl recombinant rnasin ribonuclease inhibitor , 0 . 6 μl amv reverse transcription and 1 μl oligo ( dt ) 15 primer or random primers were mixed together , and at last 20 nuclease - free water was added into the above - mentioned mixture . the following reagents were added in order . after the reagents were mixed well , the reagent mixture was put in the pcr machine to run the reaction . a . preparation of 2 % agarose gel ( 1 peace ): 1 . 2 g agarose was solved in 60 ml 0 . 5 × tbe buffer by microwave ( 2 - 3 minutes , medium power ) and was mixed with 1 μetbr well . the mixture was poured into a modeling box . b . preparation of a dna ladder ( marker ): dna : loading dye : ddh 2 o = 1 : 2 : 7 a calcium ion fluorescent probe , fura - 2 / am was used as an indicator for calcium concentration changes to detect the changes of the intracellular calcium concentration . the fura - 2 could bind with calcium ions and was with fluorescent property . the wave length of the light activated by the fura - 2 bound with calcium ions was 340 nm , but the wave length of the light activated by non binding fura - 2 was 308 nm . according to this property , a ratio of the wave length intensity could be converted into the calcium concentration . the macrophages of raw264 . 7 rates were plated in 10 cm culture dishes . when the cells glowed to 90 % saturated condition , 10 6 cells were plated in 10 cm culture dishes . after 24 hours incubation , the 10 ng / ml rankl was added into the culture dishes , and the reaction was allowed to proceed for 7 days . until mono nuclear cells differentiated into multi - nuclear cells , culture medium was discarded . after 3 - 4 washes in pbs , the total cells were collected into eppendorfs by flushing with pbs , and then the eppendorfs were centrifuged ( 15 , 000 rpm , 4 ° c ., 5 minutes ). the supernatant was discarded , and the cells were added by 2 ml culture medium and 2 . 5 μl , 2 μm fura - 2 / am , and the reaction was allowed to proceed for 40 minutes in 37 ° c ., and then was centrifuged ( 15 , 000 rpm , rt , 5 minutes ). the supernatant was discarded , and the cells were washed by suitable amount of physiological buffer and then were centrifuged . at last , the cells were added the suitable amount of physiological buffer to disarrange the cells . the concentration of the cells was adjusted to 5 × 10 5 cells / ml , and 1 ml cell suspension which was added kmup - 1 for 4 minutes in advance was put into a quartz tube . the quartz tube was put into a rf - 5310 fluorescence spectrophotometer to detect changes of the 340 nm and 380 nm wave lengths which were activated by 510 nm wave length light . after recording for 60 seconds , 20 ng / ml rankl was added into the cells , and the changes of intensity of light was detected . preparation of physiological buffer : 130 mm nacl , 5 mm kcl , 10 mm glucose , 1 mm mgcl , 1 mm cacl and 29 mm hepes were solved in 1 l ddh 2 o and were adjusted to ph 7 . 4 . monosodium iodoacetate ( mia ) was used as an inducing agent for inducing osteoarthritis of rates . 30 five - week - old wistar male rats ( national science council of the r . o . c ) with about 150 - 165 g weight were divided into five groups which are sham , mia induced group ( mia ) and three kmup - 1 given groups ( 1 , 2 . 5 , 5 mg / kg kmup - 1 ), and each group was included 6 rates . after the rats were raised in an animal room for a week to adapt an environment , the rats were orally fed kmup - 1 ( 1 , 2 . 5 , 5 mg / kg ) for a week . on the eighth day , the rats in the mia group and the kmup - 1 given groups were injected 4 mg / 25 μl , 0 . 5 ml mia into left knee joints of the rats by a 26 mm insulin needle , and then the rats were fed kmup ( 1 , 2 . 5 , 5 mg / kg ) for a week . on the fifteenth day , when the rats were sacrificed , the left knee joints were opened , and a femur was separated from a tibia . the joint which was near the tibia of the femur was taken out . after cleaning , the joints were taken photos by a digital camera . ethylenediaminetetraacetic acid ( edta ) was added by ddh 2 o and was heated until the edta was completely dissolved . a tissue from the joints was put into a tissue embedding cassette and was immersed in 10 % formaldehyde for 3 days . subsequently , the tissue was immersed in 0 . 5 m edta in 60 ° c . for a week in an oven to de - calcium . after the de - calcium bones were dehydrated for one day , the tissues were paraffin embedded and sliced which the slicing thick was 4 μm . the sections were put in a 60 ° c . oven for 20 minutes and then were immersed in xylene for 3 minutes twice . subsequently , the sections were immersed in 100 % ethanol for 3 minutes twice , 95 % ethanol 1 minute , 80 % ethanol for 1 minute , 80 % ethanol for 1 minute , 70 % ethanol for 1 minute , 50 % ethanol for 1 minute , ddh 2 o for 1 minute and then eosin for 1 . 5 minutes . after that , the sections were washed by flowing water for 5 minutes and were immersed in 90 % ethanol for 1 - 2 seconds , 100 % ethanol for 1 - 2 seconds and at last were immersed in xylene . at that time the sections could be sealed on a slide . the sealed slide was taken a photo under an optical microscopy . a preparation of toluidine blue : 1 % sodium borate , 1 % toluidine blue and 1 % azur ii were solved in ddh 2 o . c . the sections were put in a 60 ° c . oven for 20 minutes and then were immersed in xylene for 3 minutes twice . subsequently , the sections were immersed in 100 % ethanol for 3 minutes twice , 95 % ethanol 1 minute , 80 % ethanol for 1 minute , 80 % ethanol for 1 minute , 70 % ethanol for 1 minute , 50 % ethanol for 1 minute , ddh 2 o for 1 minute and then toluidine blue for 1 . 5 minutes . after that , the sections were washed by flowing water for 5 minutes and were immersed in 90 % ethanol for 1 - 2 seconds , 100 % ethanol for 1 - 2 seconds and at last were immersed in xylene . at that time the sections could be sealed on a slide . the sealed slide was taken a photo under an optical microscopy . all experimental data were presented in mean + s . e . m . and percentage (%). the comparisons of the experimental datum were presented by special marks and also were adopted student &# 39 ; s t - test and one - way anova to estimate differences between the control group and drugs given groups . when p value was less than 0 . 05 , it expressed that there was an obvious difference in statistics . armstrong a p , tometsko m e , glaccum m , sutherland c l , cosman d , dougall w c : a rank / traf6 - dependent signal transduction pathway is essential for osteoclast cytoskeletal organization and resorptive function . j biol chem 2002 , 277 ( 46 ): 44347 - 44356 . mizukami j , takaesu g , akatsuka h , sakurai h , ninomiya - tsuji j , matsumoto k , sakurai n : receptor activator of nf - kappab ligand ( rankl ) activates tak1 mitogen - 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