Patent Application: US-71488900-A

Abstract:
the present invention is directed to a method of treating inflammation comprising administering to a subject in need thereof an amount of a caffeic acid derivative sufficient to inhibit the transcription of cox - 2 . in a preferred embodiment the caffeic acid derivative is a cyanocinnamate , most preferably cinnamamyl - 3 , 4 - dihydroxy - α - cyanocinnamate .

Description:
it has previously been published that 12 - hete , the major 12 - lo end product , induces jnk in rin m5f cells [ 28 ]. furthermore , herschmann and colleagues demonstrated that v - src induces cox - 2 gene transcription by activating jnk and c - jun [ 29 ]. these studies demonstrate that c - jun may activate cox - 2 gene transcription by binding to the camp response element ( cre ) in the cox - 2 promoter . in the present study it is demonstrated that 12 - hete acts as a specific upstream agent in activating cox - 2 gene transcription . as seen in fig9 a schematic signaling pathway is depicted that identifies key elements in the pathway leading from cytokine - stimulation to cox - 2 gene activation in pancreatic β - cells . furthermore , since rat tissue does not contain the putative cre in the cox - 2 promoter , it is likely that 12 - hete regulates cox - 2 gene transcription through a novel promoter sequence . of particular interest in the present study is that 12 - lo knockout mice demonstrated a decrease in basal pge2 production compared to c57bl / 6 mice . one interpretation of this finding is that 12 - hete is necessary for basal cyclooxygenase activity as well as stimulated cox - 2 activity . since both c57bl / 6 islets and 12 - lo ko islets presumably possess functional cox - 1 enzymes it is possible that 12 - hete may regulate pge2 production at the post - translational level , similar to no . the present invention offers a very advantageous means of treating inflammation by inhibition cox - 2 at the transcriptional level . previously - known cox - 2 inhibitors worked at the enzyme level , by blocking cox - 2 activity directly or indirectly . the natural response of the body is to compensate for loss of cox - 2 activity through a bio - feedback loop , that increases enzyme production . this often leads to a “ rebound effect ” when treatment is withdrawn . the methods of the present invention provide all of the benefits of therapeutic cox - 2 inhibition , without the danger of a rebound effect , because the inhibition occurs directly at the gene level , preventing production of the enzyme . caffeic acid derivatives administered in accordance with the present invention can be administered , together with one or more suitable carriers or excipients , by any of several traditional routes of pharmaceutical administration , including orally , topically or by injection . the method can be readily practiced with doses of caffeic acid derivatives ranging from about 0 . 5 mg / kg to about 25 mg / kg [ 37 ], and preferably at about 10 mg / kg . examples of caffeic acid derivatives suitable for use in the present invention are cinnamamyl - 3 , 4 - dihydroxy - a - cyanocinnamate ( cdc ), 5 , 6 , 7 - trihydroxy - 2 - phenyl - 4h - 1 - benzopyran - 4 - one ( baicalein ), and the compounds listed in table i ( which have been shown to preferentially inhibit 12 - lo by cho , et al . [ 34 ]). the following examples are intended to illustrate the present invention , and in no way to limits its scope , which is defined by the claims . the following materials were used in the experiments set forth in the examples : rpmi - 1640 medium was purchased from life technologies , inc . ( grand island , n . y .). bca reagent assay kit was from pierce chemical co . ( rockford , ill .). pge2 ria was purchased from p . e . biosystems ( foster city , calif .). collagenase type xl was obtained from sigma chemical company ( st . louis , mo .) and used for islet isolation . il - 1 d was purchased from r & amp ; d systems ( minneapolis , minn .). cdc , 12 - s - hete , and 15 - s - hete were obtained from biomol research lab , inc . ( plymouth meeting , pa .). cox - 2 antibody was purchased from cayman chemical co ( ann arbor , mich .). horseradish peroxidase conjugated anti - goat igg was obtained from santa cruz biotechnology ( santa cruz , calif .). cox - 1 and cox - 2 cdna probes were purchased from torrey pines biolabs , inc . ( san diego , calif .). 12 - lo ko mice were a generous gift from dr . colin d . funk , center for experimental therapeutics , university of pennsylvania , pa . porcine pancreatic islets were a generous gift from neocrin company ( irvine , calif .). rin m5f cells were cultured to near confluence in rpmi - 1640 medium ( life technologies , inc ., grand island , n . y .) plus 10 % fcs plus 1 % hepes and penicillin / streptomycin . prior to addition of il - 1β the cells were depleted in rpmi - 1640 medium plus 0 . 2 % bsa for 24 hours . the cells were gently washed in pbs and depletion medium was added back . at this time cdc ( biomol research lab , inc ., plymouth meeting , pa ) ( 1 - 10 μm ) was added in certain experiments 60 minutes prior to il - 1β addition . 12 - hete ( 1 - 100 nm ) and 15 - hete ( 1 - 100 nm ) ( biomol research lab , inc ., plymouth meeting , pa .) were added to certain experiments in 0 . 1 % ethanol . the cells were cultured for an additional 24 hours at which time medium was collected for pge2 assay . for mrna experiments , total rna was extracted from rin m5f cells 3 hours after the addition of il - 1 β , 12 - hete , or 15 - hete . 12hete and 15 - hete were stored at − 70 c . in the dark and added to cell culture dishes in the dark . control experiments included the addition of 0 . 1 % ethanol alone to rin m5f cell cultures . we previously demonstrated that islets purified from 12 - lo knockout mice are resistant to cytokine - induced inhibition of glucose stimulated insulin secretion compared to control mice [ 11 ]. this example investigates the mechanism ( s ) of this resistance . rin m5f cells , cultured as set forth in example 1 , were stimulated with il - 1β and 12 - hete and total protein extracts were used for western immunoblotting with an anti - cox - 2 antibody . the rin m5f cells (− 300 per blot ) were lysed in a 1 % triton - 0 . 1 % sds buffer in normal saline ( ph 7 . 4 ) with 10 mm hepes plus 1 mm edta and standard protease inhibitors . cell lysates were centrifuged at 14 , 000 × 9 for 10 minutes and a modified bca protein assay was performed . protein aliquots ( 50 μg per sample ) were treated with laemmeli sample buffer and then heated to 100 ° c . for 5 minutes . proteins were separated on 10 % sds - page and transferred to polyvinylidene difluoride membranes ( immobilon - p ) in 10 mm caps buffer , 10 % methanol at ph 11 . the immunoblots were blocked overnight in 5 % nonfat dried milk in tris buffer containing 0 . 1 % tween - 20 . the blots were incubated for 2 hours at room temperature with anti cox - 2 antibodies ( cayman chemical co ., ann arbor , mich .) diluted 1 : 2000 in the same tris buffer without nonfat dried milk . the blots were washed and then incubated with horseradish peroxidase conjugated secondary antibodies at 1 : 2000 dilution . the protein bands were visualized with enhanced chemiluminescence reagents using x - ar film . as seen in fig1 il - 1β ( 0 . 3 ng / ml ) induced a 2 . 5 fold increase in cox - 2 protein while 12 - hete ( 1 - 10 nm ) induced a 3 - fold increase . next , western immunoblots were performed on protein extracts from porcine islets treated in a similar fashion with il - 1β and lysed as set forth about for rin m5f cells . because of the concern that 12 - hete would not permeate the islet mass , the 12 - lo inhibitor cdc ( 1 μm ) was used instead with or without the addition of il - 1β , in order to determine the role of the 12 - lo pathway on cox - 2 protein expression . fig2 demonstrates that il - 1β increased cox - 2 protein 3 - fold while the addition of cdc completely eliminated this response . rin m5f cells were cultured as set forth in example 1 , in the presence or absence of il - 1β ( 0 . 3 ng / ml ), 12 - s - hete ( 1 - 100 nm ), or 15 - s - hete ( 1 - 100 nm ) for 4 hours . total rna was extracted and semiquantitative rt - pcr was performed using cox - 2 specific primers . total rna was extracted from rin m5f cells with rna stat - 60 ( tel - test , inc ., friendswood , tex .). rna ( 1 μg ) was diluted in 12 . 5 μl depc - h20 and reverse transcribed with the 1 st - strand ™ cdna synthesis kit ( clontech laboratories , inc ., palo alto , calif . ), using oligo dt primers , recombinant rnase inhibitor , and mmlv reverse transcriptase to generate template cdna for pcr amplification . each pcr reaction was done on rna from one 100 mm culture dish for the stated experimental conditions . twenty picomoles of each primer ( shown below ) was mixed with 1 unit of taq gold polymerase ( perkin elmer , inc .) in 50 μl final volume . samples were amplified with an initial 45 second denaturation step at 94 ° c . followed by 25 cycles of 45 seconds at 60 ° c ., and 25 cycles of 2 minutes at 72 ° c .. the last cycle was extended for an additional 7 minutes at 72 ° c . pcr cycling was done with a gene amp pcr system 2400 ( perkin elmer / cetus corp ., norwalk , conn .). dna primers were synthesized in the dna / rna core chemistry laboratory at city of hope national medical center . the sequences of the primers are shown below . rat cox - 1 sense ctg gcc gga ttg gtg ggg gta g pcr products were analyzed by electrophoresis with 1 . 8 % agarose gel . the dna was transferred to nylon membranes and hybridized sequentially to 32 p - labeled probes for cox - 1 , cox - 2 , and gapdh using a random primed dna labeling kit ( boehringer mannheim ). gapdh was used as an internal control . hybridization and autoradiography were performed according to previously published methods [ 14 ]. cox - 2 mrna was hybridized with a cox - 2 probe . il - 1β induced a 2 - fold increase in cox - 2 mrna as seen in fig3 . 12 - hete and 15 - hete induced a 3 - fold and 1 . 5 - fold increase in cox - 2 mrna , respectively ( fig3 and fig4 ). the biological significance of the difference in mrna induction by 12 - and 15 - hete is further illustrated by the fact that 12 - hete stimulated a 10 - fold greater increase in pge2 than 15 - hete ( see below ). then , a cox - 1 specific oligonucleotide probe was used to measure cox - 1 mrna levels in the same experiments . as seen in fig5 neither il - 1β , nor 12 - hete stimulated cox1 gene transcription . in addition , pge2 production was measured under the same conditions , but over a 24 hour period . cox - 2 activity was determined by measuring the accumulation of pge2 in the conditioned media . cells were cultured in 24 well plates for 24 hours and subject to the experimental conditions described above . pge2 was measured in conditioned medium using a commercial ria kit . statistical analysis of pge2 levels was performed using the student &# 39 ; s t - test with graphpad prism ™ software ( graphpad software , inc , san diego , calif .). il - 1β increased pge2 production from virtually undetectable levels (˜ 5 pg / ml ) to ˜ 500 pg / ml , while 12 - hete increased pge2 production to ˜ 1250 pg / ml . in contrast , 15 - hete induced relatively little pge2 , ˜ 125 pg / ml ( fig6 ). cdc caused a statistically significant inhibition of il - 1β induced pge2 production at 1 μm ( 388 + 27 pg / ml versus 169 + 5 pg / ml ; p & lt ;. 0l ) and this inhibition was dose - dependent ( fig7 ). it should be noted that 1 μm cdc has a relative specificity for 12 - lo since the ic50 for 12 -, 15 -, and 5 - lipoxygenase is 0 . 063 μm , 1 . 89 μm , and 3 . 33 μm , respectively ( biomol research catalogue , sixth edition , p . 161 ). generation of 12 - lo knockout has been previously described [ 13 ]. all animal studies were performed in accordance with guidelines set forth by the research animal care committee of city of hope national medical center . islet isolation and culturing techniques have been detailed previously [ 14 ]. briefly , mouse pancreas was removed , minced , and poured into a siliconized 25 ml ehrlenmeyer flask with type xl collagenase solution ( sigma chemical co , st . louis , mo .). the pancreatic digest was incubated in a shaker - type water bath at 37 c . for 6 - 10 minutes . the digest was centrifuged , poured into a black back petri dish , and islets were picked by hand under a microscope . the islets were aliquoted into sterile 6 - well plates ( sarstedt , newton , n . c .) and cultured in rpmi 1640 medium containing 11 mm glucose and supplemented with 10 % fcs and 1 % hepes . typically , we isolate − 75 - 100 islets per mouse . for pge2 experiments , 100 islets per well in 300 μl rpmi medium were cultured overnight prior to additions . the following morning the medium was changed to rpmi 1640 medium plus 0 . 2 % bsa and the islets were allowed to equilibrate for one hour . then , il - 1β ( r & amp ; d systems , minneapolis , minn .) was added to the appropriate experiments . all islet experiments were run in triplicate and repeated three times for reproducibility . isolated islets ( 100 per experiment ) were brought to culture and incubated overnight as described . after approximately 18 hours il - 1β was added to the medium and the islets were incubated for an additional 24 hours . pge2 production was measured as set forth in example 3 . as seen in fig8 islets from c57bu / 6 mice generated 801 + 445 pg / ml pge2 in the non - stimulated state . while islets stimulated with il - 1β generated 5405 + 1012 pg / ml pge2 , a 7 - fold increase ( p =. 01 ). in marked contrast , islets isolated from 12 - 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