Patent Application: US-52852208-A

Abstract:
rosiglitazone , 5 - asa or structurally analogous compounds according to the general formula : or compounds according to the general formula : for the induction of camp expression in tissues having ppar - gamma receptors . such tissues include epithelia or mucosae tissue having ppar - gamma receptors and of particular interest is camp expression in the gut .

Description:
given the direct negative role of the peroxisome proliferator - activated receptor gamma ( ppar - gamma ) on the wnt / tcf / beta - catenin signaling pathway , ppar - gamma activation was examined to investigate defensin biogenesis . the tested ppar - gamma agonists , rosiglitazone ( at 1 μm for 1 , 3 or 6 hours ) and others , activates hd - 5 in caco - 2 cells , as determined by quantitative pcr analysis ( fig1 - 5 ). cultured intestinal epithelial cell lines , namely caco - 2 ( of human origin ) and iccl2 ( of mice origin ), were treated with the gsk - 3 inhibitor licl ( 20 microm ) or the phosphatase inhibitor calyculin ( 50 nm ) followed or not by stimulation with the ppar - gamma agonist such as rosiglitazone ( 1 microm ). the expression of defensin and known target genes of both wnt and ppar - gamma signaling pathways were investigated by quantitative real - time pcr . the activation of gsk3 , beta - catenin , nf - kappab , erk1 / 2 , sapk / jnk and p38 was measured by specific immunoblotting . to investigate the antimicrobial role of ppar - gamma , the intracellular replication of the crohn &# 39 ; s disease associated escherichia coli ( lf82 ) was measured upon or not stimulation with rosiglitazone in the raw macrophage cell line . incubation with rosiglitazone and other ppar - gamma activators significantly increased the expression of both alpha - ( hd - 5 and hd - 6 ) and beta - ( defb10 ) defensins by intestinal epithelial cells ( fig3 ). such antimicrobial gene expression was synergized following co - stimulation by calyculin that promotes beta - catenin degradation . accordingly , reduced intracellular replication of lf82 through ppar - gamma activation by rosiglitazone was observed . conversely , the expression of a wnt / tcf / beta - catenin target gene , cyclin - d1 , and the stability of the beta - catenin was markedly decreased upon stimulation by both calyculin and rosiglitazone . finally , licl , an activator of the wnt / tcf / beta - catenin - dependent signalling pathway , blocked the rosiglitazone - induced defensin gene expression upon co - stimulation . incubation with test substances significantly increased the expression of both alpha - ( hd - 5 and hd - 6 ) and beta - ( defb10 ) defensins by intestinal epithelial cells ( fig3 shows effect of rosiglitazone ). accordingly , reduced intracellular replication of lf82 through ppar - gamma activation by rosiglitazone was observed . taken as a whole , the results indicate that ppar - gamma activation promotes the induction of an antimicrobial gene programme by negatively regulating the formation of the tcf / beta - catenin complex . these findings highlight the therapeutical potential of ppar - gamma in complementing defensins deficiency in many gasterointestinal disorders such as crohn &# 39 ; s disease , ulcerative colitis , intestinal bowel syndrome , acute diverticulitis and for the prevention of condition such as acute diverticulitis in patients affected by colonic diverticulosis , indeterminate colitis and infectious colitis . furthermore , these findings highlight the therapeutical potential of ppar gamma agonists in complementing defensins deficiency in other mucosal disorders including but not limited to those such as ocular inflammation and infections , periodontal disease , allergic and non allergic rhinitis , bacterial vaginosis and skin inflammatory conditions and infections such as impetigo , erysipela , dermatitis , folliculitis , acne and vulgaris . in vitro studies with racemic compound 34 and enantiomers 34 - e1 & amp ; 34 - e2 materials 5 - asa was purchased at sigma - aldrich ( st quentin fallavier , france ). rosiglitazone was synthesized in the laboratory according to standard procedures . the racemic compound 34 and the two enantiomers of the compound , 34 - e1 and 34 - e2 were provided by giuliani spa ( milano , italy ). compound were re - suspended in dmem medium ( gibco ) and adjusted at ph = 7 if necessary with 10n naoh . the colon carcinoma cell line caco - 2 ( atcc htb - 39 ) was routinely grown in dmem supplemented respectively with 10 % or 20 % heat - fcs , and antibiotics . cells were grown in monolayers , incubated at 37 ° c . in 5 % co2 and 95 % relative humidity . cell were stimulated by 5 - asa , r34 , 34 - e1 and 34 - e2 for 24 h . total rna was isolated from cells using rneasy kit ( macherey nagel , hoerdt , france ) according to the manufacturer &# 39 ; s instructions . rna quantification was performed using spectrophotometry . after treatment at 37 ° c . for 30 min with 20 - 50 units of rnase - free dnase i ( roche diagnostics corporation , indianapolis , ind ., usa ), oligo - dt primers ( roche diagnostics corporation , indianapolis , usa ) were used to synthesize single - stranded cdna . mrnas were quantified using sybr green master mix ( applera , courtaboeuf , france ) with human specific oligonucleotides for hbd1 ( s : 5 ′- atacttcaaaagcaattttcctttat - 3 ′; as : 5 ′- ttgtctgagatggcctcaggtggtaac - 3 ′) in a geneamp abiprism 7000 ( applera , courtaboeuf , france ). in each assay , calibrated and no - template controls were included . each sample was run in triplicate . sybr green dye intensity was analyzed using the abiprism 7000 sds software ( applera , courtaboeuf , france ). all results were normalized to the unaffected housekeeping gene of human β - actin ( s : 5 ′- tcacccacactgtgcccatctacg - 3 ′; as : 5 ′- cagcggaaccgctcattgccaatg - 3 ′). 5 - asa ( 30 mm ), racemic r34 and 34 - e1 & amp ; 34 - e2 ( 1 mm ) were administrated by intrarectal installations for 8 days . post - mortem , total rna was isolated from whole mice colonic tissues using rneasy kit ( macherey nagel , hoerdt , france ) according to the manufacturer &# 39 ; s instructions . rna quantification was performed using spectrophotometry . after treatment at 37 ° c . for 30 min with 20 - 50 units of rnase - free dnase i ( roche diagnostics corporation , indianapolis , ind ., usa ), oligo - dt primers ( roche diagnostics corporation , indianapolis , usa ) were used to synthesize single - stranded cdna . mrnas were quantified using sybr green master mix ( applera , courtaboeuf , france ) with mouse specific oligonucleotides for ll37 ( s : 5 ′- gctgattcttttgacatcagctgtaa - 3 ′ as : 5 ′- gccagccgggaaattttct - 3 ′) in a geneamp abiprism 7000 ( applera , courtaboeuf , france ). in each assay , calibrated and no - template controls were included . each sample was run in triplicate . sybr green dye intensity was analyzed using the abiprism 7000 sds software ( applera , courtaboeuf , france ). all results were normalized to the unaffected housekeeping gene β - actin ( s : 5 ′- gggtcagaaggattcctatg - 3 ′; as : 5 ′ ggtctcaaacatgatctggg - 3 ′). male sprague - dawley rats ( charles river , l &# 39 ; arbresle , france ) weighing 175 - 200 g were used in this study . rats were maintained in laboratory conditions for 1 week before experiment . the animals were housed 5 per cage with food and water available ad libitum . all studies were performed in accordance with the proposal of the committee for research and ethical issues of the international association for the study of pain ( zimmermann m , pain 1983 ; 16 : 109 - 110 ). great care was taken , particularly with regard to housing conditions , to avoid or minimize discomfort to the animals . nociception in the animals was assessed by measuring the intracolonic pressure required to induce a behavioural response during colorectal distension ( crd ) due to the inflation of a balloon introduced in the colon . this response was characterized by an elevation of the hind part of the animal body and clearly visible abdominal contraction corresponding to the severe contractions ( al chaer , gastro 2000 ; tarrerias , pain 2002 ; bourdu et al ., 2005 ). briefly , rats were anesthetized with volatile anaesthesia ( 2 % isoflurane ), the balloon ( prepared as previously described in bourdu & amp ; al , 2005 ) was inserted intrarectally in a minimally invasive manner to 7 cm from the anus , and the catheter was taped to the base of the tail . after 5 minutes , rats were placed in the middle of a 40 × 40 - cm plexiglas box and the catheter was connected to an electronic barostat apparatus ( g & amp ; j electronics inc ., toronto , canada ). increasing pressure was continuously applied until pain behaviour was displayed or a cutoff pressure of 80 mm hg was reached . compounds were administrated daily by intrarectal instillations . for each enema , a catheter ( 2 - mm fogarty catheter ) was placed in the colon at 7 cm from the anus , and the animals received 500 μl of compound resuspended at optimal concentration in dmem medium and adjusted at ph 7 by 10n naoh if necessary for 21 days . control animals received medium alone . effect of the compound on visceral pain was evaluated after 2 and 3 weeks of treatment . all comparisons were analyzed using the permutation test for two independent samples . statistics has been calculated using the software statxact ( cytel inc , cambridge , mass ., usa ). differences were considered statistically significant if the p value was & lt ; 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