Patent Application: US-29920907-A

Abstract:
disclosed herein are novel compounds , their use in the treatment and prevention of joint and / or cartilage inflammation that provide an alternative to the nsaids and selective cox - 2 inhibitors by activating endogenous detoxifying cellular defense mechanisms that act to neutralize toxic cellular intermediate . these compounds are ppar - alpha agonists and / or phase 2 gene activators .

Description:
this invention teaches novel compositions of pparα specific ligands and phase 2 gene activators alone or in combination for treating and preventing inflammation and pain preferentially in joints and chondrocytic tissue , thereby providing an alternative therapy to the non - selective and selective cox - 2 inhibitors for joint inflammation and pain . “ additives and adjuvants ” herein refers to compositions that are known in the art of cosmetic and / or medical compositions and encompasses hydrophilic or lipophilic gelling agents , preservatives , antioxidants , solvents , fragrances , fillers , dyestuffs and colorants . “ cartilage ” herein refers to the specialized connective tissue comprising mature and / or young cartilage cells , adult chondrocytes and chondroblasts and the matrix of amorphous ground substance that surrounds a network of collagen fibers ( churchill &# 39 ; s medical dictionary ). cartilage hereby includes the cartilagenous tissue that participates in synovial and non - synovial junctions and also to cartilage of the thorasic wall , the larynx , the trachea , the bronchi , and the nose and ears . “ diagnostic method ” herein refers to a method that contributes to the ability of one skilled in the art to discriminate between one probable cause of a symptom from another . for example , to distinguish chest pain that is primarily due to costochondritis ( cartilage inflammation ) from chest pain due to a non - costochondritis etiology . “ gastrointestinal tube ” or gi tube herein encompasses all the types of tubes that are used to access the gastrointestinal tract regardless of the medical purpose for which the tube is placed , such as those placed for enteral feeding or medication delivery ( peg and ng tubes ), for lavage ( washing ), for determining gi contents , or for controlling gi bleeding . “ injection ” herein refers to the method of delivering the agent / compound , by a means other than to the surface of the skin and includes delivery to the intra - articular space and related tissue , subcutaneous tissue , to the muscle , into the veins , into the vagina , or rectum . “ joint ” herein refers to the anatomical structure that connects at least two elements of anatomy and includes synovial and non - synovial junctions . “ human mammal ” herein refers to all ages of humans from new born to the elderly and encompasses all genders . “ domesticated mammal ” herein refers to any mammal that lives and associates with humans and is most typically represented as a dog or a cat but herein encompasses any mammal though non - typically associated with humans is associating with humans . an example of the latter is the wolf . “ farm mammal ” herein refers to mammals associated with farms regardless of whether that animal is living on a farm and includes horses , cows , goats , sheep , pigs and others . “ experimental animal ” herein refers to animals used for scientific / investigational purposes such as primates , dogs , cats , pigs , rats , mice and others . “ commercial animal ” herein refers to animals that are utilized either transiently or habitually for profit . for example , this group includes cows bred to produce milk or for beef , competitive animals such as race horses and dogs and show animals such as show dogs , as well as all zoological mammals . “ oral ” herein includes any form of delivery of an agent / compound wherein the agent / compound is placed directly or indirectly into or through the nasal - oral cavity of the subject whether or not the agent / compound is swallowed . the term “ oral ” hereby includes sublingual , buccal , esophageal administration as well as delivery of the agent / compound through a nasogastric tube . “ parenteral ” herein includes any form of delivery of an agent / compound by a means other than by the mouth , such as delivery through the vein , into the muscle , into the intra - articular space and associated tissue , to the subcutaneous tissue , into the nasal cavity , vaginal canal or rectum . “ selective pparα agonist ” herein denotes a pparα agonist that has at least 5 - fold greater affinity for pparα than for pparβ / δ or pparγ . “ therapeutically effective amount ” herein refers to that amount of agent / compound that is sufficient to decrease or alleviate symptoms of inflammation and / or pain or is sufficient to decrease the probability of inflammation and pain in a method of prophylaxis . “ topical ” herein means application of the agent / compound to the skin or mucous membrane . accumulating evidence suggests that the peroxisome proliferator - activated receptors ( ppars ), cox - 2 , and cox - 2 derived prostaglandin ( pg ) e 2 participate in inflammation and cartilaginous destruction in oa and ra . ( chen , x . l . et al . 2003 . hosoya , t ., et al . 2005 . dimmeler , s . 1996 . jang , j . h . & amp ; surh , y . j . 2003 ). the peroxisome proliferator - activated receptors ( pparα , pparβ / δ and pparγ ) are a family of ligand - activated transcription factors that up - regulate target genes containing the ppar - responsive elements ( ppares ). ample evidence also suggests that the ppar isoforms and cognate ligands are differentially regulated in a tissue and stimulus dependent manner ( voehringer , d . w ., et al . 2000 . lee , m . s . et al . 2003 ). for example , pparα but not pparγ activators inhibit cox - 2 and pg expression in aortic cells . ( abulencia , j . p ., et al . 2003 ), and pparγ but not pparα expression is modulated in il - 1β stimulated rat condrocytes ( voehringer , d . w ., et al . 2000 ). the pparγ ligand , 15d - pgj2 , is reported to modulate cox - 2 in epithelial and smooth muscle cells by gene induction and via a negative feedback loop in eptithelial and smooth muscle cells . ( yokota , h . et al . 2003 . abulencia , j . p ., et al . 2003 ). in mouse macrophages , 15d - pgj2 can activate nf - e2 related factor 2 ( nrf2 ), the transactivator of the phase 2 detoxifying enzymes ( amin , a . r . et al . 1997 ). cells defend themselves against external and internal toxins by increasing the expression of antioxidant / detoxifying genes , the phase 2 enzymes . the phase 2 gene products modify electrophilic intermediates to render them less reactive and harmful as well as increasing the expression of genes that participate in the defensive arsenal . for example , the phase 2 gene , glutathione ( gsh ) transferase is a phase 2 enzyme that conjugates hydrophobic electrophiles with gsh , attenuating the electrophile &# 39 ; s damaging properties . another phase 2 enzyme , quinone reductase ( qr ) promotes the electron transfer of quinones and by this reduction down - modulates their ability to deplete intracellular gsh . other phase 2 enzymes such as udp - glucuronosyltransferases and epoxide bydrolase modify potential reactive species facilitating their excretion . the induction of phase - 2 enzymes is also accompanied by the up - regulation of gsh itself . phase 2 detoxifying enzymes share a common cis regulatory region , the antioxidant response element ( are ) and its cognate transactivator , nf - e2 related factor 2 ( nrf2 ). nrf2 is a cytoplasmic protein but upon induction translocates to the nucleus , binds to other nuclear proteins and participates in phase 2 enzyme gene activation . edible plants , the cruciferous vegetables such as broccoli , contain high concentrations of potent activators of phase 2 genes , the class of small molecules , the isothiocyanates , sulforaphane [(−)- i - isothiocyanato - 4 ( r )-( methylsulfinyl )- butane ] and its parent compound , the glucosinolates [ β - thioglucoside n - hydroximinosulfate , also known as ( z )-( or cis )- n - hydroximinosulfate esters or s - glucopyranosyl thiohydroximates ] ( fahey , j . w . et al . 2001 , fahey , j . w . et al . 2002 ). a representative listing of glucosinolates and isothiocyanates are found in fahey , j . w . et al . 2001 the contents which are incorporated herein by reference . representative glucosinolates and sulforaphane analogs are listed below . fluid shear is a critical physiological stimulus that modulates intracellular signaling in a time , magnitude and phenotype dependent manner . low laminar shear in human vessels tend to be atherogenic whereas high laminar shear tends to be atheroprotective . exposure of human aortic endothelial cells to high laminar shear flow at 20 dynes ( dyn )/ cm 2 ( 1 dyn = 10 μn ) induces expression of the phase 2 genes . moreover laminar shear flow potently inhibits apoptosis in growth factor - starved human umbilical vein endothelial cells ( huvecs ) ( dimmeler , s . 1996 ). however , low intracellular gsh levels have been linked to mitochondrial depolarization and apoptosis in multiple cells lines . ( jang , j . h . & amp ; surh , y . j . 2003 . voehringer , d . w ., et al . 2000 . in marked contrast , extensive mechanical loading of cartilage producing both low hydrostatic pressure and high fluid ( 20 dynes ( dyn )/ cm 2 ) shear results in irreversible chondrocyte apoptosis , matrix erosion , and osteoarthritis , whereas low shear (& lt ; 5 dyn / cm 2 ) is chondroprotective ( carter , d . r ., et al . 2004 . lee , m . s ., et al . 2003 ). the inventors have previously shown that high shear induces cox - 2 expression in human chondrocytic cells through a c - jun n - terminal kinase 2 ( jnk2 ) dependent pathway ( abulencia , j . p . et al . 2003 ). while not being bound by theory , this invention teaches that in human chondrocytic cells , shear stress induces cox - 2 expression , suppresses phosphatidyl - inositol 3 - kinase ( pi3 - k ) activity , which represses nrf2 mediated transcription of the phase 2 enzyme genes . this effect is attenuated with addition of phase 2 inducers and with cox - 2 specific inhibitors . this invention also teaches the unexpected finding of negative feedback loops where cox - 2 expression and inflammatory signaling is repressed by the downstream activity of pi3 - k and / or the phase 2 enzymes . while not being bound by theory , this invention also teaches that in addition to shear - stress induced down regulation of phase 2 gene expression , human chondrocytic cells ( t / c - 28a2 ) exposed to high shear stress for 48 hours also results in selective and significant down regulation of the pparα mrna isoform and increases markers of apoptosis ( bax and caspase - 9 precursors ). pre - treatment of chondrocytic cells with the cox - 2 selective blockers significantly reversed the shear - mediated changes of pparα . pre - treatment with pparα selective ligand abolishes shear induced down regulation of nrf2 , and phase 2 gene transcripts as well as the elevated apoptosis markers . shear stress has a tissue specific effect on cellular anti - oxidant capacity . high shear induces mrna expression of a battery of are - mediated genes in human umbilical vein endothelial cells ( huvec ), but decreases their expression in human t / c28a2 chondrocytic cells . cell culture and shear stress exposure : human t / c28a2 chondrocytic cells were grown ( 37 ° c ., 5 % co 2 ) in 1 : 1 ham &# 39 ; s f - 12 / dmem ( biowhittaker ) supplemented with 10 % fbs . prior to shear exposure , t / c28a2 cells were incubated for 24 hours in serum - free medium containing 1 % nutridoma - sp ( roche ), a low - protein serun replacement that maintains chondrocyte phenotype . primary huvecs were cultured as described ( goldring , m . b ., 2004 ). t / c28a2 cells were exposed to shear stress in media containing 1 % nutridoma by use of a parallel - plate flow chamber with a recirculating flow loop ( 37 ° c ., 5 % co 2 ) ( 10 ). huvecs were treated similarly by circulating media supplemented with 10 % fbs . cell viability , nqo1 activity , glutathione levels , and prostaglandin ( pg ) e 2 production : cell viability was monitored with the mtt assay ( gao , x . et al . 2001 ). nqo1 activity and total gsh ( oxidized and reduced ) levels of cell lysates were determined in96 - well microtiter plates . pge 2 levels were determined in media by the prostaglandin e 2 monoclonal eia kit ( cayman chemical ). transient transfection and plasmid constructs : t / c28a2 cells were transfected with 10 μg of plasmid and 2 μg of control with lipofectamine and plus reagent ( invitrogen ). cells were allowed to recover for 3 hours , incubated overnight in medium containing 1 % nutridoma , and exposed to the indicated treatments . efficiency was assessed by flow cytometry with pegfp - n2 ( bd biosciences ). pcmv - mnrf2 and pnqo1 / are - luc constructs were provided by n . wakabayashi ( wakabayashi , n . et al . 2004 ). igarashi , k ., et al . 1994 ), and pbj m - p110 *• myc , pbj m • p110 • ur , and pcg p110 wt constructs were provided by a . kippel ( hu , q ., et al . 1995 ). promoter activity assay : t / c28a2 cells were transfected with 10 μg of pnqo1 / are - luc and 1 μg each of pegfp - n2 and psv40 - hrl2 ( promega ) to normalize transfection efficiency . firefly and renilla luciferase activities were measured using the dual - luciferase report assay kit ( promega ). intracellular protein staining and western blots : t / c28a2 cells were fixed in 1 . 0 % formaldehyde for 10 min at 37 ° c ., permeabilized in 90 % methanol for 20 minutes on ice , and incubated at 25 ° c . for 10 min in blocking buffer ( 0 . 5 % bsa ). specimens were then incubated with fluorophore - conjugated monoclonal antibodies specific for cox - 1 ( cox - 1 / fitc ) and cox - 2 ( cox - 2 / pe ) ( cayman chemical ) or isotype controls ( bd biosciences ) for 30 min , washed 2 × in blocking buffer , and analyzed by flow cytometry . for western blots , total cell lysates were subjected to sds / page , transferred to a membrane , and probed with caspase - 9 and β - actin antibodies ( upstate ). microarray hybridization and analysis : cy - 3 - and cy - 5 - labeled probes were mixed , dried , resuspended in hybridization buffer ( 50 % formamide , 10 × ssc , 0 . 2 % sds , cot - 1 dna , poly ( a )- dna ), and denatured . the probes were added to microarray slides printed with a set of 32 , 448 or 39 , 936 expressed sequence tags ( ests ), allowed to hybridize at 42 ° c . overnight , and processed as described ( abulencia , j . p ., et al . 2003 . hegde , p . et al . 2000 ). expression ratios were derived using tigr spotfinder ( abulencia , j . p . et al . 2003 , hegde , p . et al . 2000 ). differentially expressed genes were identified by significance analysis of microarrays , and analyzed with the software tmev ( abulencia , j . p . et al . 2003 ). quantitative real - time pcr ( qrt - pcr ) was used to verify dna microarray data . incorporation of sybr green into pcr products was monitored with the 7900ht detection system . exposure of primary huvecs to 20 dyn / cm 2 increased the phase 2 enzyme , nqo1 protein activity and gsh levels in a time - dependent manner . in contrast to huvecs , prolonged exposure ( 48 hours ) of human chondrocytic cells , t / c28a2 to 20 dyn / cm 2 significantly decreased both nqo1 activity and gsh protein levels which correlates well with nqo1 and gclr mrna levels . also , microarray analysis reveals that prolonged exposure ( 48 hours ) of t / c28a2 cells to a shear level of 20 dyn / cm 2 results in a marked reduction in nrf2 and phase 2 transcript expression , including nqo1 , ho - 1 , gst and gclr . t / c28a2 cells transfected with nqo1 / are - luciferase plasmid and exposed to 20 dyn / cm 2 for 48 hours resulted in a substantial reduction of the are - driven promoter activity . addition of phase 2 enzyme transcription inducers , d3t ( 1 , 2 - dithiole - 3 - thione ) and sfn ( sulforaphane ), to the transfected cells increases the luciferase activity providing supporting evidence that are promoters are functionally intact . data represent microarray intensity ratios ( shear / static ) of sheared ( 20 dyn / cm 2 , 48 h ) to paired static controls of t / c28a2 cells . paired treatments consisted of (*) no treatment ; ( † ) 5 μm d3t ( shear ) and 0 . 1 % dmso ( static ); and ( ‡ ) pbj m · p110 * ( shear , 53 % transfection efficiency ) and pbj - null ( static ). data represent mean ± sd ( n = 5 - 8 ). the shear mediated reduction in antioxidant capacity and corresponding pro - inflammatory state ( increased cox - 2 and pge 2 levels ), in chondrocytes is reversed with phase 2 inducers and cox - 2 specific inhibitors . cell culture and shear stress exposure , cell viability , promoter activity and intracellular protein staining and western blots , microarray analysis and qrt - pcr assays were carried out as in example 1 . treatment of high shear exposed t / c28a2 cells with the potent phase 2 enzyme inducer found in edible plants , sulforaphane , sfn ( 1 . 25 μm ) abolished the shear - induced suppression of the phase 2 enzyme nqo1 activity and intracellular gsh levels . furthermore , 1 , 2 - dithiole - 3 - thione , d3t ( 5 μm ), a specific inducer of the nrf2 / are pathway was likewise effective in suppressing the shear - mediated reduction of phase 2 enzyme activity . application of high shear , at least 20 dyn / cm 2 to chondrocytes increases cox - 2 transcript levels ( table 3 ) and cox - 2 dependent pge2 production in t / c28a2 cells in a time - dependent fashion . d3t nearly abrogated both cox - 2 protein expression and pge2 production in chondrocytes subjected to high shear . transfection with pcmv plasmid containing murine nrf2 substantially reduced the pge2 production ( 53 %), consistent with 33 % transfection efficiency of pcmv - mnrf2 . furthermore , addition of the highly selective cox - 2 inhibitor , cay10404 ( 6 . 75 μm ) for 2 hours before and during shear exposure ( 20 dyn / cm 2 for 48 hours ), reduced the shear - induced down - regulation of nqo1 activity and intracellular gsh levels shear induced chondrocyte apoptosis is suppressed by phase 2 inducers and cox - 2 specific inhibitors . cell culture and shear stress exposure , cell viability , promoter activity and intracellular protein staining and western blots , microarray analysis and qrt - pcr assays were carried out as in example 1 . dna fragmentation and mitochondrial depolarization : for dna fragmentation , cells were fixed in 4 % paraformaldehyde for 1 hour at 25 ° c ., washed 2 × in pbs , and permeabilized briefly in 0 . 1 % triton - x100 / 0 . 1 % sodium citrate on ice . subsequently , cells were washed 2 × in pbs , labeled using the in situ cell death kit ( roche ), and analyzed by flow cytometry . to quantify mitochondrial mernbrane potential ( mmp ), cells were labeled using the mitoprobe jc - 1 kit ( molecular probes ). microscopic inspection of shear - stimulated ( 20 dyn / cm 2 , 48 h ) t / c28a2 cells showed cell shrinkage and membrane blebbing , providing morphological evidence of apoptosis . moreover , transcriptional profiling ( table 3 ) revealed increased expression of procaspase - 9 mrna , an apoptotic effector molecule activated by mitochondrial depolarization , indicating the onset of apoptosis . we then monitored the effect of shear on apoptosis by measuring dna fragmentation ( tunel ) and mitochondrial membrane depolarization ( mmp ). the presence of d3t ( 5 μm ) essentially abrogated shear - induced apoptosis , whereas treatment with the cox - 2 specific inhibitors cay10404 ( 6 . 75 μm ) and ns398 ( 30 μm ) resulted in a marked reduction in apoptosis markers . additionally , the role of caspase - 9 in shear - mediated apoptosis was determined by immunoblot analysis , which revealed that high shear ( 20 dyn / cm 2 ) increased the expression of both the proform ( 46 kda ) and active form ( 34 kda ) of caspase - 9 , whereas treatment with d3t or cox - 2 selective inhibitors substantially reduced expression . high shear represses pi3 - k activity that down - regulates phase 2 enzymes and increases apoptosis in chondrocytes . cell culture and shear stress exposure , cell viability , promoter activity assay , microarray analysis were carried out as in example 1 . to identify potential signaling partners involved in the down regulation of phase 2 genes in shear - activated chondrocytes , differentially expressed genes were clustered using support trees . analysis established that transcriptional regulation of p13k ( p85 ) paralleled that of nrf2 and phase 2 genes , indicating that p13k may be involved in the shear - mediated repression of are - regulated transcriptional activity and the onset of apoptosis . to examine the role of pi3k in this signaling cascade , t / c28a2 cells were transfected with a constitutively active pi3k mutant , m • p110 * and exposed to shear . this intervention prevented the shear - mediated suppression of nrf2 , phase 2 genes and the induction of procaspase - 9 ( table 3 ). similarly , constitutively active pi3k was sufficient to enhance nqo1 activity and gsh levels in static cultures , and ablate their downregulation in sheared chondrocytes . intriguingly , shear - induced cox - 2 mrna expression was markedly suppressed in chondrocytes transfected with the constitutively active form of pi3k ( table 3 ) but not the wild - type construct cell culture and shear stress exposure , microarray hybridization and analysis and quantitative real - time pcr ( qrt - pcr ) were carried out as in example 1 . exposure of human t / c - 28a2 chondrocytes to a shear stress level of 20 dyn / cm 2 for 48 hours results in a selective and significant down regulation of pparα mrna expression and concomitant upregulation of pparβ / δ mrna synthesis , while leaving intact pparγ transcript levels . pparα ligand abolished the shear - induced down - regulation of the mrna levels of nrf2 , phase 2 genes , apoptosis and shear induced upregulation of cox2 , c - jun , and jnk2 . cell culture and shear stress exposure , microarray hybridization and analysis and quantitative real - time pcr ( qrt - pcr ) were carried out as in example 1 . t / c - 28a2 chondrocytes were pre - treated with a specific pparα ligand , wy14643 ( 10 μm ) for 2 hours before being subjected to a shear stress level of 20 dyn / cm2 for 48 hours in the presence of wy14643 . this pharmacological intervention abolished the shear - induced down regulation of the mrna levels of nrf2 and phase 2 genes and apoptosis as evidenced by the abrogation of shear - mediated changes of bcl - w and pro - caspase - 9 mrna expression . human t / c - 28a2 cells , pre - treated with either a pparα specific ligand ( wy14643 ; 10 μm ) or solvent ( none ) were sheared at 20 dyn / cm 2 for 48 h . all values , obtained by qrt - pcr , represent transcript ratios for sheared to paired static controls . data are mean + range ( n = 2 ). n / a : not available . cell culture and shear stress exposure , microarray hybridization and analysis and quantitative real - time pcr ( qrt - pcr ) were carried out as in example 1 . t / c - 28a2 chondrocytes were pre - treated with a specific pparα ligand , wy14643 ( 10 μm ) for 2 hours before being subjected to a shear stress level of 20 dyn / cm 2 for 48 hours in the presence of wy14643 . inspection of cdna microarray data reveals that the selective pparα ligand wy14643 nearly abrogates the shear - mediated up - 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