CA2061061C - Blockage of coenzyme a-independent transacylase activity as a method to inhibit lipid mediator production - Google Patents
Blockage of coenzyme a-independent transacylase activity as a method to inhibit lipid mediator production Download PDFInfo
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Abstract
Coenzyme A-independent transacylase is required for the synthesis of arachidonic acid, arachidonic acid metabolites and platelet activation factor. Blocking of this enzyme inhibits the production of inflammatory mediators.
Description
F E F: - 1 ' -~ w ~~ l~l E Z:: 1 ~I ~ 1 1 FW h I h-.I F_ ~Wf I C=: C: 1-I Y~I
r_'. le:: I E
SLDCKAGE t7F Ct7ENZ'~ME A-~lIN~3EP'ENLDEi~T
TRANSACYLASE ACTI'~I~''~ AS A METHOD
Tt~ xNI~IIEI~' LIPID MEDIA1~OR PFtGDIICTI~N
FIELD ~QF 'rHE Il~'~'E
The invention relates to the area of infiarnmatary mediators.
~"he invention is bayed on the discovery that blocking a key enzyme responsible for araoialdanie aoid rennadelling, Coenzyme A
independent tcansacylase (CoA~~Ta, inhibits the production o% Iipid mediators (arachidanic acid, araehidanie acid metabolites, and platelet aetivatin~ factor (PAF)). It has been disaavered that CoA-IT
is rewired in tiza synthesis of arachidonic acid, arachidonie acid metabolites and FAF.
~AsC%~~Y~f.~UND t~F ~'IIJS I~T''~'EIdTICIN
An early event in the response of mast inilannmatory cells to immunalo~ia aotivation and other stimuli is the release of newly formed pratfucts which alter the function and biochemistry of surrpundin~ cells and %issu~s. The ensuing biola~ioal responses, as well as mush of the patho~enes~ which is attributed to inilammaticn and allergy, are thought to be dependent on the effects these products have on adjacent cells within the inilaminatory re~ian.
FED:-1~-.c.~ 4JE=: 14 : 1 :l F:fah~Ih~IEF: L:IF:n:I-I hhG~F:'tE F'. ~7E.
_Z_ Althougta many inflammatory mediators are produced, two ela5se~ of naturally oCCUrring lipids almost always appear during cell activation. One class eonststs of an endogenously formed phospholipid, PAF. The seoond class consists of eicosanoid products which are endogenously ~ormad metabolites o~ arachidonie acid (AA).
L L'~own' 9The eicosanoids include lauicotri~n~ LTB4, LTC~g and LTD, HBT~~s, thrombo~canes and prostaglandins. Both PAS' and eleosanoids are potent lipid mediators whioh induce signs and symptatns ol~erv~d in the pathogen~l~ of various in~lammatary disorders. for example, these mediators have been implicated as having an important rate in allergy, asthma, anaphylaxis, adult respiratory distress syndrome, reperPusion injury, inflammatory bowel disease, rheumatoid arthritis, andotaxie shook, and cardiovascular disuse. Salmon and Higgs ~~r, Med. dull, (19?S) ~3a~8b-298,; Piper et al, Ann. NY Acad. Sci. (1891) 6as;112-119a; ~oltxrnan tAm. ~~ .
Respir. iJis. (3891) i43a188°037. Snyder (Am. J, Physiol. Cell Physlol.
(1990) 259aC897°C?081; Prescott et al. t~. viol. Chem. (1990) 265ai738i°17884, Each ox the cell typws involved in the inflammatory response produce and secrete a unic~u~ subset o~ lipid nnediator~. The quantities and nature of the metabolites depend on which enzymes and preoursar peals are available to the cell.
Studies ire the last few years have indicated that the bioehexnistry at araehidonie acid and PAF overlap at a number of key ~roints, xn particular, it was shown in i98~ that araehidonie aoid and lyso-PA~ (an intermediate in PAP biosynth~ts) could be derived Rrom a~ common greeursor phospholipid, i-alkyl-2-araehidonoyl-sn°glyeero-FEE-..., ~ '-~~~' w~L. i a. : i w ~;Hrur~~~~: F; i ~:mm rar.:r::: z ~ F~ . ,-;;
~. ..s m$_ , 3-phosphocholine (CPC) (Chiltan et al., J. Biol. Chem. (1984) 25:12019-iz019). The association of arachidonic acid mobilisation with PAF production was supported by studies in which arachidonic acid depletion of cells is coupled to a loss of PAF production and refeeding araolxidanie acid to these cells restores PAS' production.
Suggs et al, LJ. Biol. Chew. (1990) 285:12363-°123711; Ramoeha and Plckett ~J.18ia1. Chem. (1986) 261:7592-9895'.
i-alkyl°2-arachidonoyl-OPC, the proposed common precursor %or PAF and eicasanoids, belongs to a family of phasphalipids termed 1-ether phasphoiipids, This term indicates that there is an ether linkage at the sn-i position of the glycerol backbone of the phospholipid. Over the last years, it has been determined that several inflammatory cells have high Content of ether lipfds. In addition, arachidonate is preferentially located in i-ether linked phosphalipids of these cells. Moreover, these 1-ether linked pools are the primary phosphollpfds from which araehidonic acid is mobilized during inflammatory cell activation, Therefore, inflammatory cells, in general, contain large pools of 1-ether linked phespholipids which have the large reservoirs of arachidonate. During cell activation, these 1-ether linked phospholipids release arachidanic acid which forms eicosanaids; and the i-ether phosphalipid lyso PAF is acetylated to form pAF, SUMiVIAR'~' ~~' ?'HR IN'l~~~d'~I~l~
It is an abject of this invention to provide a method of reducing allergy and inflammation, F E F: - 1 ~ - v~,'_..,., ~'.'a E =; J. =E : 1 :._ E; W r-~ ha E F: G: I F~: y r~ r~ ~ ;_ t<:: z E P . 4i -It !s also an object of this invention to inhibit undesir2.bie lipid mediator production.
This invention is based on the discovery that blocking Co-A
independent transaeylase, using selective pharmaCOlogie tools, prevents the movement of arachidonic acid into and from i-ether linked pho5pholipids and the ooncomistant formation of PAF, araohidonla acid and its metabolites such as eieosanoids.
The invention relates to a method of treating disease or disorders mediated by araohidonic acid, its metabolites and/or pA~k' by admiz~isterlng to a patient in need thereof, an effective amount of a compound which inhibits the production, activation or action of ~oA-IT. Inhibition of ~oA-IT inhibits iipid mediator produotfon as well as signs and symptoms of disease and disorders induced by lipid mediators.
The pretrcise of this invention is that blocking the movement of araehidonio acid into and from i-ether phospholipids inhibits lipid mediator (PAF as~d eicosanoid) production by inflammatory cells.
Mor~ preciseiy, when arachidonie acid is prevented from entering key common precuPSOr phospholipicl9, precursor malecules will not be formed and therefore 1P,~~ as weh as eicosanoid, formation are blocked, Similarly, prevention of the removal of arachidonie acid from salient precursors ~i,e., i-ether linked phospholipids) mear~ that araehidonic acid and lyso PAF will be not be mobilized and therefore PA>r' as well as eiccrsanoids will not be produced.
Mill another aspect of the invention relates to a method of screening chemical compounds %or potential anti-inflammatory action. In this way, chemical compounds can be rapidly and easily screened for the ability to inhibit CoA-IT and be useful as an anti-inflammatory agent.
According to an aspect of the present invention, there is provided use of an effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT) for the treatment of disease or disorders mediated by arachidonic acid, its metabolities andlor by platelet activating factor (PAF).
According to another aspect of the present invention, there is provided a composition useful for the treatment of adult respiratory distress syndrome, wherein the composition comprises a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-I~T), and a pharmaceutically compatible carrier for the active compound.
According to a further aspect of the present invention, there is provided the use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of adult respiratory distress syndrome.
According to another aspect of the present invention, there is provided a composition useful for the treatment of reperfusion injury, wherein the composition comprises a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound.
According to a further aspect of the present invention, there is provided the use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of repertusion injury.
According to another aspect of the present invention, there is provided a composition used for the treatment of inflammatory bowel disease wherein the composition comprises a therapeutically effective amount of a compound i i -5a-which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound.
According to a further aspect of the present invention, there is provided the use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of inflammatory bowel disease.
According to another aspect of the present invention, there is provided a composition used for the treatment of disease mediated by arachidonic acid, its metabolites and by platelet activating factor (PAF), wherein the composition comprises a therapeufiically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound.
According to a further aspect of the present invention, there is provided the use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of a disease mediated by arachidonic acid, its metabolites and by platelet activating factor (PAF).
DETAILED DESCRIPTION OF THE INVENTION
This invention concerns and encompasses the enzyme CoA-IT, its unique characteristics, its requirement for the production of PAF, free arachidonic acid and metabolites of arachidonic acid, and the therapeutic utility of inhibiting CoA-IT.
It has now been discovered that CoA-IT activity is required for lipid mediator production. Specifically, it has been discovered that CoA-IT activity is required for the movement of arachidonic acid into phospholipid pools from which it can be released to form free arachidonic acid and for the production of lyso PAF needed for PAF synthesis. Further, CoA-IT has been shown to -5b-be crucial in the mobilization of lyso-PAF and arachidonic acid during inflammatory cell activation. Inhibition of CoA-IT activity will result in a decreased production of PAF and a decreased release of arachidonic acid from cellular phospholipids.
1. Characteristics of CoA-IT Activity CoA-IT activity had been defined to have the following characteristics.
A. Co-factors CoA-IT activity is independent of the presence of Coenzyme A.
In addition, no other co-factors required for activity or that modulate activity have been discovered. CoA-IT activity is not ~i altered by the absence or presence of Ca2+ (0-10 mM), Mg2+ (0-10 mM), EGTA (0-2 mM), EDTA (0-10 mM), ATP, CoA or CoA-fatty acids.
B. J~H
CoA-IT activity over a wide range of pH levels was determined. The results demonstrate that the enzyme is active over a broad pH range of 6.5 - 9. The activity of the enzyme rapidly decreases below pH 6.5 and above pH 10.
C. din ti The kinetics of the CoA-IT reaction were studied with various concentrations of 1-alkyl-2-lyso-GPC. CoA-IT activity increases as a function of the concentration of substrate, 1-alkyl-2-lyso-GPC. The enzyme exhibits an apparent Km for 1-alkyl-2-lyso-GPC of 0.1 - 2 pM.
D. Other Characteristics CoA-IT is stable when treated with dithiothreitol or 2-mercaptoethanol (1-10 mM). CoA-IT is inactivated by exposure to heat or acid and is inhibited by addition of detergents such as 8-octyl glucoside, deoxycholate, cholate, TritonT"" X-100, C~2E8, CHAPS and hexadecyltrimethyl ammonium bromide.
E. S_aecificitX
A key characteristic of CoA-IT is the exquisite specificity of this enzyme for polyunsaturated fatty acids and especially arachidonic acid. Sugiura et al. (J. Biol. Chem. (1987) 202:1199-1205); Chilton et al. (J. Biol. Chem. (1983) 2~$:7268-7271 ); Kramer and Deykin (J. Biol. Chem. (183) X5$:13806-13811 ).
F E F: - 1 '~ - '_: '~ G,.~ E T; 1 ~4 : 1 ~1. E; ri h~I t-I E ~' F; :I F: r_,:
t-I t~i n: t=: :L E F' . 1 1 F. Location Within the cell, CaA-IT activity is completely and tightly associated with mierosomal membranes, Ti reatrnent of these membranes with 2 M ICCI fails to extract more than '1596 of the CoA-I'T activity, suggesting that CoA-I'1' is an Integral rriombrane component. The subGallular location of CoA-lx activity remains to be determined.
Evidence of CaA-i'F activity exista in a variety of in%lammatory cells, including human PMN, nronocyt~, lung mast cells, guinea pig eosi~nophil$ and ~hu~nan X93? monoaytic and I~IL-60 granuloayte ceps lines. There is also preliminary evidence that somewhat less CoA°1~' activity is found in tissues such as lung, liver and kidney. Lass activity yet is found in heart, skeletal muscle and brain, C. ComaarLSOn with other enzymes CoA°xx has eharaetaristfes vrhieh distinguish its activity from the activities of other enzymes involved in lipid metabolism, such as phosphalipasa A2, lypoxygenasas, cyeloaxygenases, CoA dependent acyltransierases and 1~AF acetyl transferasa. This distinction of CoA°IT from the other enzym,~.s based on eharacte~ristics ~ impartant far several reasons, First, the data indicate that CoA-IT activity is a novel enzyme activity. Second, even though a microsomal preparation is used to assess CoA-IT activity, the distinct characteristics of CoA-I'f assure that the assays znaasure only CoA°xT
activity. Finally, the characteristics of CoAmx~' demonstrate that the pharmacologioai utility of inhibition of CoA~i~ is unique.
FEF:- 1 W-~-~',_~ IJEW 1 ~4 : :L ~ F:yhlh-IEF'. ~: I F: C:f-I f~ln:h::: I E F' . 1 W
-g-Z. Role of CaA-IT in PAF Praduetior~ and AA Release The molecule 1-alkyl-z-araohidonoy,l-~GPC has been spawn to be a beoe~ssary precursor far PAF production. ~oA-lx activity plays two pivotal rotes in PAF production, eenterir~g on this molecule. First, CoA-IT activity is required for the specific ~navernent of araehidonic acid into the alkyl PC poet to produce this precursor molecule, Second, CaA-iT activity has been shown to promote the breakdown of 1-alkyl-z-arachidonoyl-GPC into lysa PAF to allow FAF production.
'This CoA-X~' mediated production to lyso BAF can be differentiated from p~A2 activity. CaA-IT activity plays a central aed necessary role in the production of PAF.
There is strong evidence that, in activated inilamcnatory sells, arachidonic acid is released from specitie phaspholipid peals. ~'ar axam~ple, in neutrophils and mast cells the primary source of free arachidanic said is I,-al4cenyi-2-arachidonoyl-GPE. Due to its unidue properties, GoA-IT activity can replenish this pool with arachidonic acid to allow and maintain the release of araehidonie acid, tt has new been discovered that OoA-IT activity is necessary and essential xor the release of free arachidanic acid and the subs~~uen~t formation of biaactive lipid mediators.
S. OoA-IT inhibition Inhibitors of ~oA-IT activity have now been dissevered and Characterized. Suitable inhibitors can readily be identified employing assay tai described below, Often, inhibitors will include an imida2ole structure, F E F: _. 1 ~ _ .a .' LJ E =~ 1 ~-~. : 1 .W F: ha t-1 ha E F: F: I F='. i '. f-I t' I C: H:: I E H~ . 1 .;
-C~.
Represontative eornpounds which inhibit CoA-XT activity in a mierosomal Cod,-iT essay (assay a) at So uM are:
1. 8thy1 &-(3,9,5-txSphany'1-2-oxa-'2,3-dihydroimidazoL-1-y1)haxanoata 2. Sodium 7(3,4,5-txiphenyl~2-oxo-2,3-dihydroimidazol-1-yi)heptanosulphona 3, Diothyl 7-(3.4,5-txiphAnyl-2-a><o-2,3-dihydroimidazoi-1-yi)heptano phoaphonato 4. 8-(1,9,5,~Trlphanyilmldaaol°2-yloxy)octanoia acid 5. 8-(2,3-Diph~nyimaiwlmldo)oetania aald 6. ~1-(2,3-Diphonylmalaimido)undaoanoia acid 7, ~thyi 3-(3,4,x-trlphenyl'2-oxo-2,3-dihydroimldaaol-1-yl)propionato 0, mthyi 5-(3,4,5-triphonyi-2-oxo-2,3-dlhydroimldazol-1-yi)valerate 9. ethyl 5-(1,4,9-triphanyLlmidazol-1-yloxy)Valerata 10, 2-(9-Carboxyhoptyi)-4,5-diphonyloxaaola li. ethyl 6-(3'mothyl-9,5-Aiphanyi~2~oxo-2,3-dihydrolmidazol-1-yl)hoxanoAto 12. ethyl-A-(4,S-Biphenyl-2°oxo-2,3-dihydroimidazal-1-yi)oatanoato 13. 8-1(1,4,5-Ttiphgnyllmidaaol-2-yi-oxy)petanoio acid, ammpnlum salt 14. 1-(7-M~thoxycarbonyihop~tyi)~4,B-diphonyl-1,2,3-txiaaoio 15. 8-(1.4,5-Txiphanylimidazol-2-yloxy)-ootanbmida 16. 1-(7-Cerboxyhaptyi)-2r3r4-triph~nylimldazola 17. 6-(4,9-Diphonyllmldaaoi-2-ylthio)ootanoic aoid 18. 4-tZ-(3,4,5-Txiph~nyl-2-oMO-2,3-dihydrolmldazolyl)nonanoic acid 19. 2-(9-fiydxoxynonyl)-4,3-diphanyl-1.2.3-txiazolo 20. Diothyl 7-(1,4'S~0xiphenylimidazol-2-yloxy)haptano phoophanate 21. 1-(6-$thoxyoaxbonyih~xyl)°2,4,5-triph~nylimidazola 22: Bthy1 8--(4,5-Oiphanylimidaaol-1-yi)octanoato 23. 11-(3.4.5-Txiph~nyl-2-oxa-1,2-dihydeoimidazol-1'y1)undaadnolc acid 34. 7-(3,4,d-Txiphanyl-2-axo-1,2-dihydroimldaaol-1-yi)hoptanitYiS.a FE~o- 1 :;_- __~~; 0.~JELr 1 -~ ~ 1 c. E~F,hIhdEF' E~ I F'.WI-i Y~1 C: H:: C E
- ~o -25. 7-(3,4r9-Triphanyllmtdaaol-L-yloxy)heptanikxilA
Z6. i~(6°CaCboxyhs~xyl)-2,4.9~tslphenyLimidazolo 27. 2-(6-Carboxyh~ptyl)-4,5-diphonyi-1,2r3-triazoio 28. i-(8-~romoootyl)-4,8-dlphenyl-1,2,3-txlazol~
29. 1-(8-Cnrboxyockyl)-2,4,5-trlph~nylimidazole 30. RGhy1 (7-(3,A,g-tslphanyl-x-oxo-2,3-dihydroimidamol-1-yl)m~khyl phonphonako 31. Z-(2-MAthnxyothoxy)ekhyl, 8-(4,5-dlphonyllmidaxal~L-yl)actanoata 3t. 1-(8-Cyandoatyi)-4,3-diphanyL-1,2r3-Cri.axolA
33. 1-( 7-Carboxyhoptyl )-3-( 4-mathoxyphanyl ) ~A r 5-d lphanyl lmidazolo 34. 1-(7-~Ghoxycarbonyihapkyl)-a-methyl-4r5-d(phanylimidaaolo 39. MAChyi 7-(3r4,5-kriphanyl-2-oxo-2,3-Aihydrolmida,zol-1-yi)-3-hApkynoaGo 39. 2-eonzyl-1-(7-carboxyheptyl)-4,3-dlphanyllmldazalo 37. ~thy1 0-IDh~nanthroE9,14-d)lmldazol-1-yl)oatanoato 38. 1-(7-Catboxyheptyl)~2°(4-hydroxyphony1)-4r5-diph~nyllmidazolo 39. ~khyl 7~t1.4,9-trlphonylimidnzal-2-yloxy)hAptano n~mthylphoephinakA
46. 2-(4-(3-CdriSaxypropaxy)phenyl)-4,5-diphonyilmldaaol9 4L. 1-(7-Caxbaxoyhepkyl)-6,9,-bla(2-chlorophpnyi)-2-phonyllmidazole 42. 1-(?-Casboxyh~ptyl)-3~(4-hydroxy-3.5-d11o8ophonyl)-4r5-diphonyiimidazolo 43. 1-(7-CatbdxyhoptyL)-2-phenyl-4,9-bi9(4-makhoxyphonyi)imidsaclo 44. 1-(10-CarboxyAocyl)-x,4,g-trlphonylimldazolo 45. 1-(7-Casboxyheptyl)-2-phenylimldazole 46. 1-(7-~thoxyearboriyl)-4-ph~nylimidazolA
47. 8-(3,4-Diph~nyLpyxazoi~~L-yl)oobanoic said 48. 1-(8-Carboxy~6,8-dim~thylackyl)~3r4.5-Crlph~nyi lmldaaolo 49, 1-(7-Caxboxyhopkyl)~2-aetylkhia-4.9-dlphanylimidazaie 50. 4~(9-(2,4,9~-'rrSphenyilmidazol-1-~yl)butyloxyabonxoic a=ld 51. 1-(Carboxyheptyl)-2-heptyl-4, 5-diphenylimidazole 52. 1-[7-(5-Tetrazolyl)heptyl]-2,4,5-triphenylimidazole 53. Sodium 7-(2,4,5-triphenylimidazole-1-yl)heptane sulphonate 54. 2-[5-(1,3-dioxalan-2-yl)pentylthio]-1-(7-ethoxycarbonylheptyl)-4,5-diphenylimidazole 55. 7-(2,4,5-Triphenylimidazol-1-yl)heptane phosphonic acid Methods of making the above compounds are within the skill of the art.
Methods relevant to the preparation of suitable compounds and relevant activity data are presented in copending U.S. Patent Nos. 5,648,373 and 5,663,053.
To further demonstrate the utility of inhibiting CoA-IT, compounds 1-3 were shown to inhibit the production of PAF (assay c) and the release of arachidonic acid (assay b) from human neutrophils. The methods of synthesis of these compounds and their structural formulas are set forth below. These compounds inhibited PAF production and arachidonic acid release completely and in a concentration dependent fashion. The specificity for inhibition of CoA-IT activity for these compounds and not the activity of other enzymes, such as PLA2 and PAF acetyl transferase, has been demonstrated. This data demonstrate the inhibition of CoA-IT can and will inhibit the production of PAF
and the release of arachidonic acid.
F E ~: - 1 ~ - ~~ 2 lJ E W ~ <E = a r E; Fn r-i r-i ~ ~: F; x F::: m Nt r~1 ~
_: f::: x ~ F . t :__, ..lz_ ~xaro~pl~ 1 iath~rl 5-(3,4.5-triphenvi-2-oxo-2.~-dih dx.~.~irnidazol-1-vi hexanoat~
A mixture of 1,4,8 triphenylin~iidazole t6.24g), ethyl 6°bromohexanaat~
d13.83g), potassium earbanate d13.2~) and 2~butanone was stirred at refiux for 6 hours. The mixture waa filtered, and tho tiltrat~ was ~vaporat~d. The residue was ehromato~raph~d on siltea gel otut~dd with ~tt~anol-hexane to give tire title compound d8.61~) zn.p.
104-106 ° C. , found: C.?6:35; 'x,6.561 N,6.079~
(C2g~Ig~~2C3) g3,equires: C,?8.63;11,6.69; N,6.16~
F' E E: - 1 ::. - ~ '~ 4~.I E _: 1 ~* : 1 ;=: F: f-"H h~a h~ I E F~ E: I F~.
n: I-i Y~ 1 C h::: I F-_" F~ . 1 i' example 2 3odlum 7-(5.4.5-triuhenYi~a-oxow2,~°dih~ydroimidazoi-l~vi~,-h~n~an~u~honate ar4 a) A, mixture of 1,4,5-trlphenyllmidaxol°2-one (15-3g), dibromaheptane (50.6g) and potassium carbonate (13.50 was heated at reflux temperature in dry butanone (750rn1) for 30 hours. ~'h~ mixture was cooled, filtered and the filtrate evaporated to an oil which was ohromato~raphed era silica gel (heXane/ethyl sestets) to giue 1,~,5~~triphenyl°3-d?-bromoheptyl)imlda%ole°°2-~ne (ll.lg,. 4696) as an ail s PIMIEt d(~D~.",13)1.2°l.d(l~l~,tri,5XC1~2)r3y(~I$,t;
~I°i~~r),5.7(2EI,t, -CH~id),8.5-7.~( t5I~,1s~,3xph)pplm.
b) A solution of 1,4,5°triphenyl-8°(7-bromohEptyl)-imidazol°~-one (3.a~D in ethanol (lOxnl) was refluxed with a solution of sodium sulphite (0.58g) in water 45m1) 8or 2Q hours. Mare sodium sulphite (0.2g) was added and refluxin~ continued for a further 2~
hours. 3'he nnl~tture was evaporated to dryness, boiled in ethanol, Illtered hot and a aparated to an oil. This was taken up in ~a small volume of ethanol, a%aess diethyl ether added and the precipitated solid filtered off and et~romato~raphed on silica gel (diehloro-F E F: - 1 ' _' - ~=. _' G~.) ~ L: 1 4 : 1 c; F: i-: twl h-1 E F~: ~: T F: n:
hl 1'I n: k: I F F~ . :l c:
- ~4 ., methanelmect~anat ~;i). 'rlm resulting oil in aneth~nnl/w~t8r 1:1 'Wad passed down an Amberlyst i5 ion exchange rein (~1a form) and evaporated to a solid. ~'hIs was taken up In ethanol and precipitated with diethyl ether giving Sodium '~-(3,4,5-triphenyl-2°oxo-2,3-dihydr~imidazol-1-yl)°he~tanesua~hanate (0.49g) as a white solid, m.p.
160°C.
Found: 0,63.47; I~,5.69; N,S.04; S,6,659~
C~gl3zgNzNaoq,S+s.59G water ~i,e~uires: 0,63,31; ~I,5.89, N,5.23; 8,6.0496 ~xamule3 ,P~ solution of i,4,5-tri~henylm3-(7-bromoheptyl?-imidazol-2-one (l.Og?, p~fuced in Exarnple 1~, step (a) above, and triethyiphosphite (1.66g) in xylene (5 ml) waa heated at reilux temperature for 40 hears.
The Solution was evaporated to an oil and ohramatographed on silica gel (ethyl acerate/ethanol).
F E Ff - 1 :c: - W ::G~., E tf 1 ~. : 1 '=~ ~f f_, hd hd E F: F7 I F:: C: E-I
f' I C: f : I E F~ ~ d ~-, .
-The resulting oli was taken up in diethyl ether, filtered and evaporated to give diethyl 7-(3,~,5-triphenyl-2-oxo-2,3-dihydro-imldazoi-1-yi)°heptanesulphonate as a clear oil (0.8g, 7596).
Found 0,70.11; H,7.3~; N,~,9496 ~32~38~2~4p ~,equires: x,70,31; H,7,19; N,5.1296 .~ssavs (a) Asst, f~A~1T ~.etivity ~Cel.l Preparatir~n w 11937 cells were obtained from ~me~iaan Type Culturo Collection and grown in itPMI-1640 media 4Cibco, brand Island, New York) supplemented with 1096 fetal bovine serum (Hyalone, Logan, 13T) at 37°C, S96C02. Cells were grown without differentiation (basal state) by any agent, such as dimethyi sulfoxide.
Mlerosorz~al ~~r~uaration C~1~ were washed with a buffer of 250 ,mM sucrose, 10 mM
Tris, i, mM E~T,~, 1 rnM MgCl2, pH 7.4 and ruptured by N2 Gavitation (790 psi, 10 minutes). The ruptured c~iis were centrifuged 1000 ~ g, 5 minutes. The resulting supernatant was aentri~uged at 20,000 X g, 20 minutes. Mierosomes were prepared from this supernatant bay aentri~ugation at 9,00,000 x g, 60 minutes. The resulting pellet was washed ono with assay buffer (150 rnM Nazi, 10 mM Na2lKPQ~, 1 mM
EG'~,A, pH 7.4), r~:centrituged and tixe pellet resuspended in assay buffer (4-20 mg protein/a~l) and was stored a,t -80°C until assayed, F E ~: - 1 '~ -- ~~ '~: t~J E Li 1 ~~ : 1 ~=~ L: Fn h~1 hd E F: F: I F: C: f-I
h1 C: h:: T E F~ , ~_ 4_~
..
CoA-IT aotLwitY
CoA-1't activity was measured in 1.5 ml centrifuge tubes in a total volume of 100 ~1. Mierosomes wars diluted in assay buffer to the desired protein concentration (6-20 ug/tube). ~'he reaction was initiated by addition at ~ 8171-alkyl-2-lyso~sn-glyeero-3-phosphocholine (CpC) (0.I ~Ci/tube) and 1 uM tinal cold 1-alkyl-2-lyso-GPC in assay nutter with 0.25 mg/ml catty aoid-poor bovine serum albumin (BSA) (Calbioehem, La Jolla, CA).
(9Ft~1-alkyl-2-lysa-GPC, approximately 50 Ci/mmol, was from MIEN-Dupont (Boston, Massachusetts) and oold 1-alkyl-2-lyso-GpC was from 'Biomoi (Plymouth Meeting, Pennsylvania). Nlicrosomes were pretreated with desired agents for 10 minutes before the additian of ~ 81,1-alkyl-2-lyso-GPC. The reaction was run for 10 minutes at 97~C. 'The reaction was stopped and the lipids extracted by addition of 100 pl of chloroform:methanol (1:2, v/v) tollowed by 100 ~f3 of ehlorpform and 100 lei of 1. M KCI. The samples were vortexed and centrifuged at higlx spe~;d in a microfuge for 2-8 minutes. An aliquot of the ehlorotoem-extracted materials w~re separated by TJGC in chlorotor~n/methanol/acetic aoid/water (50:25:8:4, v/v), visualized by radioseanning (Bioscan) and the product, I aII y Z-alkyl-2-aryl-GPC, was scraped and quanti%ied by liquid scintillation spaetrosoopy. 'With thls 'TLC System, the ~f values for synthetic standards of 1-alkyl°2-lyso-GPI: and k-alkyl-2°aoyl-GPC were approximately 0,25 and O.SS, respectively.
FEFt-1 W ~_~'~ 4.lELi 1=E :..r'4y k:W hlh~IE_F: E:IF:C:F-I f~YC:h::IE P. ~::1 Protein Protein concentration were assesssad using the protein assay yeagents horn Bio-Rad (itiehmond, ~aiiforr~ia)a ~, variety of compounds have been tested in this assay and inhibitors o! 5-lipoxygenasse (S-1.0) and eyeloo~sygenase (CO), such as indomethiein, naproxen, g-(~1~°~'luorophenyi)-5°(~'-pye°ldyl)-2,8-dihydroimidzo-~ 2, i-b a thiazole and 0-(4~-1' Iuorophenyl)-5~(~'°pyridyl)-2,8-dihydroimldzo-'~,1-b~thiazole-dioxide had no effect an ~o,~-IT
activity at concentrations up to 100 uM. The anti-oxidant HIT also has na eitect at concentrations up to 100 ~rM. Compounds whiot~
complex with phospholipids and inhibit P1,A~ activity, such as quinaerine and aristolvchic acid have no effect on GoA-IT activity at concentrations up to 500 uM, Doxepine, a compound reported to inhibit PAF release did not inhibit Cop-IT at concentrations up to 100 uM. Sodium diclofenac, reported to deerea5e leukotriene production by altering arachidonic acid metabolism, had no effect on CoA-IT
activity at concentrations up to 500 ~M.
(b) Assay for Arachidonic Acid release Preparation of hung.,an n~utroohils Human neutrophi>s were obtained in the lahoratory using three diiierent methods. Clne meth! used ieukophoresis packs irorn normal humans and neutrophi>s were isolated using the histopaque-i077 technique. The blood was centrifuged at X00 ~c g for 10 minutes. The cell pellets were resuspended in PAS composed of 13T mM NaGI, 8.8 mM Na~HPO~, 1.5 nttVl K~I~PO~, 2.~ mM 1101 (Dulbeceo~g Gibeo Laboratories, Lotlg Island, New York) and layered over F E F. - 1 w - ~. ~ w G~.i a r.; t ~~ : ::~ a m m r~n r-i ~ r=: F; x ~: ;w t-i r~y r: r::; x E
I~~~.~~~~.
histopaque°1077 (Sigma, 5t. Louis, Missouri), The pellets were eolleoted after centrifugation (300 x g for 30 minutes) and washed once in PBS. '~ha cell pellets were ea~pase~A briefly to lionized water to lyre any erythrocytes. The remaining cells were collected by centrifugation, suspended in PBS, counted and identified after Cytaspinning and staining. The final leuicaCyte prepac°ation was of greater than 9596 purity and viability.
The second method isolated human neutrophils iroan fresh heparinized normal blood using the Histopaque-i077 technique. The blood was layered over Fiistopaque°10?7 (Sigma, St. Louis Missouri) and centrifuged at X00 x g for 80 minutes. The a~ll pellets were resuspended !n 35 rnl of ;CBS and 12 rnl of 69b Dextran, followed by Dextran sedimentation at room temperature for 45 minutes, The upper layer was Collected and further Centrifugated for 10 minutes at 1000 rpm. The cell pellets were exposed brief ly to deionized water to lyre erythrocytes. The remaining calls were collected by centrifugation, suspended fn PHS, Counted and identified after cytaspinning and staining, The final leukocyte preparation was of greater tkaan 9596 purity and viability.
The third method isolated human neutrophiis from freshly drawn heparinized normal blood using the Percoll teohniqu~. The blood was first treated witty 696 Dextran at room temperature for a i hour sedmination. The upper layers of plasma were collected and centrifuged at X00 x g for 10 minutes. The cell pellets were resuspended in PerCOl1 1.070 g/ml supplemented with 5~ fetal bovine serum and layered on discontinuous gradients (1.650, 1.085, 1.090, .. . . . . ._ .~ '__.__4~~ ~ L~ 1 ~4 : ~ 1 F: r, t~d t~a E 'F: F: I F: r_': Ni f~1 n: h=: 't ~ F' . ~ _ oi~_ 1.095 g/ml) ioiiowed by centriiugatlon at 400 x g for 45 minutes, The neutrophils were collected from interfaces of 1,090 and 1,.085 and the 1.095 and 1.090 Pereoll der:sities, followed by a centrfiugation at 400 ~c g for 45 minutes, 'The neutrophils were suspended in PBS, counted and identified sitar cytospinning a;nd stai~~ning, The final leuicoeyte preparation was of greater than 9596 purity and viability, There was no difference noted In the response di the neutrophils nor in the etteots of test compounds in neutrophils isolated by the three diifer~ant techniques.
~xeatm,ent of human neutr.., aahils Neutrophils were suspended in pBS with i mM Ca2* and i.i mM Mg2* at eaneentratlor~s of S to 20 x lAS cells per ml, Cells were added to test tubes and treated with the desired compounds for 5 to 10 minutes, then challenged with calcium ionophere A29187, 2 ~M, or vehicle control, PAS containing 0.25-i ang/ml 1~SA. Alter S to 20 minutes, the reactioa~ were terminated by addition ax an equal volume of ehlorotorm:nnethanol (1:2, v/v) to the samples.
'2l~glAraehidonic said (SQ, i00 or 200 ng) was added as an internal standard and the lipids were extracted by addition ox equal volumes of chloroform and dLstilled water. The samples were vorteaced and centrifuged at high speed and the chloroform ia~yer removed to a alear~ tub~.
Assay for tree araehidonic acid 'The chloroform extract far each sample was evaporated to dt°yness and the material resuspended in hexane, The hexane was passed through a Silica solid phase column (500 mg), washed 2x with F'EF7-1'~-~n:<: 4.IEr~ 1=E:.._.._ WWhlh~IEF: WxF:C:H f~ln:Er:'tE'. F'.~:4 ~~~3~ '~~i~
-Za-hexane az~d a fatty acid enriched fraction eluted with he~cane:ethy!
ether (1:1,, v/v). Solvents wer~ removed from the samples under a stream o~f nitrogen then the samples were eanverted to pentafluorobesa~yi esters using pentafluorobenzyl bromide and dilsopropylethylamine in aoetronitrile. Solvents were removed and samples w8re suspended in hexane. GC/M8i analysis was performed on a suitable instrument, such as a Finnigan ~YAT Tack ?Oa GC/MS/1~S/DS
(Sass Jose, California) operated as a single stage quadruple system or a Hewlett~Packard 5$9a (c) Assa fo Product on of P atelet~ tivat n F'aetor 1PA
Preparation of human neutrons ' Blond was obtained f rom normal humans and neutrophils were isolated as described for the araehidonie acid release assay, above, The final leukocyte preparation was of greater than 9596 purity and viaaiilty.
Treatment of human neutroohils Neutrophils were suspended in FIBS at concentrations of S to 20 x ~aS sails per ml. Cells were added to test tubes and treated with tlze.desired compounds for 5 to iU minutes, thcxi challenged with calcium ionophore A23~19?, 2 ~M and 2a-30 uCi of (sH7aoetio acid (NEN-lDupont, Boston, Massachusetts). ar the vehicle a8 1PE5 with 0,25-1 mg/ml of the. A~f tar 9 to 20 minutes, the reaetiorrss were terminated by addition of an equal volume of ehioroform:methanol (1:2, v/v) to the samples and the lipids were extraetecl by addition of equal volumes of ehlorofarm azrd distilled water. The samples were F a ~: - 1 ~y - ._--.: ~~ G.a E =. i ~x : ~ .__ F: ~: r~ i r..i ~ r: ~, z ~:
:_: Hi r~ i :~: r.:: z ~ ~~ . _ ._.
~21~
vortexed and centrifuged at high speed and the chlorotorxn layer removed to a clean tubE.
Assay ior_ P.A, The chloraiorm from each tube was evaporated to drynea and the material suspended in a small volume of ohloroiorm or ehloroiorm:methanol (2S°100 ul) and the total material spotted on a Silica TLC plate. The plates were deveidped inn c:hloroinrm/methanoi/
acetic acid/water (SO:ZS:B:~;r v/v) visualfzeef by radiogcannin~
(BioSean) and the product, C 3~ 1 PAF, was scraped and quantified try liquid scintillation spectroscopy. 'With this T1,C system, the Rf value for a syntk~etio standard of PAF was approximately o.33.
5. Assa or se Benin ehe iaal eo ounds far ten ia1 anti-initammator~r a~e_tion An assay method for determining the inhibitory activity of compounds for !?AF and araohidocaie acid production is also eneompa~sed by the invention, The method eornpris~ (1) measuring the inhibition of the CoA independent acylation of lysophospholipids in broken cell preparations of said compounds; (2) measuring the inhibition of PA1F production its aetiv&;ted ingla:nmatory cells of Said compounds; and/or (3) measuring the inhibition of araohidonie acid reXease ire activated intlaanmatory cells of said compounds, The activity a~d the eornpound is determined by inhibition of at least 209b of the activities of CoA-1~, PAF or arachidomic said r~leas~, This gay method provides a means wherein chemical compounds can be easily screened for CoA-T"1' inhibiting activity.
FEF:- 1'c'.-W.' L.IEL~ 1 ~1. :.._ _ ~'i=,hlh~IEF: F~ I Fi W'1-I 1~l n:F:.'. I
E F'. 'i=.
~zzr Therapeutic use inhibition of CoA°1T and the simultaneous reduction of PAF and eicosanoid release from inflammatory cells according to this invention i5 0% therapeutic benefit in a broad range of diseases or disorders. The invention is use%ul to trs:at disease states both in humans and in other mammals.
PAF is a very potent gasteric uloerogen whose endogenous release may underlie or contribute to certain forms of gastric ulceration. Intravenous infusion of PAF at doses of 20°200 pmol kga°l,~rnin<°l,a~ into rata has been reported to result in the formation of extensive haernorrhagie erc~ions in the gastric mueosa.
Psoriasis is an inflammatory and proli%erative dis~ase characterized by skin lions. PAF is pro°inflammatory and has been isolated from lesioned scale or psoriatic patients indicating PAS' has a role is the disease of psoriasis, There is also increasing evidence for a potential patho°physiological role for PAk' in cardiovascular disease.
In this regard, recent studies in angina patients show PAF is released during atrial pacing. antraeoronary injection ox PAF in pigs induces a prolonged decrease in coronary flow and, in guineas pig hearts, it induces regional shunting and ischaemia. in addition, PAF has been shown to initiate thrombus formation in a mesenteric artery preparation, both when administered exogenousiy and when released endogenously, More recently PAF has been shown to play a role in brain ischeania induced to animal m~els of stroke.
disease states which benefit xrom the inhibition of CoA~IT
include adult respiratory distress syndrome, asthma, arthritis, F E E. - 1 '~: - ~ '._ 4J E =: -* ~ .: -6 E: F: h-I by E F='. E: T F='. :_: f-I Y~ I :;: h::: I E F' . ' i -z3- ~~~ ~~~ r.
reperfusian injury, endotoxie shock, inflammatory bowel disease and various inflammatory skin disorders. Compounds which inhibit ~oA-1T, by virtue of their ability to antagor~iz~ the actions of IaAF, as well as CoA-TT, are of value in the treatment of any of these conditions.
xn therapeutic use, the CoA~tT inhibitors are usually administered in a standard pharmaceutical composition. ~oA-Ix inhibitors and their pharmaaeuticaily acceptable salt9 which are active when given orally, can be formulated as llquids,,for wrxample syrups, suspensions ar emulsions, tablets, cagsuleg and Lozenges. The choice of form for administration as well as effective dosages will vary depending, inter ai , on the conditiar~ being treated. The choice of mode of administration and dosage is within the skill of the art.
A liquid formulation w111 generally consist of a suspension or solution of the inhibitor or pharmaceutically acceptable sail in a suitable liquid carriers) for exa,mpie, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or colouring agent.
A composition in the form of a tablet can be prepared using any suitable pharmaceutical aarrierts) routinely used for preparing solid formulations. Exampi~ of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.
A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active tng~redient can be prepared using standard carriers and then filled iota a hard gelatin capsule; alternatively, a dispersion or F E F: - 1 ~,~ y t 4~.~ E L: 1 4 : ~ -1. F: ~, N h-1 E F°. 7E: I F: C:
y r~1 r_: k: I E F' o ~: .~
suspension can be prepared using any suitable pharmaceutical earrler(s), far example aqueous gums, cellulases, silicates or oils and the dispersion or suspension then filled into a sof t gelatin capsule.
Typical parenteral eompositians consist ~f a solution nor suspension of the inhibitor ar pharmaceuticaiiy acceptable salt in a sterile aqueous carrier or parenterally adceptabie oil, for exampio polyethylene glycol, polyvinyl pyrrolidone, lecithin, araehis oil or sesame oil, Alternatively, the solution can be lyophfl)sed and then reeor~titute with a suitable solvent dust prier to administration.
A typical suppository formulation comprises a eo~mpound of structure El) or a pharmaoeutiealiy acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glyeols, gelatins or cocoa butter or ether low melting vegetable or synthetic waxes ar fats, Preferably the eornpositian is in unit dace form such as a tablet 4r d:ap8u18o Each dosage unit for oral administration contains preferably from 1 to 250 mg land for parenteral administration contains preferably from 0.1 to 25 mg) of the ~oA-1T inhibitors or a pharrnaeeuticaily aCeeptable salt thereof calculated as the tree bas~, The pharmaceutically acceptable compounds will normally be administered to a sub~eet in a daily dosage regimen. For an adult patient tlzts may be, for example, an oral dose of l~tween 1 mg and 900 mg, preferably between 1 mg and 250 rng, or an intravenous, subcutaneous, or intramuseular does of between 0.1 mg and 100 mg, pret~rabiy between 0.1 mg and 25 mg, of 'the CoA°1T inhibitor or a . ~ ~. . ~ < ~ ~ 4.~ ~ :~~ ~ ~~ : < ~ F~ H r-i ~n ~ ~: ~ t ~~ c: Hi r~ i n:
f::: i ~ r~ . w ~a pharrna~coutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per d,~y.
'the shave description fully disatos~es nrie invention im;iuding preferred embodiments thereof. Modil~ca~tions and improve;nents of the emlaodim~nts specifically disclosed herein are within teh seopu of the following eta~3ms. ~tith 5ut further elak~rar'~m, it is belie~~ed that one skilled in the art can, using the prececf,~.~~ desc~riiation, utilize the present invention to its fullest extent. Th~>re'ure, the Examples herein 2~re to be construed as merely 'ttustrativ%; and not a limitation of the scope of the present invenrl;;n in a~~~ gray. The ernbodirnents of the invention in which an exolu~i~~~: propa:~ y or privilege is claimed are defined as follows.
r_'. le:: I E
SLDCKAGE t7F Ct7ENZ'~ME A-~lIN~3EP'ENLDEi~T
TRANSACYLASE ACTI'~I~''~ AS A METHOD
Tt~ xNI~IIEI~' LIPID MEDIA1~OR PFtGDIICTI~N
FIELD ~QF 'rHE Il~'~'E
The invention relates to the area of infiarnmatary mediators.
~"he invention is bayed on the discovery that blocking a key enzyme responsible for araoialdanie aoid rennadelling, Coenzyme A
independent tcansacylase (CoA~~Ta, inhibits the production o% Iipid mediators (arachidanic acid, araehidanie acid metabolites, and platelet aetivatin~ factor (PAF)). It has been disaavered that CoA-IT
is rewired in tiza synthesis of arachidonic acid, arachidonie acid metabolites and FAF.
~AsC%~~Y~f.~UND t~F ~'IIJS I~T''~'EIdTICIN
An early event in the response of mast inilannmatory cells to immunalo~ia aotivation and other stimuli is the release of newly formed pratfucts which alter the function and biochemistry of surrpundin~ cells and %issu~s. The ensuing biola~ioal responses, as well as mush of the patho~enes~ which is attributed to inilammaticn and allergy, are thought to be dependent on the effects these products have on adjacent cells within the inilaminatory re~ian.
FED:-1~-.c.~ 4JE=: 14 : 1 :l F:fah~Ih~IEF: L:IF:n:I-I hhG~F:'tE F'. ~7E.
_Z_ Althougta many inflammatory mediators are produced, two ela5se~ of naturally oCCUrring lipids almost always appear during cell activation. One class eonststs of an endogenously formed phospholipid, PAF. The seoond class consists of eicosanoid products which are endogenously ~ormad metabolites o~ arachidonie acid (AA).
L L'~own' 9The eicosanoids include lauicotri~n~ LTB4, LTC~g and LTD, HBT~~s, thrombo~canes and prostaglandins. Both PAS' and eleosanoids are potent lipid mediators whioh induce signs and symptatns ol~erv~d in the pathogen~l~ of various in~lammatary disorders. for example, these mediators have been implicated as having an important rate in allergy, asthma, anaphylaxis, adult respiratory distress syndrome, reperPusion injury, inflammatory bowel disease, rheumatoid arthritis, andotaxie shook, and cardiovascular disuse. Salmon and Higgs ~~r, Med. dull, (19?S) ~3a~8b-298,; Piper et al, Ann. NY Acad. Sci. (1891) 6as;112-119a; ~oltxrnan tAm. ~~ .
Respir. iJis. (3891) i43a188°037. Snyder (Am. J, Physiol. Cell Physlol.
(1990) 259aC897°C?081; Prescott et al. t~. viol. Chem. (1990) 265ai738i°17884, Each ox the cell typws involved in the inflammatory response produce and secrete a unic~u~ subset o~ lipid nnediator~. The quantities and nature of the metabolites depend on which enzymes and preoursar peals are available to the cell.
Studies ire the last few years have indicated that the bioehexnistry at araehidonie acid and PAF overlap at a number of key ~roints, xn particular, it was shown in i98~ that araehidonie aoid and lyso-PA~ (an intermediate in PAP biosynth~ts) could be derived Rrom a~ common greeursor phospholipid, i-alkyl-2-araehidonoyl-sn°glyeero-FEE-..., ~ '-~~~' w~L. i a. : i w ~;Hrur~~~~: F; i ~:mm rar.:r::: z ~ F~ . ,-;;
~. ..s m$_ , 3-phosphocholine (CPC) (Chiltan et al., J. Biol. Chem. (1984) 25:12019-iz019). The association of arachidonic acid mobilisation with PAF production was supported by studies in which arachidonic acid depletion of cells is coupled to a loss of PAF production and refeeding araolxidanie acid to these cells restores PAS' production.
Suggs et al, LJ. Biol. Chew. (1990) 285:12363-°123711; Ramoeha and Plckett ~J.18ia1. Chem. (1986) 261:7592-9895'.
i-alkyl°2-arachidonoyl-OPC, the proposed common precursor %or PAF and eicasanoids, belongs to a family of phasphalipids termed 1-ether phasphoiipids, This term indicates that there is an ether linkage at the sn-i position of the glycerol backbone of the phospholipid. Over the last years, it has been determined that several inflammatory cells have high Content of ether lipfds. In addition, arachidonate is preferentially located in i-ether linked phosphalipids of these cells. Moreover, these 1-ether linked pools are the primary phosphollpfds from which araehidonic acid is mobilized during inflammatory cell activation, Therefore, inflammatory cells, in general, contain large pools of 1-ether linked phespholipids which have the large reservoirs of arachidonate. During cell activation, these 1-ether linked phospholipids release arachidanic acid which forms eicosanaids; and the i-ether phosphalipid lyso PAF is acetylated to form pAF, SUMiVIAR'~' ~~' ?'HR IN'l~~~d'~I~l~
It is an abject of this invention to provide a method of reducing allergy and inflammation, F E F: - 1 ~ - v~,'_..,., ~'.'a E =; J. =E : 1 :._ E; W r-~ ha E F: G: I F~: y r~ r~ ~ ;_ t<:: z E P . 4i -It !s also an object of this invention to inhibit undesir2.bie lipid mediator production.
This invention is based on the discovery that blocking Co-A
independent transaeylase, using selective pharmaCOlogie tools, prevents the movement of arachidonic acid into and from i-ether linked pho5pholipids and the ooncomistant formation of PAF, araohidonla acid and its metabolites such as eieosanoids.
The invention relates to a method of treating disease or disorders mediated by araohidonic acid, its metabolites and/or pA~k' by admiz~isterlng to a patient in need thereof, an effective amount of a compound which inhibits the production, activation or action of ~oA-IT. Inhibition of ~oA-IT inhibits iipid mediator produotfon as well as signs and symptoms of disease and disorders induced by lipid mediators.
The pretrcise of this invention is that blocking the movement of araehidonio acid into and from i-ether phospholipids inhibits lipid mediator (PAF as~d eicosanoid) production by inflammatory cells.
Mor~ preciseiy, when arachidonie acid is prevented from entering key common precuPSOr phospholipicl9, precursor malecules will not be formed and therefore 1P,~~ as weh as eicosanoid, formation are blocked, Similarly, prevention of the removal of arachidonie acid from salient precursors ~i,e., i-ether linked phospholipids) mear~ that araehidonic acid and lyso PAF will be not be mobilized and therefore PA>r' as well as eiccrsanoids will not be produced.
Mill another aspect of the invention relates to a method of screening chemical compounds %or potential anti-inflammatory action. In this way, chemical compounds can be rapidly and easily screened for the ability to inhibit CoA-IT and be useful as an anti-inflammatory agent.
According to an aspect of the present invention, there is provided use of an effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT) for the treatment of disease or disorders mediated by arachidonic acid, its metabolities andlor by platelet activating factor (PAF).
According to another aspect of the present invention, there is provided a composition useful for the treatment of adult respiratory distress syndrome, wherein the composition comprises a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-I~T), and a pharmaceutically compatible carrier for the active compound.
According to a further aspect of the present invention, there is provided the use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of adult respiratory distress syndrome.
According to another aspect of the present invention, there is provided a composition useful for the treatment of reperfusion injury, wherein the composition comprises a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound.
According to a further aspect of the present invention, there is provided the use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of repertusion injury.
According to another aspect of the present invention, there is provided a composition used for the treatment of inflammatory bowel disease wherein the composition comprises a therapeutically effective amount of a compound i i -5a-which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound.
According to a further aspect of the present invention, there is provided the use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of inflammatory bowel disease.
According to another aspect of the present invention, there is provided a composition used for the treatment of disease mediated by arachidonic acid, its metabolites and by platelet activating factor (PAF), wherein the composition comprises a therapeufiically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound.
According to a further aspect of the present invention, there is provided the use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of a disease mediated by arachidonic acid, its metabolites and by platelet activating factor (PAF).
DETAILED DESCRIPTION OF THE INVENTION
This invention concerns and encompasses the enzyme CoA-IT, its unique characteristics, its requirement for the production of PAF, free arachidonic acid and metabolites of arachidonic acid, and the therapeutic utility of inhibiting CoA-IT.
It has now been discovered that CoA-IT activity is required for lipid mediator production. Specifically, it has been discovered that CoA-IT activity is required for the movement of arachidonic acid into phospholipid pools from which it can be released to form free arachidonic acid and for the production of lyso PAF needed for PAF synthesis. Further, CoA-IT has been shown to -5b-be crucial in the mobilization of lyso-PAF and arachidonic acid during inflammatory cell activation. Inhibition of CoA-IT activity will result in a decreased production of PAF and a decreased release of arachidonic acid from cellular phospholipids.
1. Characteristics of CoA-IT Activity CoA-IT activity had been defined to have the following characteristics.
A. Co-factors CoA-IT activity is independent of the presence of Coenzyme A.
In addition, no other co-factors required for activity or that modulate activity have been discovered. CoA-IT activity is not ~i altered by the absence or presence of Ca2+ (0-10 mM), Mg2+ (0-10 mM), EGTA (0-2 mM), EDTA (0-10 mM), ATP, CoA or CoA-fatty acids.
B. J~H
CoA-IT activity over a wide range of pH levels was determined. The results demonstrate that the enzyme is active over a broad pH range of 6.5 - 9. The activity of the enzyme rapidly decreases below pH 6.5 and above pH 10.
C. din ti The kinetics of the CoA-IT reaction were studied with various concentrations of 1-alkyl-2-lyso-GPC. CoA-IT activity increases as a function of the concentration of substrate, 1-alkyl-2-lyso-GPC. The enzyme exhibits an apparent Km for 1-alkyl-2-lyso-GPC of 0.1 - 2 pM.
D. Other Characteristics CoA-IT is stable when treated with dithiothreitol or 2-mercaptoethanol (1-10 mM). CoA-IT is inactivated by exposure to heat or acid and is inhibited by addition of detergents such as 8-octyl glucoside, deoxycholate, cholate, TritonT"" X-100, C~2E8, CHAPS and hexadecyltrimethyl ammonium bromide.
E. S_aecificitX
A key characteristic of CoA-IT is the exquisite specificity of this enzyme for polyunsaturated fatty acids and especially arachidonic acid. Sugiura et al. (J. Biol. Chem. (1987) 202:1199-1205); Chilton et al. (J. Biol. Chem. (1983) 2~$:7268-7271 ); Kramer and Deykin (J. Biol. Chem. (183) X5$:13806-13811 ).
F E F: - 1 '~ - '_: '~ G,.~ E T; 1 ~4 : 1 ~1. E; ri h~I t-I E ~' F; :I F: r_,:
t-I t~i n: t=: :L E F' . 1 1 F. Location Within the cell, CaA-IT activity is completely and tightly associated with mierosomal membranes, Ti reatrnent of these membranes with 2 M ICCI fails to extract more than '1596 of the CoA-I'T activity, suggesting that CoA-I'1' is an Integral rriombrane component. The subGallular location of CoA-lx activity remains to be determined.
Evidence of CaA-i'F activity exista in a variety of in%lammatory cells, including human PMN, nronocyt~, lung mast cells, guinea pig eosi~nophil$ and ~hu~nan X93? monoaytic and I~IL-60 granuloayte ceps lines. There is also preliminary evidence that somewhat less CoA°1~' activity is found in tissues such as lung, liver and kidney. Lass activity yet is found in heart, skeletal muscle and brain, C. ComaarLSOn with other enzymes CoA°xx has eharaetaristfes vrhieh distinguish its activity from the activities of other enzymes involved in lipid metabolism, such as phosphalipasa A2, lypoxygenasas, cyeloaxygenases, CoA dependent acyltransierases and 1~AF acetyl transferasa. This distinction of CoA°IT from the other enzym,~.s based on eharacte~ristics ~ impartant far several reasons, First, the data indicate that CoA-IT activity is a novel enzyme activity. Second, even though a microsomal preparation is used to assess CoA-IT activity, the distinct characteristics of CoA-I'f assure that the assays znaasure only CoA°xT
activity. Finally, the characteristics of CoAmx~' demonstrate that the pharmacologioai utility of inhibition of CoA~i~ is unique.
FEF:- 1 W-~-~',_~ IJEW 1 ~4 : :L ~ F:yhlh-IEF'. ~: I F: C:f-I f~ln:h::: I E F' . 1 W
-g-Z. Role of CaA-IT in PAF Praduetior~ and AA Release The molecule 1-alkyl-z-araohidonoy,l-~GPC has been spawn to be a beoe~ssary precursor far PAF production. ~oA-lx activity plays two pivotal rotes in PAF production, eenterir~g on this molecule. First, CoA-IT activity is required for the specific ~navernent of araehidonic acid into the alkyl PC poet to produce this precursor molecule, Second, CaA-iT activity has been shown to promote the breakdown of 1-alkyl-z-arachidonoyl-GPC into lysa PAF to allow FAF production.
'This CoA-X~' mediated production to lyso BAF can be differentiated from p~A2 activity. CaA-IT activity plays a central aed necessary role in the production of PAF.
There is strong evidence that, in activated inilamcnatory sells, arachidonic acid is released from specitie phaspholipid peals. ~'ar axam~ple, in neutrophils and mast cells the primary source of free arachidanic said is I,-al4cenyi-2-arachidonoyl-GPE. Due to its unidue properties, GoA-IT activity can replenish this pool with arachidonic acid to allow and maintain the release of araehidonie acid, tt has new been discovered that OoA-IT activity is necessary and essential xor the release of free arachidanic acid and the subs~~uen~t formation of biaactive lipid mediators.
S. OoA-IT inhibition Inhibitors of ~oA-IT activity have now been dissevered and Characterized. Suitable inhibitors can readily be identified employing assay tai described below, Often, inhibitors will include an imida2ole structure, F E F: _. 1 ~ _ .a .' LJ E =~ 1 ~-~. : 1 .W F: ha t-1 ha E F: F: I F='. i '. f-I t' I C: H:: I E H~ . 1 .;
-C~.
Represontative eornpounds which inhibit CoA-XT activity in a mierosomal Cod,-iT essay (assay a) at So uM are:
1. 8thy1 &-(3,9,5-txSphany'1-2-oxa-'2,3-dihydroimidazoL-1-y1)haxanoata 2. Sodium 7(3,4,5-txiphenyl~2-oxo-2,3-dihydroimidazol-1-yi)heptanosulphona 3, Diothyl 7-(3.4,5-txiphAnyl-2-a><o-2,3-dihydroimidazoi-1-yi)heptano phoaphonato 4. 8-(1,9,5,~Trlphanyilmldaaol°2-yloxy)octanoia acid 5. 8-(2,3-Diph~nyimaiwlmldo)oetania aald 6. ~1-(2,3-Diphonylmalaimido)undaoanoia acid 7, ~thyi 3-(3,4,x-trlphenyl'2-oxo-2,3-dihydroimldaaol-1-yl)propionato 0, mthyi 5-(3,4,5-triphonyi-2-oxo-2,3-dlhydroimldazol-1-yi)valerate 9. ethyl 5-(1,4,9-triphanyLlmidazol-1-yloxy)Valerata 10, 2-(9-Carboxyhoptyi)-4,5-diphonyloxaaola li. ethyl 6-(3'mothyl-9,5-Aiphanyi~2~oxo-2,3-dihydrolmidazol-1-yl)hoxanoAto 12. ethyl-A-(4,S-Biphenyl-2°oxo-2,3-dihydroimidazal-1-yi)oatanoato 13. 8-1(1,4,5-Ttiphgnyllmidaaol-2-yi-oxy)petanoio acid, ammpnlum salt 14. 1-(7-M~thoxycarbonyihop~tyi)~4,B-diphonyl-1,2,3-txiaaoio 15. 8-(1.4,5-Txiphanylimidazol-2-yloxy)-ootanbmida 16. 1-(7-Cerboxyhaptyi)-2r3r4-triph~nylimldazola 17. 6-(4,9-Diphonyllmldaaoi-2-ylthio)ootanoic aoid 18. 4-tZ-(3,4,5-Txiph~nyl-2-oMO-2,3-dihydrolmldazolyl)nonanoic acid 19. 2-(9-fiydxoxynonyl)-4,3-diphanyl-1.2.3-txiazolo 20. Diothyl 7-(1,4'S~0xiphenylimidazol-2-yloxy)haptano phoophanate 21. 1-(6-$thoxyoaxbonyih~xyl)°2,4,5-triph~nylimidazola 22: Bthy1 8--(4,5-Oiphanylimidaaol-1-yi)octanoato 23. 11-(3.4.5-Txiph~nyl-2-oxa-1,2-dihydeoimidazol-1'y1)undaadnolc acid 34. 7-(3,4,d-Txiphanyl-2-axo-1,2-dihydroimldaaol-1-yi)hoptanitYiS.a FE~o- 1 :;_- __~~; 0.~JELr 1 -~ ~ 1 c. E~F,hIhdEF' E~ I F'.WI-i Y~1 C: H:: C E
- ~o -25. 7-(3,4r9-Triphanyllmtdaaol-L-yloxy)heptanikxilA
Z6. i~(6°CaCboxyhs~xyl)-2,4.9~tslphenyLimidazolo 27. 2-(6-Carboxyh~ptyl)-4,5-diphonyi-1,2r3-triazoio 28. i-(8-~romoootyl)-4,8-dlphenyl-1,2,3-txlazol~
29. 1-(8-Cnrboxyockyl)-2,4,5-trlph~nylimidazole 30. RGhy1 (7-(3,A,g-tslphanyl-x-oxo-2,3-dihydroimidamol-1-yl)m~khyl phonphonako 31. Z-(2-MAthnxyothoxy)ekhyl, 8-(4,5-dlphonyllmidaxal~L-yl)actanoata 3t. 1-(8-Cyandoatyi)-4,3-diphanyL-1,2r3-Cri.axolA
33. 1-( 7-Carboxyhoptyl )-3-( 4-mathoxyphanyl ) ~A r 5-d lphanyl lmidazolo 34. 1-(7-~Ghoxycarbonyihapkyl)-a-methyl-4r5-d(phanylimidaaolo 39. MAChyi 7-(3r4,5-kriphanyl-2-oxo-2,3-Aihydrolmida,zol-1-yi)-3-hApkynoaGo 39. 2-eonzyl-1-(7-carboxyheptyl)-4,3-dlphanyllmldazalo 37. ~thy1 0-IDh~nanthroE9,14-d)lmldazol-1-yl)oatanoato 38. 1-(7-Catboxyheptyl)~2°(4-hydroxyphony1)-4r5-diph~nyllmidazolo 39. ~khyl 7~t1.4,9-trlphonylimidnzal-2-yloxy)hAptano n~mthylphoephinakA
46. 2-(4-(3-CdriSaxypropaxy)phenyl)-4,5-diphonyilmldaaol9 4L. 1-(7-Caxbaxoyhepkyl)-6,9,-bla(2-chlorophpnyi)-2-phonyllmidazole 42. 1-(?-Casboxyh~ptyl)-3~(4-hydroxy-3.5-d11o8ophonyl)-4r5-diphonyiimidazolo 43. 1-(7-CatbdxyhoptyL)-2-phenyl-4,9-bi9(4-makhoxyphonyi)imidsaclo 44. 1-(10-CarboxyAocyl)-x,4,g-trlphonylimldazolo 45. 1-(7-Casboxyheptyl)-2-phenylimldazole 46. 1-(7-~thoxyearboriyl)-4-ph~nylimidazolA
47. 8-(3,4-Diph~nyLpyxazoi~~L-yl)oobanoic said 48. 1-(8-Carboxy~6,8-dim~thylackyl)~3r4.5-Crlph~nyi lmldaaolo 49, 1-(7-Caxboxyhopkyl)~2-aetylkhia-4.9-dlphanylimidazaie 50. 4~(9-(2,4,9~-'rrSphenyilmidazol-1-~yl)butyloxyabonxoic a=ld 51. 1-(Carboxyheptyl)-2-heptyl-4, 5-diphenylimidazole 52. 1-[7-(5-Tetrazolyl)heptyl]-2,4,5-triphenylimidazole 53. Sodium 7-(2,4,5-triphenylimidazole-1-yl)heptane sulphonate 54. 2-[5-(1,3-dioxalan-2-yl)pentylthio]-1-(7-ethoxycarbonylheptyl)-4,5-diphenylimidazole 55. 7-(2,4,5-Triphenylimidazol-1-yl)heptane phosphonic acid Methods of making the above compounds are within the skill of the art.
Methods relevant to the preparation of suitable compounds and relevant activity data are presented in copending U.S. Patent Nos. 5,648,373 and 5,663,053.
To further demonstrate the utility of inhibiting CoA-IT, compounds 1-3 were shown to inhibit the production of PAF (assay c) and the release of arachidonic acid (assay b) from human neutrophils. The methods of synthesis of these compounds and their structural formulas are set forth below. These compounds inhibited PAF production and arachidonic acid release completely and in a concentration dependent fashion. The specificity for inhibition of CoA-IT activity for these compounds and not the activity of other enzymes, such as PLA2 and PAF acetyl transferase, has been demonstrated. This data demonstrate the inhibition of CoA-IT can and will inhibit the production of PAF
and the release of arachidonic acid.
F E ~: - 1 ~ - ~~ 2 lJ E W ~ <E = a r E; Fn r-i r-i ~ ~: F; x F::: m Nt r~1 ~
_: f::: x ~ F . t :__, ..lz_ ~xaro~pl~ 1 iath~rl 5-(3,4.5-triphenvi-2-oxo-2.~-dih dx.~.~irnidazol-1-vi hexanoat~
A mixture of 1,4,8 triphenylin~iidazole t6.24g), ethyl 6°bromohexanaat~
d13.83g), potassium earbanate d13.2~) and 2~butanone was stirred at refiux for 6 hours. The mixture waa filtered, and tho tiltrat~ was ~vaporat~d. The residue was ehromato~raph~d on siltea gel otut~dd with ~tt~anol-hexane to give tire title compound d8.61~) zn.p.
104-106 ° C. , found: C.?6:35; 'x,6.561 N,6.079~
(C2g~Ig~~2C3) g3,equires: C,?8.63;11,6.69; N,6.16~
F' E E: - 1 ::. - ~ '~ 4~.I E _: 1 ~* : 1 ;=: F: f-"H h~a h~ I E F~ E: I F~.
n: I-i Y~ 1 C h::: I F-_" F~ . 1 i' example 2 3odlum 7-(5.4.5-triuhenYi~a-oxow2,~°dih~ydroimidazoi-l~vi~,-h~n~an~u~honate ar4 a) A, mixture of 1,4,5-trlphenyllmidaxol°2-one (15-3g), dibromaheptane (50.6g) and potassium carbonate (13.50 was heated at reflux temperature in dry butanone (750rn1) for 30 hours. ~'h~ mixture was cooled, filtered and the filtrate evaporated to an oil which was ohromato~raphed era silica gel (heXane/ethyl sestets) to giue 1,~,5~~triphenyl°3-d?-bromoheptyl)imlda%ole°°2-~ne (ll.lg,. 4696) as an ail s PIMIEt d(~D~.",13)1.2°l.d(l~l~,tri,5XC1~2)r3y(~I$,t;
~I°i~~r),5.7(2EI,t, -CH~id),8.5-7.~( t5I~,1s~,3xph)pplm.
b) A solution of 1,4,5°triphenyl-8°(7-bromohEptyl)-imidazol°~-one (3.a~D in ethanol (lOxnl) was refluxed with a solution of sodium sulphite (0.58g) in water 45m1) 8or 2Q hours. Mare sodium sulphite (0.2g) was added and refluxin~ continued for a further 2~
hours. 3'he nnl~tture was evaporated to dryness, boiled in ethanol, Illtered hot and a aparated to an oil. This was taken up in ~a small volume of ethanol, a%aess diethyl ether added and the precipitated solid filtered off and et~romato~raphed on silica gel (diehloro-F E F: - 1 ' _' - ~=. _' G~.) ~ L: 1 4 : 1 c; F: i-: twl h-1 E F~: ~: T F: n:
hl 1'I n: k: I F F~ . :l c:
- ~4 ., methanelmect~anat ~;i). 'rlm resulting oil in aneth~nnl/w~t8r 1:1 'Wad passed down an Amberlyst i5 ion exchange rein (~1a form) and evaporated to a solid. ~'hIs was taken up In ethanol and precipitated with diethyl ether giving Sodium '~-(3,4,5-triphenyl-2°oxo-2,3-dihydr~imidazol-1-yl)°he~tanesua~hanate (0.49g) as a white solid, m.p.
160°C.
Found: 0,63.47; I~,5.69; N,S.04; S,6,659~
C~gl3zgNzNaoq,S+s.59G water ~i,e~uires: 0,63,31; ~I,5.89, N,5.23; 8,6.0496 ~xamule3 ,P~ solution of i,4,5-tri~henylm3-(7-bromoheptyl?-imidazol-2-one (l.Og?, p~fuced in Exarnple 1~, step (a) above, and triethyiphosphite (1.66g) in xylene (5 ml) waa heated at reilux temperature for 40 hears.
The Solution was evaporated to an oil and ohramatographed on silica gel (ethyl acerate/ethanol).
F E Ff - 1 :c: - W ::G~., E tf 1 ~. : 1 '=~ ~f f_, hd hd E F: F7 I F:: C: E-I
f' I C: f : I E F~ ~ d ~-, .
-The resulting oli was taken up in diethyl ether, filtered and evaporated to give diethyl 7-(3,~,5-triphenyl-2-oxo-2,3-dihydro-imldazoi-1-yi)°heptanesulphonate as a clear oil (0.8g, 7596).
Found 0,70.11; H,7.3~; N,~,9496 ~32~38~2~4p ~,equires: x,70,31; H,7,19; N,5.1296 .~ssavs (a) Asst, f~A~1T ~.etivity ~Cel.l Preparatir~n w 11937 cells were obtained from ~me~iaan Type Culturo Collection and grown in itPMI-1640 media 4Cibco, brand Island, New York) supplemented with 1096 fetal bovine serum (Hyalone, Logan, 13T) at 37°C, S96C02. Cells were grown without differentiation (basal state) by any agent, such as dimethyi sulfoxide.
Mlerosorz~al ~~r~uaration C~1~ were washed with a buffer of 250 ,mM sucrose, 10 mM
Tris, i, mM E~T,~, 1 rnM MgCl2, pH 7.4 and ruptured by N2 Gavitation (790 psi, 10 minutes). The ruptured c~iis were centrifuged 1000 ~ g, 5 minutes. The resulting supernatant was aentri~uged at 20,000 X g, 20 minutes. Mierosomes were prepared from this supernatant bay aentri~ugation at 9,00,000 x g, 60 minutes. The resulting pellet was washed ono with assay buffer (150 rnM Nazi, 10 mM Na2lKPQ~, 1 mM
EG'~,A, pH 7.4), r~:centrituged and tixe pellet resuspended in assay buffer (4-20 mg protein/a~l) and was stored a,t -80°C until assayed, F E ~: - 1 '~ -- ~~ '~: t~J E Li 1 ~~ : 1 ~=~ L: Fn h~1 hd E F: F: I F: C: f-I
h1 C: h:: T E F~ , ~_ 4_~
..
CoA-IT aotLwitY
CoA-1't activity was measured in 1.5 ml centrifuge tubes in a total volume of 100 ~1. Mierosomes wars diluted in assay buffer to the desired protein concentration (6-20 ug/tube). ~'he reaction was initiated by addition at ~ 8171-alkyl-2-lyso~sn-glyeero-3-phosphocholine (CpC) (0.I ~Ci/tube) and 1 uM tinal cold 1-alkyl-2-lyso-GPC in assay nutter with 0.25 mg/ml catty aoid-poor bovine serum albumin (BSA) (Calbioehem, La Jolla, CA).
(9Ft~1-alkyl-2-lysa-GPC, approximately 50 Ci/mmol, was from MIEN-Dupont (Boston, Massachusetts) and oold 1-alkyl-2-lyso-GpC was from 'Biomoi (Plymouth Meeting, Pennsylvania). Nlicrosomes were pretreated with desired agents for 10 minutes before the additian of ~ 81,1-alkyl-2-lyso-GPC. The reaction was run for 10 minutes at 97~C. 'The reaction was stopped and the lipids extracted by addition of 100 pl of chloroform:methanol (1:2, v/v) tollowed by 100 ~f3 of ehlorpform and 100 lei of 1. M KCI. The samples were vortexed and centrifuged at higlx spe~;d in a microfuge for 2-8 minutes. An aliquot of the ehlorotoem-extracted materials w~re separated by TJGC in chlorotor~n/methanol/acetic aoid/water (50:25:8:4, v/v), visualized by radioseanning (Bioscan) and the product, I aII y Z-alkyl-2-aryl-GPC, was scraped and quanti%ied by liquid scintillation spaetrosoopy. 'With thls 'TLC System, the ~f values for synthetic standards of 1-alkyl°2-lyso-GPI: and k-alkyl-2°aoyl-GPC were approximately 0,25 and O.SS, respectively.
FEFt-1 W ~_~'~ 4.lELi 1=E :..r'4y k:W hlh~IE_F: E:IF:C:F-I f~YC:h::IE P. ~::1 Protein Protein concentration were assesssad using the protein assay yeagents horn Bio-Rad (itiehmond, ~aiiforr~ia)a ~, variety of compounds have been tested in this assay and inhibitors o! 5-lipoxygenasse (S-1.0) and eyeloo~sygenase (CO), such as indomethiein, naproxen, g-(~1~°~'luorophenyi)-5°(~'-pye°ldyl)-2,8-dihydroimidzo-~ 2, i-b a thiazole and 0-(4~-1' Iuorophenyl)-5~(~'°pyridyl)-2,8-dihydroimldzo-'~,1-b~thiazole-dioxide had no effect an ~o,~-IT
activity at concentrations up to 100 uM. The anti-oxidant HIT also has na eitect at concentrations up to 100 ~rM. Compounds whiot~
complex with phospholipids and inhibit P1,A~ activity, such as quinaerine and aristolvchic acid have no effect on GoA-IT activity at concentrations up to 500 uM, Doxepine, a compound reported to inhibit PAF release did not inhibit Cop-IT at concentrations up to 100 uM. Sodium diclofenac, reported to deerea5e leukotriene production by altering arachidonic acid metabolism, had no effect on CoA-IT
activity at concentrations up to 500 ~M.
(b) Assay for Arachidonic Acid release Preparation of hung.,an n~utroohils Human neutrophi>s were obtained in the lahoratory using three diiierent methods. Clne meth! used ieukophoresis packs irorn normal humans and neutrophi>s were isolated using the histopaque-i077 technique. The blood was centrifuged at X00 ~c g for 10 minutes. The cell pellets were resuspended in PAS composed of 13T mM NaGI, 8.8 mM Na~HPO~, 1.5 nttVl K~I~PO~, 2.~ mM 1101 (Dulbeceo~g Gibeo Laboratories, Lotlg Island, New York) and layered over F E F. - 1 w - ~. ~ w G~.i a r.; t ~~ : ::~ a m m r~n r-i ~ r=: F; x ~: ;w t-i r~y r: r::; x E
I~~~.~~~~.
histopaque°1077 (Sigma, 5t. Louis, Missouri), The pellets were eolleoted after centrifugation (300 x g for 30 minutes) and washed once in PBS. '~ha cell pellets were ea~pase~A briefly to lionized water to lyre any erythrocytes. The remaining cells were collected by centrifugation, suspended in PBS, counted and identified after Cytaspinning and staining. The final leuicaCyte prepac°ation was of greater than 9596 purity and viability.
The second method isolated human neutrophils iroan fresh heparinized normal blood using the Histopaque-i077 technique. The blood was layered over Fiistopaque°10?7 (Sigma, St. Louis Missouri) and centrifuged at X00 x g for 80 minutes. The a~ll pellets were resuspended !n 35 rnl of ;CBS and 12 rnl of 69b Dextran, followed by Dextran sedimentation at room temperature for 45 minutes, The upper layer was Collected and further Centrifugated for 10 minutes at 1000 rpm. The cell pellets were exposed brief ly to deionized water to lyre erythrocytes. The remaining calls were collected by centrifugation, suspended fn PHS, Counted and identified after cytaspinning and staining, The final leukocyte preparation was of greater tkaan 9596 purity and viability.
The third method isolated human neutrophiis from freshly drawn heparinized normal blood using the Percoll teohniqu~. The blood was first treated witty 696 Dextran at room temperature for a i hour sedmination. The upper layers of plasma were collected and centrifuged at X00 x g for 10 minutes. The cell pellets were resuspended in PerCOl1 1.070 g/ml supplemented with 5~ fetal bovine serum and layered on discontinuous gradients (1.650, 1.085, 1.090, .. . . . . ._ .~ '__.__4~~ ~ L~ 1 ~4 : ~ 1 F: r, t~d t~a E 'F: F: I F: r_': Ni f~1 n: h=: 't ~ F' . ~ _ oi~_ 1.095 g/ml) ioiiowed by centriiugatlon at 400 x g for 45 minutes, The neutrophils were collected from interfaces of 1,090 and 1,.085 and the 1.095 and 1.090 Pereoll der:sities, followed by a centrfiugation at 400 ~c g for 45 minutes, 'The neutrophils were suspended in PBS, counted and identified sitar cytospinning a;nd stai~~ning, The final leuicoeyte preparation was of greater than 9596 purity and viability, There was no difference noted In the response di the neutrophils nor in the etteots of test compounds in neutrophils isolated by the three diifer~ant techniques.
~xeatm,ent of human neutr.., aahils Neutrophils were suspended in pBS with i mM Ca2* and i.i mM Mg2* at eaneentratlor~s of S to 20 x lAS cells per ml, Cells were added to test tubes and treated with the desired compounds for 5 to 10 minutes, then challenged with calcium ionophere A29187, 2 ~M, or vehicle control, PAS containing 0.25-i ang/ml 1~SA. Alter S to 20 minutes, the reactioa~ were terminated by addition ax an equal volume of ehlorotorm:nnethanol (1:2, v/v) to the samples.
'2l~glAraehidonic said (SQ, i00 or 200 ng) was added as an internal standard and the lipids were extracted by addition ox equal volumes of chloroform and dLstilled water. The samples were vorteaced and centrifuged at high speed and the chloroform ia~yer removed to a alear~ tub~.
Assay for tree araehidonic acid 'The chloroform extract far each sample was evaporated to dt°yness and the material resuspended in hexane, The hexane was passed through a Silica solid phase column (500 mg), washed 2x with F'EF7-1'~-~n:<: 4.IEr~ 1=E:.._.._ WWhlh~IEF: WxF:C:H f~ln:Er:'tE'. F'.~:4 ~~~3~ '~~i~
-Za-hexane az~d a fatty acid enriched fraction eluted with he~cane:ethy!
ether (1:1,, v/v). Solvents wer~ removed from the samples under a stream o~f nitrogen then the samples were eanverted to pentafluorobesa~yi esters using pentafluorobenzyl bromide and dilsopropylethylamine in aoetronitrile. Solvents were removed and samples w8re suspended in hexane. GC/M8i analysis was performed on a suitable instrument, such as a Finnigan ~YAT Tack ?Oa GC/MS/1~S/DS
(Sass Jose, California) operated as a single stage quadruple system or a Hewlett~Packard 5$9a (c) Assa fo Product on of P atelet~ tivat n F'aetor 1PA
Preparation of human neutrons ' Blond was obtained f rom normal humans and neutrophils were isolated as described for the araehidonie acid release assay, above, The final leukocyte preparation was of greater than 9596 purity and viaaiilty.
Treatment of human neutroohils Neutrophils were suspended in FIBS at concentrations of S to 20 x ~aS sails per ml. Cells were added to test tubes and treated with tlze.desired compounds for 5 to iU minutes, thcxi challenged with calcium ionophore A23~19?, 2 ~M and 2a-30 uCi of (sH7aoetio acid (NEN-lDupont, Boston, Massachusetts). ar the vehicle a8 1PE5 with 0,25-1 mg/ml of the. A~f tar 9 to 20 minutes, the reaetiorrss were terminated by addition of an equal volume of ehioroform:methanol (1:2, v/v) to the samples and the lipids were extraetecl by addition of equal volumes of ehlorofarm azrd distilled water. The samples were F a ~: - 1 ~y - ._--.: ~~ G.a E =. i ~x : ~ .__ F: ~: r~ i r..i ~ r: ~, z ~:
:_: Hi r~ i :~: r.:: z ~ ~~ . _ ._.
~21~
vortexed and centrifuged at high speed and the chlorotorxn layer removed to a clean tubE.
Assay ior_ P.A, The chloraiorm from each tube was evaporated to drynea and the material suspended in a small volume of ohloroiorm or ehloroiorm:methanol (2S°100 ul) and the total material spotted on a Silica TLC plate. The plates were deveidped inn c:hloroinrm/methanoi/
acetic acid/water (SO:ZS:B:~;r v/v) visualfzeef by radiogcannin~
(BioSean) and the product, C 3~ 1 PAF, was scraped and quantified try liquid scintillation spectroscopy. 'With this T1,C system, the Rf value for a syntk~etio standard of PAF was approximately o.33.
5. Assa or se Benin ehe iaal eo ounds far ten ia1 anti-initammator~r a~e_tion An assay method for determining the inhibitory activity of compounds for !?AF and araohidocaie acid production is also eneompa~sed by the invention, The method eornpris~ (1) measuring the inhibition of the CoA independent acylation of lysophospholipids in broken cell preparations of said compounds; (2) measuring the inhibition of PA1F production its aetiv&;ted ingla:nmatory cells of Said compounds; and/or (3) measuring the inhibition of araohidonie acid reXease ire activated intlaanmatory cells of said compounds, The activity a~d the eornpound is determined by inhibition of at least 209b of the activities of CoA-1~, PAF or arachidomic said r~leas~, This gay method provides a means wherein chemical compounds can be easily screened for CoA-T"1' inhibiting activity.
FEF:- 1'c'.-W.' L.IEL~ 1 ~1. :.._ _ ~'i=,hlh~IEF: F~ I Fi W'1-I 1~l n:F:.'. I
E F'. 'i=.
~zzr Therapeutic use inhibition of CoA°1T and the simultaneous reduction of PAF and eicosanoid release from inflammatory cells according to this invention i5 0% therapeutic benefit in a broad range of diseases or disorders. The invention is use%ul to trs:at disease states both in humans and in other mammals.
PAF is a very potent gasteric uloerogen whose endogenous release may underlie or contribute to certain forms of gastric ulceration. Intravenous infusion of PAF at doses of 20°200 pmol kga°l,~rnin<°l,a~ into rata has been reported to result in the formation of extensive haernorrhagie erc~ions in the gastric mueosa.
Psoriasis is an inflammatory and proli%erative dis~ase characterized by skin lions. PAF is pro°inflammatory and has been isolated from lesioned scale or psoriatic patients indicating PAS' has a role is the disease of psoriasis, There is also increasing evidence for a potential patho°physiological role for PAk' in cardiovascular disease.
In this regard, recent studies in angina patients show PAF is released during atrial pacing. antraeoronary injection ox PAF in pigs induces a prolonged decrease in coronary flow and, in guineas pig hearts, it induces regional shunting and ischaemia. in addition, PAF has been shown to initiate thrombus formation in a mesenteric artery preparation, both when administered exogenousiy and when released endogenously, More recently PAF has been shown to play a role in brain ischeania induced to animal m~els of stroke.
disease states which benefit xrom the inhibition of CoA~IT
include adult respiratory distress syndrome, asthma, arthritis, F E E. - 1 '~: - ~ '._ 4J E =: -* ~ .: -6 E: F: h-I by E F='. E: T F='. :_: f-I Y~ I :;: h::: I E F' . ' i -z3- ~~~ ~~~ r.
reperfusian injury, endotoxie shock, inflammatory bowel disease and various inflammatory skin disorders. Compounds which inhibit ~oA-1T, by virtue of their ability to antagor~iz~ the actions of IaAF, as well as CoA-TT, are of value in the treatment of any of these conditions.
xn therapeutic use, the CoA~tT inhibitors are usually administered in a standard pharmaceutical composition. ~oA-Ix inhibitors and their pharmaaeuticaily acceptable salt9 which are active when given orally, can be formulated as llquids,,for wrxample syrups, suspensions ar emulsions, tablets, cagsuleg and Lozenges. The choice of form for administration as well as effective dosages will vary depending, inter ai , on the conditiar~ being treated. The choice of mode of administration and dosage is within the skill of the art.
A liquid formulation w111 generally consist of a suspension or solution of the inhibitor or pharmaceutically acceptable sail in a suitable liquid carriers) for exa,mpie, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or colouring agent.
A composition in the form of a tablet can be prepared using any suitable pharmaceutical aarrierts) routinely used for preparing solid formulations. Exampi~ of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.
A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active tng~redient can be prepared using standard carriers and then filled iota a hard gelatin capsule; alternatively, a dispersion or F E F: - 1 ~,~ y t 4~.~ E L: 1 4 : ~ -1. F: ~, N h-1 E F°. 7E: I F: C:
y r~1 r_: k: I E F' o ~: .~
suspension can be prepared using any suitable pharmaceutical earrler(s), far example aqueous gums, cellulases, silicates or oils and the dispersion or suspension then filled into a sof t gelatin capsule.
Typical parenteral eompositians consist ~f a solution nor suspension of the inhibitor ar pharmaceuticaiiy acceptable salt in a sterile aqueous carrier or parenterally adceptabie oil, for exampio polyethylene glycol, polyvinyl pyrrolidone, lecithin, araehis oil or sesame oil, Alternatively, the solution can be lyophfl)sed and then reeor~titute with a suitable solvent dust prier to administration.
A typical suppository formulation comprises a eo~mpound of structure El) or a pharmaoeutiealiy acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glyeols, gelatins or cocoa butter or ether low melting vegetable or synthetic waxes ar fats, Preferably the eornpositian is in unit dace form such as a tablet 4r d:ap8u18o Each dosage unit for oral administration contains preferably from 1 to 250 mg land for parenteral administration contains preferably from 0.1 to 25 mg) of the ~oA-1T inhibitors or a pharrnaeeuticaily aCeeptable salt thereof calculated as the tree bas~, The pharmaceutically acceptable compounds will normally be administered to a sub~eet in a daily dosage regimen. For an adult patient tlzts may be, for example, an oral dose of l~tween 1 mg and 900 mg, preferably between 1 mg and 250 rng, or an intravenous, subcutaneous, or intramuseular does of between 0.1 mg and 100 mg, pret~rabiy between 0.1 mg and 25 mg, of 'the CoA°1T inhibitor or a . ~ ~. . ~ < ~ ~ 4.~ ~ :~~ ~ ~~ : < ~ F~ H r-i ~n ~ ~: ~ t ~~ c: Hi r~ i n:
f::: i ~ r~ . w ~a pharrna~coutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per d,~y.
'the shave description fully disatos~es nrie invention im;iuding preferred embodiments thereof. Modil~ca~tions and improve;nents of the emlaodim~nts specifically disclosed herein are within teh seopu of the following eta~3ms. ~tith 5ut further elak~rar'~m, it is belie~~ed that one skilled in the art can, using the prececf,~.~~ desc~riiation, utilize the present invention to its fullest extent. Th~>re'ure, the Examples herein 2~re to be construed as merely 'ttustrativ%; and not a limitation of the scope of the present invenrl;;n in a~~~ gray. The ernbodirnents of the invention in which an exolu~i~~~: propa:~ y or privilege is claimed are defined as follows.
Claims (11)
1. Use of an effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT) for the treatment of disease or disorders mediated by arachidonic acid, its metabolities and/or by platelet activating factor (PAF).
2. The use of claim 1 wherein the arachidonic acid or PAF
mediated disease is adult respiratory distress syndrome.
mediated disease is adult respiratory distress syndrome.
3. The use of claim 1 wherein the arachidonic acid or PAF
mediated disease is reperfusion injury.
mediated disease is reperfusion injury.
4. The use of claim 1 wherein the arachidonic acid or PAF
mediated disease is inflammatory bowel disease.
mediated disease is inflammatory bowel disease.
5. The use of claim 1 wherein the arachidonic acid or PAF
mediated disease is rheumatoid arthritis.
mediated disease is rheumatoid arthritis.
6. The use of claim 1 wherein the arachidonic acid or PAF
mediated disease is endotoxic shock.
mediated disease is endotoxic shock.
7. The use of claim 1 wherein the arachidonic acid or PAF
mediated disease is asthma.
mediated disease is asthma.
8. The use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of adult respiratory distress syndrome.
9. The use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of reperfusion injury.
10. The use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of inflammatory bowel disease.
11. The use of a therapeutically effective amount of a compound which inhibits the production, activation or action of Coenzyme A-independent transacylase (CoA-IT), and a pharmaceutically compatible carrier for the active compound in the treatment of a disease mediated by arachidonic acid, its metabolites and by platelet activating factor (PAF).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US83385092A | 1992-02-11 | 1992-02-11 | |
| US833,850 | 1992-02-11 |
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| Publication Number | Publication Date |
|---|---|
| CA2061061A1 CA2061061A1 (en) | 1993-08-12 |
| CA2061061C true CA2061061C (en) | 2005-12-06 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2061061 Expired - Fee Related CA2061061C (en) | 1992-02-11 | 1992-02-12 | Blockage of coenzyme a-independent transacylase activity as a method to inhibit lipid mediator production |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2061061C (en) |
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1992
- 1992-02-12 CA CA 2061061 patent/CA2061061C/en not_active Expired - Fee Related
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| CA2061061A1 (en) | 1993-08-12 |
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