GALAjCTO§K*PRpNCaEl> POLYSACCHARIDES IH= A f ORMCIUTM ⁾ N MW ANTraRϋiT€ THERAFlES

DA^JSλϊRUUNU .-Qr ϊ Hh IN v iiN-.I. iUJ\-

This patent applk&tϊoa 'claims, pnoriiy :pf ^' Provisional Patent Application Ser. -No. 60/74.7,313, ftj&d May !Cή 2&)6 ^: aαd δntilk^ ^i; Ck\laotose ^; 4 ³ .ronged P^ly^cpharkk^ Iri a l:H>m.n?latio.a Ik AntiSbs^tjel ^' herapies, ⁵ '

Fibrosis- Is p inlerynsciiMe result of mi a:paihάSogkal process itself leasSittg fro«ϊ tlss« Injury tfeugh us eseapsulation- by «xfeκe-lfuH^ taøx P i pMkoWgwm sh'Mikm ^: of scan: tissue, ^■

kjαj-y leading Io tibrosb Occam in response .{« ^• a. variety of άmmie iosuits incKkiiπg •slcpho! abu:ss _; drugs _s toxins, viral hepatitis fe ^: - aud C, some metabolic diseMes., .foreign objects, such as s^ail mi«eral or -organic particle ^' s ^' (e.g., /Is th« kugs), o«Mihg dironic and/or peπnέuiM itesm. .imiatiotv,. or other- Hepatic of •gtilmpnafy sbπoHpi-itifes. The gdv^ncecl stage of -Hbro^fs is ^' oiretøis, defined ^' by tfe. presence ^' oftsHcapsulsϊecl nodubs,

εhrpβk is a sv^i:erøi(;.res|>pήsic to _. ekϋhic irijuijv dwefeptog through a.smo^αf MgMy c∞rdii-uited .mofes^lm" events, c^ifectb-'dy called filiyogenesis, Ia one «κ^mp ^' k% fiteasi^ develops as a result -of chrome maiήmaϊ.mi .1rverMjuθ ^; ' _' ^' ^ steps- smmediaieis? following, eferonk- m<^).m8H^.;i!ver kψϊty- tφ^αi & process ^' sailed. ^iMiMioif^ w-hiέ ^' h in turns ^: are ssaj events ^' of ^aetivatϊqs' ^* of hepatic- stellate cells, Ths nest, .step <>C sleJMe cells ^{' •} a«tlvati<>n ;is ^w p.eφ^ι^tk>.n ^r _? Mύ ύxs. leads, .to preiiieratioπ, ! ^r !.brø|:eϋέsi;s mi -iϊiβlήn

d«grøφ&oxi> E ^' sc-h ofthese ^' evsnts ixi aeoorøpaaied % specific .mdiecαfe p&fes,.&ueh as cøliagen I ^: (a mmkm: on fibrosis},; alpha 1-smooϊh .rausefe m$n (® maλ« on &&ivaikw of sfeihlte edls) _f bέta BiXrJvrscepfor {a.iaatikct oh prøJtfefat.ϊ>sι} _? -røal¥JK ^' tsetalI{)protrø^ι«s and ώeir iehibiiors^iMP^, MME9 _S TϊfyiPt φd TMFk (markers όii matrk ^' <f«^3al?on}, cytokine TFC>pl .{a marier.o.s fibrogeiiesis),

DevelopBieRt of fibrosis can be βv&li&bd by (fe .qiianliteilye 3«vel of Um- r^pecllw lϊiarkits ¹ , Rsdsstion of ilbfosls Can. ht evato ^' .«d b _v v- .the dsccosfse' of Ac* • lev^l ύf. life s^speciive iBaπcorx φrlng vai-lpαs- staged, of fi&tφis. Fibrosis -.can fc. r^dncød _: :and rδwrsed. Furlfermoj.'^ s^eπ tlie- :a ^' flvιιncM stag^ of ^: Ebro^ιs, drrkjsb, sau $st> be

≠MMAKl Oh 1: IiE -IlγV.bRf, Ii ⁾ N

Ifi one embodimest, ntethods ind ^: toπφosHk∑&s are ^' _. provixkHl that rekfe U. ih$ sάmimstmϊtύB of a gakctopoiysaccbrøde iniicomliiMto witii ; _: J απiifikotlc compound to- a subject in a-fonriu3M ^' iατvin which fibrosis /is reduced.

In asxϊtiKϊs i'mtsodhncπL a »κ|.hod:is prpvicfed ibrtetbg ^■ fi ^' hmsf&ln % anfef^l; by admiπ.iskβiϊg aii afϊe;όlivό tee- of iι:aitι!ϊbrό.iic ∞ώpo<.aκito-?.he- ^' sub)«&t i.o «.:ϊimmlatvAω αmiaiaiπg =s . _. strhabic polysaccharide, wHldj may m&lude,.. bit & s.β( |i.φited -to _s i. •lalaettmuύαnan, rhamnøgϊiisMurQβaB, ^irabkogatoa^ wmbtøing the., polys^cebariite ^' with tikidlt^frø dαse ^' .of a sWitsbie cό ^' mpόUπd i& fomi a .mlxM-a; uxiά uάmmhl& ^' mgibz

ln _. snother embodiin^rt, ϋ\ύ- present iήyemiόβ relate to a m&ik iøά .for.cfeilvermg w. efectivδ .dose of M adπusture .of a. .Mώable |al8ctosc-oøiϊ?δmlng |x>!ysacd)iiπ ^' de and. a «.ompotJrid4"e3ec3;ed ftom rise gtϊ>uρ oø):^istuig ^: 6 ^' f1mtiix.jdies specifk; to cell-st^lace ami/or Intracellular: (i).fl-FDøF receptors _. ; ψ) sympibphym; (uiyzyt§Bιilv} CCRI. ttc-cptpά; (v) couiiecdve tis-sue growth fac*«r; (vi} a!ρ!m l-δjnøot!i -.muscle acim:- ?λ?O| nvdϊk vM8.ticip.tht€mas£$ MMP 2 ύiά M.MP< ⁾ ;. (viii) matrix ?«etaS!oprøie:iiiasδ kiήMtβre ^' HMPl mά TMP3; (iκ)αrjtegτiτιs _. !; (x) TF(5"j|l; (xi) ^dothqiiϊi reøepoι;«|tøgατiists; wd {pϊ}:< ^' xόlh$&ιi%frάli&$k ami degrøύatM ^' πio.dιtottig^δm|^>ua(|s;.(xiri} ξi&m ^' . svsithesls aiπi • ^• dφiκistϊθ]i.me4ώaφE cømiitttrøφj SBdtxiy) tyrøfe kln^s ^;

!jvmfc&^' embόdmrøt _s the parent invention relates Ib aaethod-for ik?!.?^er.mg an. $!&eti;V$ dosφ: of rø admi^iure. of & suitable gakctose-feoπtøimrig poiysaccMrkle fed. a compound selected {torn ibe gκ) _: up cpusistmg σt (\) IL- .1 ; (Ii) IL-ICl; ^' (iiϋ) aαti-ϋvegiB

omnpøyβds; $y) Mitlerdis alpha O :(alplm. 5); and (^ kφfi-ie striate .odl sεi&sf icm kilUfeitmg. $ompou:n<fc.-

•Iβ Motlw embodimi'iit _j th&. present l^veatipw rdsfelo a vMifecl fe.-Hejwri.ng- as effective dose of-axv admMtws øf. a. ^itablfc-.gsl^c ^' foge^cor.ttøtbg. polysacafeaside. _. ar.u];u eom.pousκ1 sdsetfcd fmrn % group consisting: of: (i) ^Itamis- E; (Ii)- alpha-Iipio acid;, (lii) letmn.dπ.βe; .{JY) Sliyrsadw -anά -Sijyrti^rift tfem^tivos; a«l (v} _: Tbalidorside aml ^" fhalktaόiRie amiTόgs.

ID jmothsT erπhodirøsni.. (he'pres^pi iiiyailIpXtfeiate^to a metBoii i;br de.Ii,v#ri«g ^tt/effecUve ύ<m M M a<lπ.«xtiϊfe..of a suitϊibls.gakclosc^oMaining pol ^y saceimncfc asxi a eøp;vpθϊ«κi. §#!όOTd Smm the. grρ«p ^: »p§.hstk)g- oK (i) pmeϊne; f«) li ^» c?srøer (ilj). L- methionine; (iv) S-adenosy! .feøthiomne;- (V)- S-mettiyl εyskdns; nnύ (v-i)- N-scetyl

In another gmboiiffient, the prøeπt itwefjdόn relates -IQ a nMhύi f® t άφ\ ^ι ermg.m QMϊiiti\ ^! ^ άosz oi ^' mi adaixiar^ of a ^: suitabM galactos^coatiktiiig polysaccharide, and a coQtpQrøa selected &i>ørthe gmtφ consisting φ (}). coppeε chelating cmnpotimis.; (ϋ) jMc#gmo.ms; (iil) |^ -amifio-.projsi όω.i tdlcs j (iv) Ivj?&- V εytMc m?ei»tide

mmpcmhάs;- and. (vii) MrMl .compouads.

&.aSQ&.c OTbsK%TOf _. ,. |He.-pr^ssT.H .iBvenlio!3 re ^' Etes to a ^' meifeod ^' -fe delivering Bi siliϊctive dose of- asi admlxiure- ύf a suitable. gδJacrαse'Conminmg iKilysacdmπfc mύ & cpmpomiβ. jelecied- ^' ItαήV the group ctmsfetrag ^fV (l) <iexfran- sulfate; {μ) penlmim |)ϋtysullafe; (in) gkmdroitin atlfefe; (i^)-hepmfl..su!.fats?;,and-(v) hqmrm,

.DϊSF£ϊUξ[> DESCliπiøH OF TW INVENηpN

0φ;ited έmbodi.ώδats ύfthe present iϊiveβiiørute 4be!osed ^; lwmn; knvwαji is Io be understood lhat%cs-ib?losed siijfeodrøgϊvβ? φ rørφ" :iH;ύsfeiύv© of tht jhwαftibn thai: may bo embodied in vario s fornix.. In. addition, eaph ^" of the cciimpfes giveo m •com«fiϋn ^■ wilh tlie vi^rioutejtϊbpφ ^' ai^itai ^' tte iiivfetion ^re mtead^i w be iltets:ative _f and ^' are -not τMτi.ctlvS; ther&fer^, specific -strueterai. and fuπe^longi <tetaife dilckmxi Ijareiή are ftot to b&- ii^tarpreted as- limiting; htit .met&ty ^' i\s ■& υ.$ψm<i&ϊtMW& basis- & leaclsmg β»e skilled .uvfheM to-W.iousJy oiniplojf the present ihvpMioω.

The :E>lbwϊκg ferms shall, b^ε the Meaήlags ^' iMfcaleci ^: hersin.a&! kttki dair xaqurøϋ oφefw^. by the coϊvteκ.t:

''1^" shall Iϊ

"EHE ⁵ ' ghai.! mem BagfφrMi-Odm Swarm.

^■ ffi%m Soluble- b.ra«:c]jδέl Eagle's .Mimma!

^CMF^BS^^hali ∞aπ ^' Gs ^{v "'} - mύ ^' W& ^* -Bmψh<>$^te&Mfm$. $άlm ≠l ?;2<

"BSA" ύiM &Wάt\ Bøvke. Ssruffi _: .A3b«mm,

"g?i!UA ^S; φalI me?pp]8«itopv.rpo!^ uropig acid also called pikϊipt>ni« mxl

«i gvλai!" ^" s AiisM iϊieaη ^' gδlaetose.

%:®r" shall mm maimose.

"gW shall meaπ ^' gHicQse,

^* W §ba|1. m$βiι slkm^

' ^* jd<>-': slisll. ffiw idose.

^* %1" shall meaiitatose.

^w gul":sli^rmsad guiose _*

*tara" shall meat siabihose,

"xyf -BMp mmx xylose,

⁴ p,s.i ^w shall Bic^fi..psrøe,

•Isor shal! rπeto. ss

"rhs" shall me«is rhamnαse.

^■ qyϊβ" st.ι&u nieaa qmsoyOse,

^(< 2-d>-.rfl> ^w shaJ! rae«i 2-di'θxJ-rib«se>

"AdrairtJstratioxi" ^' refers lo orftl, or parenteral incJudirsg ^' infr<?vem>iis _s . ^ybcuiaiiep-us. \ϋp\$φ. _s ^usde-rmjil, Ipirattermal; (ransmucosai i«lrap©ritp«eal _r inlrsntuwukr, ύ^ra&φsύlari iptsraofbhal, intocapdiac, i^nstr8cheai, subcui^eous, subeudc&tej inkaariiøukr _y subca|)suiar, subaraδltaoid, ^' kvlraspInaK -epidural and.

^Siϊbieoi" is deiϊi^ed here -and in, the ^: claims as a. mamrad mcl.adi.ng s MmM m

dogs, &%% _: pig,s\. cow>\ $fkg\% gβats, horses,- raϊs _s am! raiiie .and himmis-, Xhe :\mm- *$φ}<scf φεs nQf. ijxcHick: an iadtviduaJ OiJU:.is _: no.t:.maϊ ψM rtspects.

*fMmnt -ύvdl m$tm a fπmiaa sμb]ecf:wkx h^ pr&sested at a elM _' δai^fmg with 8.pπibu:Uϊr aympfem ft sympiø.niδ $uggέM«g the- seed fe treatment

^<!' πtjμ.m«s\l of fthrδsis" refe ^' to prognostic- treatment σf -subjects at hϊgb. risk- of <teve!όpmg ^: -SL finite m cirrhotic .carøiitbtt as. wφ -as., subjects- vtfhρ ii&ve already- developed- a. fibrøtlc or drtlkHic. eomiiison; regardless .αf foestkM ^r type f HSSMQ- in-, wliieli \}}$ fjbmk ψciπbotk addition arises.

'TrMUrø?τ" may ^tho b& applied u> the reduαicm αr μrøvejπtto ^' B-of ateormal cell pmliferMiøτit _r cell ^ggFegξitioη rø<l. fcelS di^|?e^al. ^' {m€ξ^tei.s) ,

"<Dvjτk>sIs" iφ-ή Io .any tissue dtiofdδr, iachxjing suoh celh<)ar di ^' sorciets -md ^y dlftgv but n^: limited %. -is^a! .$.rrhe$j& ViW- eii^hosis^ tin^rian eirrh<>^s, iyng.. girrh^sis, ^• gas^Mϊjfelrøi- or siaϊiadv cirrhosis. I'he term ^ϊMjftsif- refers M ^' M ^: adv&Geed sfag^ «f ^■ fibrosis, defiled % the ^e^eiic^ #f encapsulated npdute; umd event«aiiif &mceι- ^' , Fosr purposes of tins -gpeqifreatios and claims, ^cirrhβ^s" k mnήάomά to be a tv|.x? of fibrosis,, and Is iπG ^: .Uκlcd wiiiii π tft§ ήiesrøiπg of the term ^fibfo-sis" usai herein,

^■ ^fibrosis'- Tefsrs ^' to any tissue disorder,- -iacludbg,- biit- .BOt limited ^' % s«ch ^' e.4k4tr disonte 3.«, .for ixεHϊφk\-drrkκsis, Kidney fibrosis, liver fibrosis, . ovumiit iFo.ro.si« _? hiύg α\)roύ% _! gmtnmtcstittiu or- s.tømaeli fsbr-osis. Fibroids, ^' The ^: mm " ^' fxbtQisfa* refers -to both. the- pivih ^' oiogicsl process ^' bMmg fe&'m. tissue -hijμry. ϊhrαisgh its ^' e«capsuiatidn .by _: ^stracdjui|r..ϋtairk _! #d .tføζ result of the |?rø^es& _s whid? sϋ a ^thelo^c^ fammtiøn ^' of .scδr tb^ύε, FibroMS-fe-t SySϊαMc- mspcmse-to dϊrome-hihifywcie^liSpmg tko^gli scenes- of iijgbiy eoϋnlmaied iaol^lpr $v&% φtiϊ&βφ called libmgenesis. tiw sϊ^p.8- imm^l _< felj -MipwHig: chronic Jlyer ^■ J«jurv represeBt ^' a process called 'ihUia&m'',. ψhiφ m MmsiWsj ssrly-fvfeiUέ ^' .ϋf -^ctivaUOπ'' of tepime ste!ia& csϊls> 1% .»;? mp όfsMlate ^■ cdls. activation js ^' ' ^; perpetxiat5on ⁿ _s ^' snd".£his leads Io pmiiferalibπ., IfBrogeBeskand .matrix deanulatiorii

'^dφjiar.&aϊkerA of εk>dh§ϊϊύesl markers ^* _* or "Bfemarkcrs", or ^** Marke$f refes k) IncBviάpal møkeuks-. αf. bioiogioa! .øsigm, -which c® hp. uimύHϊø &i $ "rtetbuf of specific metabolit eveαts«.TIϊrøfe ^' -w©ats are. accumpaaiad ^" bv. feπn&tloh .of ik ^> *m$tεkm ^' \ ihϋ quantUativa level of which, aaft^ffcen be used ^' as fo ^: h)dϊαrtioό. to advsnϋemόat of ύb event. .Examples of ssch markers in fibrosis are eoii&gαxl (a,røad'<ϊf on fibrosis), .alpha. U- &rfiύϋύϊ ύ\ύm\φ άcϊin (a tMtker -όJi 8«f.h'8lϊoJ- of ^teUate- toils), bøta ^^OB-røceptdr (a marker .otv prolsforafen), røstri?; ^■ .me^lk^oteiMses tmd tbdr- Ihh%u'φ& MMP.2, MMf?9, ^" OMFl atid 'fMF2 (mafeB o^ matrix ..degradation), cjjtofdse TF0-|II (^ fti&ifes ¹ . cut

^■■ "Aφηkfαrt*' Meafis moreiliaB one compoaέnt mixed togeξfe tcs Ikffi a combination, Fi?)? pmpo$?s o ^' f the prø»ϊϊ lπy^t?.κm. ^idxakture'' means the mkxure of two ^' βr more «»mpo«mfe at ®y time ^: prior ox subs&gusnt to- or. concomitant witti, administmikrø.- •

^ϊ! Oθpoiym;ώaH$5f r.φ.rs fepatl^l.α? cαmρkte.-%drolysis of fe _: ρqlysacφaπv|e- backbone ^' occύrxm$-M example when ^• &e-;pd-ysacfcftarκle h treM.ed. ci>«mica11y resiikuig ^' m fmgnmM of re-duoed ϊ?rzs \vheB α>mpaτed;wi?Ii. tfie ^' original po.lysatehsfkie,

"EiTecttve- <3ose" ^' re&rs. to a --dose oϊ iv cόmøoutKi mόføt admixture that improves φe svπϊptαβrø of the sublet o.t the kmgevity of the^ύ^jφ, su!:ll:πsg frάm _; qr tit _. high risk '-of siiffeplng. txαni eiττlk>sisi Dw dBetrvε. dose, iff emko4mitøfe -of ^' this invention _: can. be qaaiilitδtJΫelv sMiκ.d ψ-MM - mnmt of a p(>iysi.røclijui^e.«oi'«|muϊϊd j ^χ dfalaisiei:«4 ύløae .or ^¬ is a solxSixs 9fi\h θ sk>s§ -.o|.» pslillb^fic- compound aώBittkttτed h, s- subject ibr iτe^.iiri| fibrosis that d«"«sέ^ a l&v.d &iϋ chosen molecular marker hy..at feasl about ^: S jx ^! r ceui

ft

:.morκ:}sa"€h.aridδ. -derivatives, monosaα'lwiφ? analogs, -pgays» Inclu ing ^' ?k>s« _> vMφ farm the iiκMdual Units m m oligosaccharide at # polysaccharide,.

"Moαosacύljaride" ..refers to po!yhydτoxyaid«hycl« (δkiose) or polyhdrosykctone

^■ mςiydes up. ^' to ami. IiI sψφ.&mk§ t&vM linked yia.glvcosidiQ btmds.

"Polvsacchavidε" lefόrs- 8) miyrøφis- fbrmόd from about: ϊO lύ over J 00,000 srøimmte units imk\^ to «.3c ^' h other .by feemiaόetaJ or .glycosi.<|i(; boπda> The pohsaocharidg. may -he either- a srtesigtii chain, singly branched or multiply br»Hch<?d wBereh.V θadi feicfi ώay liavo .uldiiiόual ^• sεcojK ^' Nt -b.nmeli.es, mύ the moDosacdtsrides mav%'sta&dard ]> ^• «» ^• 1> e> ⁵ clkm?.gatslK the-gpgBøse (jS-mem&red ring}\or fcraiiose (5- m ^' erøtere*! ώ^g} ^' fotiϋs such: as l>ihϊcffise-:and D-gaiactose,..reg|}ectlv-el^ or they may k; cyclic- sugar, de vatives, &r. sample ammo sxii4prs;sUdh as ω-glucosjimm^ ^' doosy sugars such as D4«i:c>sβ.t« L-rte«ϊrø6se, .sugar phosphates such -^ β^πk^e-S-phospbai^ Sigar ■acicis such as- 'D-gak>όii#<>JH.c add _s or mtϊftf'dmvati^d sugfe .^u^h- .as ^^leέtyKD- .ghøasajmige _β N ^: ^cφlyIϊMsurarπlais _: agd.(si$lie iiύά%. or ^" N-sαl;Fato-D'<g|a.θQsp;iine. ^■ Wirøi ^■ isόfeαi Cram 'Sfitufe, ixjlysacgbtπifø- prepaiattes compπse- .πTQ1« ^: U!.ϊ.-S-: -tbu rø .Isetsrα ^' gbifcβus- hi mδi.eciitø; weight _; Pp]> ^j ;si^o&baricfe irit?hκfe among mher. ε-ompoMilSj

.dBJnήygafeβtάrøis deffi ^f itd\ ^? .es,- and giilacϊBf.amfeuiQøsisδtørOn TO! galaeto-

11}

"Bssktens" πirønis %, mφ-fch&m of a pdysacdiadde, or lite £hmn osgmaimg jfem- ths imlrn-- φάl -of a. siankg ^ϋlysεtcchaπαe^ ftpiαg $ϊlεfetokfe .rπeuefes seqjjMfMiy Imkcύ by eitfe-f -ec us: f) gbeosϊdie teds. A backbone may comprise •addϋio ^' nai -moπø^cd^ϊidfc xaoieti'es _: ς©mτscie<$ thereto ;at. vadϋus jpd&røs alθng the

^!V Est8.fiβeatiori ^>s røfε?s to the prøseπce- of Kjdihyfeters.: or 0lh©r..ss ^: ter,gs'opps _: at ite- tuirbosyh ^' c add po#i?oπ of lfee la'oiiic-acid-ώo ^' ieliέs'of a saccharkie.

ύ.ϊve^ik>ϊ); _t 'my of" the ϊstaiufe! pu! possible deπvalives of monαsaseteife (supπ _* ); Includsng- -biα Sϋt limited to ^' ^feos^ODOsacohaddes, d^eoKf m^os^hariides, wjβt ^' ■ύ&Moh _f sugψ acids, .sugar esters^ sugar .ethers^ sugar halides _v mftino suga;rs _s ^' sug«r ptesφlvaies;. pymao^e .sad :fea«ose cyαlie- Ibmis, _; open ring ^' forms, splfenk . sstes of sagars, .glycosiάie deπvativ«s,- glycols, giycόleneSj- kefe sϋf δ^s _> ilifetø sagars _. , pixHceted. sugars, act-tils mah as ^• b&αsάlMeirø and ketals stich as isopropylkienes. nitrfe sugars, N-. •acoiyl sugars, N-sceiyliHuπmuc acid and aαtibioik sι?gars. mψi m nq|snmj;c.m ajid dihydπ^ojirimyciπ ^' -

εftκ:s.øy-' of a aiitiiibrotk eoϊnporød, reform- to the ^: re||ϊtrøship bel.w« ^• & .ϊδkit^^t δflectivs-d(Be afκl M eXle-Ht of toxic side felTsctS; ^; ElH«y øf all-mm|K>und is increased if s ^uiifi^iptic eπci point am be thieved, by aφϊ.ύøislføfi!m of -a ipwr dose or a shorter dos8gg-?sgrøgω. If ^' tonicity son be docm®e<i a aalifi&κ>dc comppus^-csn. kr sjimύliste^ti ^■ cm a longer dosage t'egimea όx eveα chϊOfck&lly. wϋh creates: .patient cpϊBpϋpee: ao<t improved XjX5«^|y o.f _. life; Fyrlhet\ £lsc;reaa^| ^' økidti ^f an ødmpound rabies the practitkmstf tx> iaerease the dόsjige to-aαhieve the; α?ftiilbκ>t!« ^dpoiut ,so«»\ ^■ Qf to «cMevj*-a higb«?r ^; i3βϊlfφrotici endppwh-

U

rnsdisi. &g. _s ^' hxrø-iu albumin dremss-Siήkec! gdaiin pόtvptrpiufes, eoiitmgS _s saiibasϊeriai ami thllteg$i φsήpøuijφ, Jspt-oraiCj e;g.., sddaim _: elϊ!άri& W sodium- glutamatex. ^• mid ύnϋψtiwi 4«ia}'mg compoymi% wyά the MH iϊrάi are phpiologkally $røj>ali^« ^' . Tk?nst of sueh nwάm mύ-.mmpMxiάii far phafiMooutkally active sybsia ^: aces:ij?ivel) Isiwn. in the mi, JHβϊ&My, ύϊt. caMei is. suitabfe for o.ra|, hrttavciibus^ iflimiiιiseιsk«\ &tfeeuMøø(&,, pamtferai spinal dr epidsnsl sd^kilstnύion (e.g., by ii\?eii|θ-ti o:rlxrfμs)δή}. pepeπdiπg, cm ife τøιne Qf administetura, the active compound can be «uatai b .a. .røieώi Ia protect tte soprpqμad _. B:a«). .th# acfidp- of peiφ pd other piarøl c<nuii(;i.dhs

Is> one embodiBJeat, the collagen . .marker is πrøsuml md qyai.ititat.ed ^' by rnferpHometry. lώig !b.«x pcrosirnis red. stained slides per aiiEmai. Nine. pictures έire.feifeα fan<:iøsi!y p.e.r^]|ά% fer- a t{ϊtøj BcVpictøef .per aBJinai, which- afe then έ?v^hiated ^" «siϊ>g-t.h$

BIC)QfJAK ^{■ '} J TV" .iBiagc aaalysts program-

Iβ aaotlier embodrøtcπt the following markers - $$M 1-mwφ mpsα.le.sctin _; .!.)eti PpOF-xej*pU». W$im ^cφlet, MMPI, MMi>2, _: MMPlI ₅ ItMFI, /FMH s»a ^oll^gen I ■- ^..pses^tl ^: atiήg UMA ύhimntά by mver^ irssriscfiptioE from mipA,

<$ ϊmm Ikertissive,

^■ sύl>iect ^: .tQr ireiϊ&5g .fihrofe h defined &$ mm decreasing a level of a eϊrøsen inόϊeeulsf .markst ^' .by .20 per smt

. ^' hianqilkf embodiment llie effeeli.vo. aim&mi of M aiirnitύsi-efed po|ysacέhaτk1e mmpoumtis ddlried.as dressing $ φoββi molecular .marker betwςrø ^' 5 _. sμκi 50 pdr εesi am! .higher, depending- on an advaiiceoient of the disease

In. one -.embodiment, -m eilgctiv^ . amount of n polysaccharide ..eampQun^i iulmimsktred . ^' dtane or jrt a mistta? xwib a. _. dose όfawdfttodή cόmpound:idrøfø.fe.torα! ^: -m a subjesf for

per cent,

Ia jffct am>|her enste^iinent sa> -efefe arøcami of an αάmmkicmά ρύ\ys%®ch&ήdψ mmpαimά m ύtilmύ ύs. ^' άm&άsϊαg &chόse« mobcifa marker .between .§■ and 50 p& ce^t ^■ SUK! .higher,, cleperidmg on ^ advan.cemέnt of the disease-

In. ^' ariQUiiT eiTikxIiment ^' if an induςsr of fϊkraslsς -SUCb is csrfen .teiπtdjleϊide . {sobc-tήaπeousi-y) or tMoaeetamidt- (iiim^peptoTieaMy) admifMMared sJθBδ _f fuses ^' foππatrøi of the biochc^Mcai markets biifa corøtot^βt.admmiStøtioϊi αF&e inducer .and. a syitø&k* ;polysacchari<ie inciiidiϊig h≠. net . limited iό .pkκtoø:isnnan, rhanliiogalacturoBan.-; (jf:.sf4b,«K>galactan,- dees. not lead t\\!b.r.m.aiiDδ.δfthf pmε niarker,. or ^' leads to femmliøό -M the. -Stnne M&br bat in a teducδd Mioaatj, . it can be snggesfe! Itøt ssk1 ρoi} ^: sac^li.a.dde.pm¥8.«its Gγ .SIOWS^øWH fibrosis _*

In v€t another srnb^lme^ if iø pϋϊymxchwrkk m . sάmiπ.is!«^d at tef^r stages .<?f d«iv^loprø«?53| -i?f iibjMϊs,. and the administration eaaseϊs-reHiiαtioh :df the ^:: .feγsl. of tr)s? .bioehmuc&I irasfeirs c®ip ^' amd lϋ that hi control on to thai U5. i\vi same expodki^sjtaJ -kaimal... it ς&π be saggssted tliai the polysaccharide fa- able to averse ■ or slow dSwn

In yeϊ άtioihe? CTlχ3C%πent if sφ<t frøfyssedmuie is admmist&fed at Jai ^' er sieges of ^■ fedo^πem $f i%om sod -the sώϊ.πni^raf:iQii .»« reducing of iM .level ot.φo Mecherøieaϊ itmrkets-.t-όmp&t'd to that M control -or Io that in the same experimental amp&L ^' ami jliex? kibse^ueilt ;$§aj$)eafahCe of the markers ooii^cides with i\m m\m io IieallK qf ^' a subject vάύ% vtspςεt to this.paHie«.iar p^fhøjqgy _* it. esn fe syggestsq ihai &e--

^■ fe ^' yei CUKYtW embόάraest, if: mi pøl^aoohsride is ^• .siimϊmsterecJ -at the; iafe siagesX^t librosp, which oan ^' tedsteratmβd ^: as cirrfests, and the admniis-t^lion causes a feduδtkm .of die kvsjj of. fee ^' biocli^πήcal mpte'ij eompared. to iliat is coαuol ar to. that m. ^' the sasxte experiment mύmiil. it can be siigfsstdl that tkf pdlyέaecfeϋMe js-^ie-ϊϊ> slow ddwtt or

In yet anoilw eaib^diiseiit,- if said poiysaccfaiiide is tdήϋrtisιcr«d at. later stages of deveJopmeift .of fibrosis, mά the admkilsltrafion ^' causes ::redιsc$ioi? αl ^' ϊhe bvel of the- biochemi-cai tn^rkers conspat^d to thai in sbtrtrøl or iύ ihkt In the ssme «xpedra^it&t Bmnuύ by at least approximately S%, .h.eas fee suggested that ills polysaccharide ? ^' s able iα reverse dr slow -do^ir fibrosis.

fe »ns; eτ$feρdlErøf: (sf lfes patørtt _. a method 1% trδafiftg a ii.bsx>si^ m.a Object is dfecle.s&l,. røϊSpri^ng- iidmi.ms ^' teriήg ^' parenteilh: k& effective .<fess of a. fieϋtrai or a catiόβk- polysaccharide φ«λ|)øun£i ύi.aa admixture wiώ a %se of a compiiM to said mifcjeicr .i.n r^«α ϊkumaf m mkϊ to teal: .tlkosk

Itx mwikm' ψmhoάhxmii the {%.>vβ. method is dfec-iosedy/wherein the librpsis is uf. Uic liver, Ia-yet.atipili.6f eaibodimem lbe metliod. is-. disclosed, Wherein the iikøsb Is -qf the lufjg, In &M\hoτcmbξ)άhmm thφ tύmsh is of tks lϋdάeys;

fe aisDiter embodiment, flue p%saeeharide compound m a rhamnogakeuuxmιm> M imoih^r.embθ4ϊϊπ«ι.it the polysaccharide corapόmi is an arablnαplacJaπ. In another embodiment the g^iaόtomanriari las a .røeleduiar w^gfe ^' between ICLOOO mά 460,000 D, Ii) 3.YHiBk.? ernbodiifceiϊi the: .ratio of rn.ao.np.se to galactose IB a gatecferasmo&o is -UsA,

In ^" Another embodiment fhc .xafe pf mannose io galactose in .sr g&l{$etomarø.uMS 1.2; 1. M mother et&bodmieisi a mαlscuJarwdght olHhe rtemiftgaiόέt.o:rofta« is. beiweeή. ^' 30,000 and ^' 40p,0δ( ⁾ _. D. hi anolher αttkxti ^' jηeJii the mih of galactose. t.o rharøsose m the rhaffinogalacutti is 3,?:L lKsaafeer effltodtemnti &e-mdo «!.f|alscbse to rhrøiose in the rham.migaktctaή b {setweea 0,4 ^' mύ 6;7 io L In another mbodisneni th.o ^■ a?abmo.gafef^a M§ * τπαl«scHkr- w?ight. betw^^a 20,000. ip.« ^' 200, ⁽ KKl ϊ>. 1» aftothstr δøtboU.Utie»t ,th& -ratio of galactose fø. aralϊήϊos© m tbe ^rabinugakictase is i>emeεύ 12 ^' aad ^,0 to- ] ,

In a forlber embodiment of the ttwbttkm, gakctd-rhai^nogalacfύranafe (GRi is a b.mnc)κκl hetm>ρό1ym<jr of aHerhafitig. l,2- ^: iiBk$d rδaaias.>se &nά ^" 1 ^tlnfed QύA ^' residues thai eames neytel ^' sM&»cl^ins <£. prettemvBivπliy .1 ,4rb-l>-gakii:.f.oae ami/or ! ^' _f $»&-.L- αmMfiose itsidik^ attaόfed to the rhairøosε residues of the KOt bafckbona OR. sid«-

Bldi.KriDg ..fucόsg, x^lόsø, ^' asil rijrøόsέ, ^' Thssfc are ik& ϊβiHτt%41« fe^ofen^ot^i use as

i ^' ή &tm ^: mήh&UmmυL |mφarafe>a iαvøl.ve.d ^: πκklιfyfegsamraly uccurrijtg _. -polymes- to redύ& the mokciite weight fo ^' p i\w <ie _. sifαt ^; saήge, adiiistmg tlϊe alkylated gfø'«p^ (Vfeffl^.hoxvkfiøn or ^feacetftatiøn), aittl adjysti^g side skim .oβgαsaeeharieies φr optkήufji./eiBcacY, Pin .exarnpk, nαύpiJ. pcslvsiccliarife ^' is^ay Jpv£ g siioleculsr wdghp

range ύf beiweM about 4u$fHM,U.00,G(M) ^: mϊh f\ϊ\ύ\ϊρte ^' him\a\& o£ ^' mmhmάm, Mt exanipk, brsnδte comprm! of 1 to 20 ij^Epm-ehatici^s ^' øf giyeøsfe, axablπose, galactose ete _> , ®aά iiwύβ benches may .b§ amπetlt^i fe the lmkbose vis fsswtmt .rw>uosact'hariil&? such _: as &mttόse;. ^' dfee- molecules miiy "felhei ^4' uickϊde a single or e^έϊiji oCuranie $$iά $«cch#kie backfepne'-tivat-niay- ^' be.eslcrifk-d fτoψ mϊiith as'.a ^' bout 2% to a$ m««h ϊiS:aljøui: ^; -3Cii^, ^' O^iBal^te bf^acte ^: tliMsseIy$s may liiivδ.m.ulϊip!e feticfes ^■ of sacchasid^s, tfe muitijiie feuiehes optio^Ilv ^• ffichkHEg.se#:sl siiteMil#s έ»ul:f5tiuraf m^hsrkfc (kri natives or«s&| ftjaiisly hyάrø ^' phβi?k eϊjtife,

λ»-δ-Mfpρ|δ f-f a galaofomajiir;® is a. polvsaccba-kie- prepare.ci Sonii.gw g^m-ami having s si;«e- isk tfe rang«.β£ 2000-600,080 1> Dr iό the τm$& of/SKiSOO K? 41 S ₅ CK)O I.) or in fe range of B0,ø0iV2-ø{.V000 Il hi specific exampfe, tbe gaia ^' eterøaaasϊ- pvaf hav-e- au svemge ske Bi^kculsr wdglit. of 50,000. D or 21.5,000 B. In a .further easlKX&røy ofϋήe bveiHioπj ibe.gakcto.mkrøaH ?j? isolated from jxnvder όfotainfed iron.; tte seed of tlte. plant Cyamoμφ teύ-agmwkώa .

1«. a farther i?mbodimeni _» ,a ^'' ϊϊiai)røogai|t(JUjr9nsιi«? ks ftrool^Uto we%!t mag§ of 2 _f 000- iϊ? 20pj)pp 11 _. . ^' fe S|^ci!& ^■ qfafflples _* the λamnαg^actαpinaMte may fcwe m sm&gβ ήzs- ^' isoleoitar weighi: of 34^000 B or B5,θOø D MU h obtήtsM through φ&mϊQaU m^mmc _% pkyφύ t$φlm&w$. ^■ άhii,pnø£$ύpn ϊτόm iMictic ^u|>s?arøi of Ci&is ϊkltød sp^pom^ ør søyfeiω h«ll or alteraafϊvel) ^i: p3^c«sιsci l)m«rsHprl>«l pectin.

In further eBibot1ϊmέ.«ts. of the inveajidji; a eaket&mannan -is- ^' usfed Id reLfuce fibrosis .under a a^tiø.brøiic action of.aa atiiiBbro?i(;.α>j»po«iκi wfen it is missl wiϊli the. røaipdund prbs Io administration. Ilie ga!aclϊ.nnatmaa may be s- $4 A D-gufackittmmn: ^■ Møreovssr, the g&ϊ&£i?o.q5£røϊum m&y .mclud.e a røiio of 2.0-3,0 isaunase ?.b 0;5-i.5

.galactose- The r$k> ά£ marøosέ tQ galactose may ^ 2.6 rrrøiQse to 1,5 galactose or 12 inenupsδtø-0;9 ^' -gώeiαse .or-1J.3 ^' -maimose ^: b i p:teomu>r 2;2, mam^M I galactose..

compound Is the mixture may be in -the range,- of .0..I: I -w/w to tøύ w/w/ in rø. er ^χ ]bt>ώt)«nt of ih«? ^' lϊϊs ^? ^τtio% adffiinisMtio.ii of the øiMurø .reianlts in s i&iuc-i^l ήbrøs^s {as dettxπrøv&I by a reduced ieyeJ αf tbe-speeilc maiifeϋ) of jareater than 20% eompargd to host st&Y&mi of thernpy.- in another fembfcdimenl,; the gaiaet^di^saεdrøde in. combinitio^ ^• with.3ftli4ibτoϊk- driigs reduced fibrosis b^ great&y. than 50% .compared- vvitJi. .Standard øaxev

i.. feihar δϊftbόdϊπ-jait of tfe inyet}liQi- pttivides a -meiliod lor yse in treating. |t fibrosis i«dιutrni> but. aoϊ .limited tϋ\ of the liver. ih&.lu«gs _s ike kiteySj thce^ tlieslcov.

In o.ne.eijifoodisKjiitj an example of ^.polysaccharide includes;

-5

ipd 6§l«.q|.pma?iBaji ixom G.usrϊgυm

to ^< m vmhoάumM ^' of ^: .tlie- tnv«Rtϊpji _# Oaiaαt»-ζl5Mi^ogak?|røήl#s (QB$ is s bπmefe≥Cl &tsϊs^όlymtr -of- alternating- ®-]:; ^: 2.4mk^ χ]Mmκ>s$ -tod a-X44i«kid CMC re^idUei tJmt.ca^kiS ^• όθtjtm! .siiJe-oliaife.Q.f predømirtahtjv iAhφ-gal.aetøse ^tfer L$φ- L-arøbmαse- røsickes attached Io ^' the rhaniaiose.xesiducs: of the RCH baekBone. GR side* εMiM may be «kcopite(l with 4rat?ais>sy! ^' residues {arablBogalsδtffi 1 ^" ) or' infer Sugars, including: fucQS« _f 'X^los«j _> s^xl mmws?«. Thesι; sire _. φo jtfefecl to ϊn corømerφi W as- ^' peetio .øMerml

,1a- o«e eHsbødrørø^ _. sojvil}Ii?/«%m _: H;s!ly altered gai^ct^H|)arøiH)S&^cfe#nφi*:ar ^g :|wpafeα by mousing siafirøliy ϋόcumng jκ%τners to ^• fλoe.te.-røotectslai- 'Wøight for tfe ^ι tetøζ! m»g^ _s reducing., the aifςyl$4ti. group (de-meHrøx^tion or de -acetylatioa) Ih'ioT to c-lKmical ^' .modifioation _í the-mvturaf polysaccharides may hav.e s ^' møleeitlar yveigh?, .range- of betweeα about 40,.0(H)-I _f O(H\O0O -witli mul%b braπcfe øf sacchandes, fo? δy;tøφ}e; braB€hδs comprised of 1 to 20. møpsa$ctøid.eκ Oi' ^' glucose, srabiaose, galactose && _> and ifese braaches ihay bfe .odtmecttxl io die backbone via Hώut^il .m«βosas;chs»d&s..8uch a^ thirøHoss, lheso rϊώl^cqles miψ fύtMf iimfaάβ n ύngte or

ta ^.iπiich-as about 39%, TSw multiple brarscte themselves may have mutφte bmiiches ^• of sfsccfeffjfe. the multφte bfaacte-iψπpnJiO^ tδdydifigjse ^' ύtriil ^coharid^ JBigi ^' nsMpi ^• sacehsmfe ^rhr^Ves 6it^iήg.miiJaly:- ^: hydκφhobic ejϊtities,

Wo descnbo ebemicsl .modliϊcairø- prø««8ϊxrø« ih≠ involves ^a eim'πuϊ.ϋc, ρtw.κid« tød -Imrisstioπ ^' .meials or peroxidd/asc»rMt«:ses«iøn Qϊ the glvcøskivc bonύ m. the- polymer b&elφ&m -or ■aJk&line'τedϊicti've---d«gd-l>;πτέris5afiόh . or cpokolϊcϊi bid smaiks\ branched polysaccfcsidie molecules, using coMroJkd cpπdilions of time. temp«rs?ure ψ4 hxf;k\\ ^: rre-st«Mts f see ^' Example ^: 1* %, 3),.. The first purlilc&tioa procedure ig tm.ϋtH>B: of

tils polysacclMtkk- wish alkaline ύύύJm chelation solution to: -remove protein^ pigment

Tte lbl!øwi.«g are ...merely- .lilusiMϊvs examples αf the ^' productitos of polysaccharides that & ^• § -not. meant to lmiit the iiweπiios,

EKampie ! : Method of Mbdify i π.g . Nat «r|i 1 }y OceimiβgiP^v^cdm^ck?

■A /St.ai1i*.\g poiysafichsrida. ψ treated wiffi IXY, mdktioB or suφελckcl in 70%: alcohol to$i?tts.iit:fe φoύ 4B |κnp; tα. reduce nikrø|) ^: iai ^oiU ^' &niuH^iqα.. All _. ftøher steps &v£ ^' -øfs.ϊicli50tti] Mύm: Sδiji-sterile coridiiioos. Afte: •ϊrradi^øn lk ^? poij'sdeclu^ide Is •slowly dissolved in. άhύlhά wafer and the amount of lotai carbbhycirate is d«tώtui«αi by the -phenol suJfipc $cκ1 _: «)eikκf (Fidk;f yt.at, CJwrhosls Re«,, (19/3), vαl. 41, pp. 3(>42- 3956.}

A soitrαtw In distilled wafer fa. ptψmi-ά øi 20g/L θf pøi^ilcβva^dOi, starting, for- example with USF ^' psciM ^; (Pd%}:ls iøk iβά:p- ll ifMjt$t%$ ϊό pB 5.S with 1 M sόtfήii ^ή : pcdngfe TBe |κ>lysaechaήde; _; ; solmκ«ιs; js _; . then -3irte.tiba.tod ^' M fi.R iempφKilWilv &r «xami>to; 20 ^' "C aBdipø.mshie.ϊ^.κi. _' θf astwyre-are-ail^xϊtø|^.fe as-iή-exaTπple (Piguit

iψJz c-Mimφa slow. sdssk)β of Li ^' SF fso{ih-at|)H -£5* .at ϊθ asM fPigur* 0- ^'' HH? half time ^φitcd to φv«bl? %.-;sf»edfic fαkiity wss .1.5dJ2.h ^: (Etgt»e 1). L-->;%ε(Mmte at ^• same cmidMos-mdi coiK-ffltetkmli&tee.d iaste-r seisslpα- Bv Ik ifemosl iinumtfc^Pi^t ^• Mϊwevet, was prøifucecl- by tofflbinalioft αf S- ώM &scw\laie mά. S. mM -HMJZ \iWck ^■ ήrøed fepr sφskiπ, SeqiientM &4$&m ^' :θf ^■ sscofbaι;e^ρ _. poJy _; &'δC«har5& so!u&πval.rea4y ..cantrini.ng even- LsiM ^' &jCh caά^d &rther:slimuktiQή άir mission. ^' l%«-M-β md ®φϊxi\--$f;

scission ^as deii'ππmed by .the dose øf ascorbate .added, time of addition jSϋSt- M;/);? .jKtdilϊcm, pU mxά imnpmwm.- Ttee. mdicat€ ttet &n. imxiή rmcψm pmφteύ tk? Scission ρybctsS--A ^' :.pE of CS _* gave the shortest.4ύ\y andy&e gnMssi tøiβ bi-smmmi. 'Thy? ^U- #ifeπclxl ofϊect gf«?eorbaie- ^' #peήd.ed 6B ^" HjO≥ of iishydroascϋφt!o ^'' |>iX5dufee<i- by tlio ^; ?«aeuøτ)s (bslow).

^' AH» t-Oj.-. AíHA or

Whets Mf? is isce^at^ mid^A is deli^dfMscorbatg

For sxavπgfe digqsπon of 20 g/L σϊ lMf pmήn mtύ 10 mU li2O?; or- 10 rnM or lfe^ eθH^imaMvπ .of both at 5 mhi eacli- iadicate a.lwsr. reiadoo between digestion, time ami the ^: vtss©meter .measurements, as b^iαwvthe kiαetie« was also well ce.r.re.kvl«d vy5tivrøubat|oi).tøfήj>δϊature.

This m#ϊρd vmi b$ fartfe enhanced in pre«tce of .v«da!s like Ca ^':r , Cu' ^v αr F^. ^' Tbe.aibst }ϊk≠.y .so«ω*f fejMvagefe Feite teaetiott (IlαlBwell. B. -arid CMfcn4$v I M- C ζl ^x )W} Methods. EmtfwM, IMi h--Z3), wέlεh _. geβe^tes- foamd QII _V iceq-aiies H2O2 δiκi the fsdm'tά fόrra of.afeassitipB ^' riVeiaLio% C«*\repomd bdvr| 60 ϋmts more

AJi iher|κ-jlygaepkv4tte sam|3:|es usually, containα! measurable traces of 1% €U MKI ^" Zn m. pp.B kwd., The C'y aixi Fs cøήlent wss _. ϊarølar in ssveral. comrørck! pαiysaceharides ^' (ts m|)M^;J»HfeOT€:θf-A):afjd ^niUarly sπdyUdai gϊ^de«eorbie add

€c>Ma?πe<i low ppni tebss of C\f mi Fe. ϊKereioit,. whm mi Im)Mm mύ$l am deliberately adsted* :.ihe fuwkm isaelions -would be fei#k.

εximψU 2; Metfed of Mbdii^jjg-Natø^il-y

'Obe pU. of the ppiysacόh8ride-soiutio«: is: iucr^asgd ύ>--pH 10 w?th _? % 2Xi«ωple, 3N NaCli A^-shorrinαiktios at S ® S0 ^Q Cfbr 30 to fiO ^' -nnjuaes _* iθ fe> 30 ?i etibanolk addαi astcl _. the pπlϊet! rølypα^aride- is .precipi.Meci , This mmoved proteins Md pigra&Dts associated ^dclicemtiereiaLly avaϋabfe piysaeetarKle^ li^-pftlys^dhkride Is ihea dissolveϋl to a 20 g/L aad tlien m) mήύ is added, for exaropb iriiόuoϊoacδliε aoid at fed eopentiillon of iXOl. to. Lø M :|ias; b«8B φssκκl$i|if# tø gl^§ ^' S bosteikd ck--

^•■ >-?.

|x>ivιmntάιtkm,- Other addsor όorobhHitioλ of them like sulfuric,, HOi ₅ Acetate propionic:. add, θirTOø.hrøώl»n.ic--ae.i<l.-«ar. bβ usαifø shorten the hydrolysis timstaπd irppr»vg-th«? yiϋlά ϋf ύ -desired' -.s&ucføHs of Bfaisbtxl polysactharnie wite& oxi ^' dstimi Alter ^• :|pp?t!p!πate time 3ϊ##FV4%. for sxampie a imie qάui-se $χύm ϊil xήhMits to 4S- hours, at teτφ.euιture ^' of 15 to 12,1 ^C, the soluifovt- is neutraibed Io pH 3 to 5. cooled lϋ 4 ^ύ € ami ^• «raf.tfjf«gecilo .remove, any iiBolublo msiMr. Tiltui tbo supema&m ^ πeutralixed io a ^' &ai ρl:l of ^tbcϊut 6.0 to E-O with. IK HaOB foi: example, 20% ethaMnasaded ¹ & recover the soiubk |x>lysaech;5ri<iδ: Ilaiio of polysaccharide to miά, iy$μ σf rød _> eo.B?eofc!tk>» _} .pi-! teή|ϋeratιιtt- am! time mtαrysi are ^' ssketed so- to .geuαaiψ -a solobfe branched |X>jysaoc:haπdii' that has a aw-kx'-ular wight ^fSO k.D _s <>β _v .kli>. _v 75 kp,;90 kϋ _f . UlS kD _t ϊ2 S. M3i 150 tO, !7-5 kD, aϊiϊl up ^' to If)O Rft. The rssalth-ig.soiilbuj: bmBChed _. poiysacchsrkle pϊocfet. CaH lurtber-wasMd with !?0% etHaiiol <$r witfi l0O% ^: acetone ra prαvkfc« ^: final. dx> ^> po\v<fe Tte«apQ.«. the aolubfe braiiched. polysaccharide Is røo.hϊbilix«d in water to a fc»I røneentiMion of about 5^15% by wesglit for analytics] -ideαtlfscatian, efficac-y or Mκtcθk>|kal sUkik _: S; Xliδ soKifcle. ^' bravMiδd fκ^lysacchafi _; de #:ψ b& fαrtlier diluted for t« according iύ mnhαάhmntύ of the hivzntiόn $π wliicii conc^ritrations -of 0.0140% may be p5 ^: øvkfe4to.e«ik .Dqiendirig;.øn φe desired soluble bMiched ^■ p.Q^sscchaπde-coiiiposidQn mύ mpkαύ'^ weight

$άMmp& 3í Method .^fMMfy-ftg;Natu^1^Oecurrf% ^' Fόlys^cetøds&

•life pB -βli-fe pol^sa^cbarMe paluilpft Is racreased'-to jsH ^' 9 with, for e^mpk ^l NaOIL Alter short ieααlmtbritU § to.3OC -for 30 lø 60 mϊπutesj 10 %-# ₄ aβol,i« adfeiiiml tlio: |wi%4 .pøiysaccljarϊde .is precipitated This .removed ^rotem^ -aαii. pigssy&is sasoelakxi. w!& commercially avsriabfe pαiysacfeSmrii!^.. Tkm mάming αompouπd sαch m a sodnivή bdrøbyιtridK _j. iilhiύm bovipyάrsψ^ senium cys.πoboro%dri'di\ sόdύtiB lriac<5ϊox)iχ)]χ>hydfid® or other bojxibvdmte salts, to eosate t session by αfoims reductive m«ehimism of the glyceric- bøiscis. liiis fόriB .fragmδπts ^f a si≤si άm^poπilkig Ri a

5- 3r

fepeaώg $ubUλit.(U,:S, Pαf No, 5y5S4 _? 3S6); Again terøjrøi'ύfe ami ύma cmb& used \a shøβep the \ψk{ύyu$ iime _. mύ impioyp- t&e-yjteld όξ -pόlyMffMfφ: λftor appropriate ύm& iri.terval.Sj- fbrexaπap&s a femrcoiSBe fmnx Mhrnmfc fe 23 tours,- at temperature- of 2S ϊp75*C*-ihe-^)ι3^lϊoπ js έbαied/ϊt) 4 ^? ^ ^: :sώ &eϊiinfU|ό tø _. .rem<>vo-aay iasblubie. matter, Then -tlie superoamπi: .b: ne«fefe<i kr a ϊiual pH..(?f «b«M δjβ wkh IN HCl % example, ^■ $Wa. ψxh&sϋl te ύizhd © τβchvhέ ύm &ϋh\bk pάlysae.d'j^klφ, -l^iϊo ^' af polysscekiritte to red ^' uo^ ^' ve eøβipMJ^. ty^-ete^mpoiwjd,, c^nceptmdai. pϋ ismpέJiliire sjxi limb jmervai sre sebcte^ go k* ge^κifo s sølu ^' bi.φ branc*W-pc»i-y^^l»Me;tfeat-.ha8..t..πi(>totlaf w^Igivt of SO Jt0, ;6O, kD, 75 kD _R 90 \ψ, 105 kD, ϊ2^ J[0, 150 !d>, 175 kD _f and ujxiσ 26D kD. Tfe;ri^a!fesg-$αfebfeb.mHS-h^d |.κ>lysaQcha?.ide pro<.iue|; c;^i: fmiMf t^tionats ^' m$i 28 to l\ψ/v etkiBδi aad ^' IMally with- 10d^ό:δc«ton& ^: iϊ) pirovid^.s- fetal dry _. powtot ωδrøφoϊi-tte sd!πl>|e; ^: brai|ched pdlysacskmdcis iieSfii.ubUized ^' in vrat^;(ό-a Tm$\. tpύcmii\%{mn ύiφMt S~ϊ$% Ijy weighC for assalvivca! yentlfieatioa, eSicacy or toskotegica; smtiies, T3ie. sokibie braMφed ;pø!}iMe&nds _: tmj ^" be- txitiUm diluted- ^" hi .use according ^' Io «mlx?φrnents- of the i&ve-fl.?i<H\ kvvvliieh ooω€^itratløi ^' is Qf 0,OJ-I(JH .ma.V ^' b.e pnwided tα- cells. ^Depending øή the desire4. sohible bmachcd -p<iiy$8c.chan<$«- eόmρ(>&ύio« uxϊά moJeculsr wight.

The Mge-t mokrølar' weigfe- range' feV the chemical.!}' soluble brrøched pόly^€€lMideS ^' is hi lhe rango cvf 50.to 200 ld>,.

As fe feftiperatαre- (or p.H) ϊnmuϋse^ u-^o-oaUed ^-δϊimlmuioin stalls. The A •e&ninadon. 'k pnsmύ .of alkaline fcorohydraies results in caatroiied process o.f chain cleavage ac5c-ommodak;d ^' with loss of viscosity and. gelling p ^v opsrties _y which M« xwasά. to

2.4

Example Miτάh$ti)\mmx&m

Ftvr.exaπφk. _: |>olysaochari<le ptodseed fr&rø 6w Bnm X powd.er fkκ»- sedfer-øϊ

We provMe a method of treating, a sυbjέd wcth a anfiωbrøtJc «ό3npouβci -Hmi in$xp't®<%$ %k§ .«:!S«9.cy ^f up -vaxdfibx&ut- cøλippxmcf, The meilio ^' d Tάpires; ϊhψ, co- admirasiratio.iv of the comp&unc! with a polysaccharide, In su,kli.tioo4o imm^smg eiϊloaey, tfee.αvξUfenmistration- όf ύ p^^aς&hsπde with a a»tt.δbrøtlo cotϊipøwuέi ^■ «% ^■ iecfoce the; l^xk% - of ite cfjmpoittuC. h: t-mhύύhmniP of %" Hweήtkn\. th« poi.^k-charkle, gakϋctovaaω.n&iv _; has ^' bseri sho ^' w\i tø bfe effective in. increasing' the efficacy ύϊ ;mtifil>R>tic compounds; when coadmuiistei^d witli thex:om]M?ήd ^' s. Although the e:s-«mp.i«?s p.«>vided hernia desα-ibe ihg. beneficial effects of plactoramina^^ we do x&ϊ excruife the possibility: that. piher-pdy& ^? se«Mari#s iiuvj: M\>$ I sϊύύfai efe-ct _* TM li.ierMs? in ^.fflc-aey »«ικ §τke. itwa- s s^Jiotgistic- eflect beϊweeδ t|jø • .galactom.Mi^iπ Md tBe pitlibrøtie cδϊ«pøά«d. ^' i:akture»

Both the polysaccharide ,asd the asjtifi ^' brotic εβijipoujϊd tøay itφarδtely be- femiukited, m a dxy.form for ^' exampfc- as s jλ>w(ler _v ^' or in ..s !igmά ^' :.fomτ, Ia 4 ' pfiϊfeδd embodiώδMj _; tlie polysacQbaride and anlifibrgtic cq.mρound are mixed. pnOr ^) sdfeirasirati^ _^ Th« mb.tifo may- be-ipihe fomi of a llquki a p.owφ;r qr &n 4«røfi _f

2S

Qm of αrφMry skjjj fa- the. art can deterroiiie- ami p&scribe tk sfibcϋve amoust of . ^' the antiflbrotk imposition required ^' teed <?n c&r&al $?mocαfc. In. gesiersi & suitϋfeie ^• daily dose of a: compDsitbn of Hie invention will, be that amoimt -øf tfe cfiirøpsiϋαOj wl)loh.b ώo Jϋ^std^se ^' elfάϊivδ to pϊόduύo aMtiβbϊpiie^Seά.

(τa!s«tό.msh3'iώ it k polymer ih&t may dώewr ^' m a fin'mγ .όi am- ranges: Mo^o'v^ tte gal.actomaj.man- may ixt^isriyMfeed or h-y.drolys<?d to-resuJi in fragmei^s σf the «alive mαiectik or s^ay b« reacted to .piwkfe chemically modffied ^: ihr.m$ όi ^" ife.miiive molecyie,- _. BmboiJhtients of .the bveMϊdn ^royitteM gaiaetpmtean paving -a.moLeeuiat- weight in the ranfo .of . JO^OO _' δOO.øόl) -O, The.g^ ^' iacfeHjanπpi nmy furilier have % m%& in b% ^.pg$.of 90-4!#CK> D or 4i(ϊ,0θβ-2θάid0ø l>, or 50 _? 0øδ~E0 _? øOO -3D. ^' Example i uώl» ar, ^• gak^brøiαrøπ _. wJth ^ avftsrag^ nit>føeute wδj.glil $$.65,ODO D w:Mb Exampje 2 istilixcs a galactoininu^tt with an jreeragfe molecule weight of 83,800 D.

The.r;3t::k> of .matraose lø g;ϊ!acu'?$<j røay vai-y aocαrding.to the .$m^ce _; o.f έhe/gaisctomanι\an and the -isolation ptocodut$.- Irx embodiments of tfe iftvenlioή, "the :gakctomή«nab .may hαyφ a msBpsse fθ gaiactoe x ^* atio of 2.(^11,Ov .mannose; 0,5-J .-5 galactose ϊlfet miio -.of n^!K.«e to.§s^bc4'«:e tria.y ^' be-2-:d:J.^ or, 2,2: i, lή Example 1 _? tl?e ratio

.•of rMrøciss fo. piiiϊtose k j;?:.l ύtύ \Ά Mκmyφfa % ihφ selected tiiϊύ pf ^' τm$m&ϊ- tκ> galactose- ip- ^' the gaiact»ϊaan«aβ is,2.2; ^' l.

m^nn^ω-bsckboπs _? vήϊk ύ\& skit. numl\tύtMn ^' box\nά ^' via Ct 4 j.6-rglycosi6e linkages.

The active galacto-polisac.chaiide- is ptiύhrøϊd fern -powder .resdify ^' available fr&ήlϊiϊo wood øf ώδ Iwrøti ImJ primarily Laπx-oε&dentaϋs (WeSteαvLarcB). Tlic process- in general fallow the preparation listed «bffve for .fkim?κ>gαIa.etuϊθMto anti glϊlacleϊBaϊlBsπ. The fergsi mόbtntiar weigjit i$ 20,00& to.- ?O _ξ OOO B ^itli «i least. 5% i&xmh%ύ ϊβϊαCϊύ&s ύάp ..chain.

The Ibltowing drøBs.fe«s ami examples of assays utiϋxsά to"demorøtfst«-«^]cac.y ami eB!sc-i|v«j 4PSS _: are ^ be. _. used as illusWlve Samples. a??d $w wnt.tM%φ i& HmIi the pjteeM ms^BtkM fe file exampte: ill usϊfaleti

Hxampfe L liyt^ iαπg, ICkfey and Plasma Dmdbulloft of Radlokbeied CrHlis?e.d) gέi!acCx>m;ϊπ»aπ ^; iø B^Uiiy- ^' MaltJ,Athyinic ^' NCwiii ^' Mke.

Twelve msie NCr-βfi- atliymk niick? mke (Charles .Ei vers Lak)M<Hi$s, Eakagh, f^ ^' C) were acciin^leS is urns ^' labwatory oάe ^: we.fek ^' |irid.r to-e>φerimeλtøt'ioa, The astmuUs were hoaxed in miαrøϊsαtør .cages,- four ^' per cap, and røktg a 12~hoox.iight/i!aτi cycte..λH animals. ^' received sι.eπlbf.ed ^p wat.er:sBd- sMikurofeit lboil^lφ^w/, ϊfie.-aβø.τiξife we.re. observed .ά≠iy χ\M clmk^L- sign ^' s .watt Motal, The ύύφ were, healthy mύ had iiόt. teim |)ϊgγ].αusly used in..dte impemMital pft5c§doi'«sv ^' Ihe,isic«- vwm ^' mαi,tomked ^' aad ^' -wspu πφle ^: ar fe :ir?itiati<>.« of tit.alMe.«L

Tdiiaiiøn .of ώe- gBlaotomaϊmau -was perfomiM as .fbiiαyys-, tS,E-5B| of th$ -gaiactαmsnnaπ. was dbsoLVdd )hS;!)-mL of wafe ^' aftd exposed to SS ^' Cόdes-of iriϊktm gas itilM jjreSd^Cfi pf F(1/B;ϊVS<)..J cak ^' !yst: (t2C} rήg, totafly itisokible in. wafer}. After oac Mir the gas supply was ^; mxnovcd, ftfe esiaLysi was llferei! aw?i)% siβd ^' tte aqueous • .xoluϋon of- tk:

ga&c&smsonsn wm bψάp^mM p i^Mis ϊβp^άϊ^ {fam-Mi, adding wafer), wM *κ* labile tritium, was found. Total yield of thcs-tebded gsl^ojmtmrø.wss 3 J mQ, spedftc ?a#o^vji|y ^s 300. μft/vήg;

AU twelye i.vske Were gtves .a single. iήtravrøo.us injection of cold or tri&tte ^' d ^: g$actcat?&nπ&> (φitiier 6,.p.r60 .mg/feg) on ϊhø. sanie da^. Ncnt-labe-I&f gal ^' apføsπawm was;, feπnulated in saline, and Mtiatsrf galactdmanna^ wiss- ^' added Io ihs soiutkm ;κ> that eaefe afiirnsl fcc$ivecl IO |iC, of nidsoictiyity.. All closing solatioms (1OiI μL «a#) weav^om-ied h (bplicata. Sk. mice were mjeclM with.fi ^' mg/kg. eolation; and six mice ^• - with 4Q mg/kg solution.

Two mice from eacfr gi'oUiywtTe bled Hi, (i ti&ύ H W% iikr fejectioii, and plasms was prepared. Animsjs WQFC ihm rzmfmά; |jveπ*\ l#8e^s am _? lungs. wsre _; coilscfed, ^■ wαghάl aή<i :flαsh-lh>2eaibr mrtb«r analysis,

λiisr weighing \hurs were dissoK ^y ed in .Wl triL of Soϊuene 350 -mύ. ixicnbtMi ^' -first for 4 hrs at 5D ^b C _? ^" mύ ai roam lemperatαre _. , utiiil tissues- were sohibilhed. One aiL of & resulting suluibs was counted. \ι\ & mnύlhrtiύn counter m a smgie stimple. l^assd on tissue weight ββd d-uϊ srøipk vøhime. lho tiάraber of ^Q of.trSda^i _. ga!ac(O.mas.uu5s.t per grant, oftis^ς/wδs c&kulatqdL

E^kliiep x#.rε tMttM ^' fe tlte ^" sφ^e ftiaft«% but dis^plwd In ϊwό fήL-of S.o!u ^' ^io.- Afef th«- Itpi^; was,sαfeNli'#»ti«i ?Qem t^mpmvCure, 15 mL.oi Safety 3<>I-Vφ $eir$βatk>π i]akiwa.s ^■ added wiά $Mtφ\&$ were jncύbat"&3 oveftøgtit. Five raiv of the tesUidtig saiufeo w#n> diluted ,ia. 15 nil... of Safety Sdlve- and Qouπtάf iii ai sc[ήti]:la|ifk.v cduπie): as 43 singlfe sample ^' ,, itmga wet€ txs&ysd m tfic same manner but..d^Bi>!ycd m one ml, βf- t.okmw. fbsπ®

' samples (SO uL eaeli* v^re gWtd. ciireef into §$$άy. Sbh'g .mά counted as a ^' sϊήφ

Thφ data- are yh&wn m ^' l ^' abk L

Titble ^' l

I).Mdl5«tyfβ..i)fπjifrøbl)de<l gaiact«iftaaήa» ..(at.6 t>r &d ni%lk$. ni tismpSi n$ ink®

llio .statfetieai eyalSalicm- ^' was baspd on ^' fexpclimenss ^: tό irac^s and ψuiπiifv ^: the Uφekc! gatoefomannan in orgass^lsssues fjiver, 3dffey$v _. ^$ _* ^ pferpa}. Six mm were treated with plactόraatem at 60 ^: mg/¥g' with eacft of 2 mice sacάlked ^: at K.6, and 24 hours; six mice werø' iteafed with gafectoffl(«ϊru3.n at 6 mgf^tg with e&eh of 2. VBϊCφ saαifeed at l _s δ _f akl 24 hours, as iie.s«ifed above.. Tlie percentages o£ "U-- ^' galk:to.inann»;m rø^Brøcl TM pv%p.a,{jxCi /gram) was "usjsd .fox aaafees; plasms oαtαam? CμCi /mi), was eosmteslferø the. entire sample.

The fcssmmenT ^• o.P-.radiokfce ^' t uptake .was cka.lie,βgj.ng wi!.iτι «aiy iwo .ft>k« per ^eatrft ^g nt^ Sacrifice iϊtm coήύύnatmt mύ ^' ottύshψl -όutifes; To δddrδss Ik; jiunfbey ^' df-mke per- group, polling, tests -were perfbrmed for: ihμ twp- piaotesiomui (reaϋaoat groups; Um wo-ald ρofenda ^: .!ly incr«iisc. sϋtnplό size pec group to four. To address possibltt^outls-s, .all

outcoines for ^aah ^' in.divM\ώl mouse was .visually εoπφajtd ta ite otb&r mke hi ύiϋ $wϊ$$Aκϊi$mmiAhn$ αvmbiptlom .the four possible xύø puteomss wf eø&lκfe§d in ihw evaluation .uwx ahy aigma-tefccl riik; % ^r ϋ\M hm$ exehxted mnm or bo.th.

SλS (VfeRtfoo ^' &U Csry, NC) M;^ usfcd- Ib dt arfyrø, PROC AKOVA ^s iml fa* pooling teste, wliils I^RCX" FRI^ -was used iφ estimate mes® BTS4 staεida^ dpmtkms.

It was observed fcl gala?;tomaτ\Mn fts&ϊf. binds ^' Id liver, kidney, lung, -and phMrm,- imά ύii Tj ₁ Oi r«?5.ch iiijte ø#i!j« hiudingj $..g, did .net- rt ^s acl.rsatuπ>lioj) of the binding belv-vseπ: the € ήi|4g am! 60 !«$/ ^' k.|\ dose ^' s. Whm 6 mg/kg:(with a rektiye rsάiόacih>?%' tti ^: .1 X>j a.tκi $0 ;nig/k|- ;(wxk a re!|tiv8. radioadiwiy of ^: 0;l) doses, of g&hzi&mmimixi \s'ext ^' admimster«4 th«-ajnoιmϊ of bound ^' :radiowiive galaciomannaK was t-he same; that i% _: ^: th^ _. tøoum o.f ^' boim ^' d galfielcnsaaiuto iaέ^ass^ .? Opioid -Ibr the 1 t ^l -!imes lughep dosό, ^'

The distxibώiffή of radioactivity in Whole tissues as well as per weight or ^h.ime (in plasma) was practieaity iifeώicai lbf 6 ^; and φ mg/kg of. gataetøit&nmπ* hm^ tfce iiespeciji'e.data were pooM for Tήbk L Qy^Mfee _: #to.k ^' T.έφi-e! ] ^re avc5rag-ρ fθf -β)ur ^' -

%-rgtϊIaetθBiarmp øUξsmatJon (KJOT μjasiM, _. Mdneys, and lungs, in Um- vioβus graiφs was relptfv^iy sføw. -^x average -of apprόximilely ^: Sg% Qf me one-hour πKϋoa.cth'lty was detected at 6 hαurs, Eimm&iiøπ of "'H- |a!actόJ.HMnan fen the limr was- more graiαa! ihss in -other tissues;. and en average, more than 50% oT the radioactivity defeeted at one hour after «yeoϊiρ:p -WsHtSJ pϊφeat at 24 tlGr^s, Tliis ftah feφ .c ^' orø^rsd _* f# exampfe^

with clearance of 541:irowac _: ii _: -from the Iker _? whidi was eiimiiiated to 1 -M after 24. fe, a?κ! from -.the lungs -.and tb.e.kidn.e}^ w!%tv» etirauiatgci m B.6%arϊd 3JB% ₃ fespectively^for-th<5 same period

.Example 2, Asking tlie imtifibtote ettoic?- of a mødH%! gatacfc tiismriogakcttutmMe compounds in LX2, jμv: immortaiimi Jiumaα:be|.uttie. sfgiϋate .cell line

Tht* antil&otb- activity of ^: :a ga(a^po))^<^tøri<fcs wafdetsiπsmed m rr!|>!i&a;fe at fi® .coBtairtrjilQωS mά fee tiαie poittts (4 _S 12: and 24 fes) oi hψφs stellale cell Pi^C;) pøϋferMi^j- δfti|jløvi«|,tfe meϊljød. of 3H/nip.M^s ^' mcorpόmtlλ δs .&dl as.

* A||άaJ-srøθotlfBu«cie aciϊ»:(a;maφer on aciivstiwi øf s^Oate cells) * ^' ϊkta FDδF-rocepfcr (a marker »n prαliferMioύ) yM?2, MMP9/EM-P1 ώ<i l11vi!% (τswfers on raatrbfr dfcgcadation)

Two taofs |K>.ksii«clitd«ies..serveei.aa refe:eBce.dønipoimds. Thev w«r% a-gal«ol»- ^• ϊMmfi^gMaε^i'omfe feήt eitru^ and a gafaclόrøaϊtfirø fVόpi g usr gimi;

IXS ce!is _? ϊiή imhϊamϊwά hummi h^ik stύMύ .cέll line, wer^. έαόubatod. at WJQC h m #me&p.hs$s .øf 5% <X32 m Dαfeee^'s pόdiliecl ϊpiniaia! ^isenlial tnψdmm CGlbco 1:1^1,1 j.§ t^αluiølogfcs, RcckviUe, MI)) erømiokg 1% fetal. ^: cslf s«rϋm (:FC.'S}, 2 athiol^ of L-g1u&ttUne.i&κS 5(HKHt J^xaL/of jpenicjilin/SOCJO-g/ω^L «tϊ§pmm.yεm for 1 ^■ %% 2 dap ^' baforέ sδanmg expedients; They wαra then serum star^cl -v^sth 0;1.% BSA (02% VQS) -ibr 24 hoA*r§i ami tr>sakd vvi.tii.eacN qt! the- three pαtysacchaήdes; - m. _: & Iblk^w ^' mg-

.mg/mj or4pf>& ^too^ft^ogalacttroiiate- O.l4l ^: 5 ^g/ml. S>r 1.2^14, 48 mύ ^' 12 hours, 'tin $XM& coneentmtioi^ of each. mcidilM. pQly^cehar&lle- was aφsted aecojxiiog. fσ oogoiήg dst&dtirmg the

To ciHiiimi thai %:■ bypolh£sk$4 reductions IE iϊbtogenk markc-rs werc? ngi

of-thc.MTT solyfiqn ζlm$ ©f STT in I ml ^' _: βF PMEM) -wrøϊo δdtel to each wsll of 24- weil plate, aαd iaotϊbated'ώ 3 ^; ? ^ti C fo I ht- After the hmfetioa, &e mtiάh ^as dborøied am!; tpp μl. δ.f N-pjiopaaoI iacktetUo teacli, wall. Aβέr S-lO-.mmuiiSS of rβsctirøj 30 μl of tho.^vkiϋoπ v\^s ialceii irβra each well ^: a«:d ^' tran^n^d ijito^Mveit piale mά ik> CV.O, vvas read wUh ELiSA Rsader at 570 ψn.

^■ l;o mdkms thai tfe eSk;l& of the ppiy ^' saeeharklex; -were specific for fibmgenk? gene e^pressioa, cell ^' prolifcraliάn- was assessed in Ui« pϋrespce of the- poiysa«charifc _i ^ss«ssm«πt- <>f prøli!emϊκm. ^ a- wk^biϋ indicator o! ^! sϊelMcs cdJ acέkξitkxt), mά xhm: iimψoimih ^' U\M. recfooσ proliferation m:e expeoled .to" ^' ϊtsdύce tte overall ^' mmife of- librάgrak cells dϊimig livei jnjiity, whl«h might, be erroneously Uiken .ss a _. specific.

Cells.- were, sertμn stpved, fhervirøib$«d i» _; «ie<!.HMii <^n&ιistπg M thyαudiqe &&- |ireyfoysi.v.48ϋ5Cfib^l(22}.. AlUn- vvashmg tbe.ce.Os: witlvfiold- ^' PBS S timeSj 50C) μl-.of 0..25 N NaOlϊrø % WS In %M$ά ^: io ea<# mil «f ^: .24-wϋϊH _. platte eaul. tscamferrM to SebtilMion vials ^; eojDtaiϊϋag- 1.00 μl oJ; W. HCi- ^' ϊJra/Srπ! c*:C S-v.mti!l«tkχα Soluøw ^: N ^' ih&ϊ. ΆMM tδ ^' tlie s ^? iai aiid Vttrtejiea. :{1*M: is .røad HiSefetilk&m C'auutcsr ^' (LSSSOl Liψki

I ^' fe.antillbrogfflic acitivhy of -the polysacshimdβs was measutsd by Itisirei&st o.π ^pr^siao ef established fibsαtfc markers, by QinutfifsUive- real time RT-FCR of ii ^' broifc: sλf f»F.:N^ UNA from LX2 cells Ws^ es.toic.fed and py$5ed using ^" th§- Qkgeή -RHAδasv fsifø.j4di Cδnesntralfd:ftl ^■ was ^: then rbέasiired m Ufc -Sckiκee -UV/VTS spectophøiømetsr I)H _. .§3.0 Bekmatu TMϊϊ! KHA was rewrsfc inunscπlxtd t» co.!t\^fe'fe«?ata"y DNλ ^' .(Ci ⁾ NA) ^' xmng thø HcYorss iWaeripiion System by Krømega (Madison, Wl) (SpOTfFM PowerSeiipt ^aM' DoaM^ FrδFihm^l Søigki- SM>ts _v CUamt^ VMA ^' ). Qr\<i mkx'øixm) of WA. in I ^' l. μl. p£uυcles$fe~ftw.w^?- was ^' .stkicd fe 5.5 μi ^' of Kk- revetse; ir&riSC%Lase-.b«Ob\ 1.0 μi biT:2S m.M.MgCS2,.2, ^; 5 μi of )ø IB ^' M dNTIK :1 μl αf .rmvdonvprimer, ^' 9.5 μ! idl' RM^e.iiifeiMtor andrO.E _. μl of AM^ iey^se .tøiscnφteέ (ofee;

μg ^: d? IH>werScι1pt Reverse. TKpscrijatkse. 20. μM liaddom. termer prlmeri. 20 ^' μM Oϊig!>CdT)LS pπmsrs. lOujg/iml BSA,.1M IXIX !OmM <JNTP mix, IDX re^pto bψa:, cfyopκAφφmi mά sUibiikα^), TB<j rόjjclfon was perfomjed for U ⁾ Mis at 25 ^W C (aω.πfeδikg} ₅ # ϊffiaai42*C (c;DHA-synffiesis}, and ^' 5 miπ tt ^CC^Kyjiiδ vleamϊirailoB), SO mm -m 42 ^? €, 10 mi« «t 70*C b^ FγG-200; Pettier Thermal Cyder.

Real-time quaαtitaiive PCIl was amilyzed hi triplicate ψά psBmsά. -wth AB]: P∑isiϊi -7S ⁾ (KHiT Se.qiu^uit DeiecϊJϊ>n System f-AppB.ed Bbsysleπm, Føsler City, CA) Ughi€^cliϊrR^p, Iξøche, USA, One jπicfolii^^fqDNA was- used ψmki fGR reactiM, Iflatimvm ϊaq litø p^yjmss^ S^ber g«?sj ^χ _> l&(imM.#3 ^' I? ^' .(Invi.^gei) ^3rp,:08i?)sbs4 CA) l^Fαfyiims% 4N1 ^? P, .DlVβiθ _v 2^M M|βl, JOX Taq Buffer, Nl ² MaO, Zyfeo Or&εBs aid the S«<>rescaEC& signals was cφtured dcaingvttaclv of &te.4ø(45.) cycfe, i« prøpodkm to: the quaBtMδs of tiouble-siranded DN:λ (deoaturatloa 1.5 s at 9 ^' 3 ⁰ C _x aβfteaEβg 4 S s- ύ 56 ⁵⁵⁵ C and exteπsiori 4i>: *$ ^' at 12^C) (1 cyelέ ^' for derlaiutstiόή SmM at 9SOC) ₅ 45. cydes _: foF amβlificalϊoϊi. (J.0 seg. ai ^S^e, I ₁ O ^c at 55 ¹³ G. 12 sfess αi 72 ⁰ C), 12 rø & -^.O-fβt Wut)g..OJfvc, awi'iø sec .at 40<>C for. Cooling, _: β^tectϊon ol ^' Ae ^' PCE

products by agarose g$ ^leettophbresas ψp usαi to confirm the fecune^eiiy of the 0NA products, OAFøll-was-ysecI m-a referøioe,geoe for iκ>πiMfeaiiqn _v mi water was be used as .negstive CøIϊ&ΌL

^■ Relative c|u?i»iii§fion was calculated v$iu$ the coφpayativo threshold eyeie (CT) method s$ feaibscl in \hφ Um BuIkHm _^ ABX PIIiSM :B00ϊ ^: If Sequent Oetee^απ System (Rejaiivt qκMtii5catiDi|- ^ialyses was done using tfee .ϋght€ydeE 4SO R«!aτivS: Cmaiitjfe;«ioκ S&ft.war« suppifed ^* ϊψ tho cαnφanv} _* . CT iπdicafeS #e feliαikas! cycU? Mipber -at syhich tkϊ _; atmϊunrof anipliiϊeci target gesies r§a«Mts | llxed ihrcshold wKMu %? ipear plias«-άf geas? _. ϊioϊplilk-atiαRx and is _. inversely related- U? lfee^ ^buHcfeee of ^' .mRNA-tmitscripis m iheinfiifd sample. Mean; GT øf ^uplkitts (fiτρJicat-θ| ffiδiisrøemejits \v»r« wsed to cskpkfe i&CT as the dif&reBςeφ CTυόϊ ttrget .and. Kftirepce (GAPDH) gsvrø. .δCJ !l>r :§mh -sample v^.s compa^ lev ibe _; coiτespoadlng CT of ife coatxol experiment and exρi'ess«4 as MCT. ^' Relative guantiatfkm was expreSsld as feld- iMusλioβ or rejg^M!^ φϊ the gepo of jfttest. compiifM ^; f» the όdBtrol condition aecordmg ^• to-, the fόitnuia 2 ^U -M.£T.

A figure, below (ϊ%we # 41 §hows that tile _: ga!δct<><im«im^p!actumsate KJR- 30D) "sup^sS^s the fibrosis ftiarksrs- expression between 5(M- mά W& while the gahicipmέinβξia (DA^ ^f λ:iSfAT) $?κ.t th^ -gai^tei-fliafi^.wgalactu3fan4te (OR*2^δ). ^' decrease fibrø&k t»srters! oxpϊ^s-sion foι.» i) to. 4ϊ ⁾ fk Ilmse _; result mcli^te s. significant aMtβlmniό dfect of tks ^' ^lysa^charids^ psjifcύlariy- that x*f .-sppie .odgm _f ai hivhmvi lepaiic .stellate αelis, asvd suggigst tlkst the- poly-saccbsjides cm pxάsoi from- mύ ^ea ?©v.&rs<? tfe.e pmgi^ssiim =»f liver libyøsis>

Markers øf ^' Fibmaenesis

The tβwl\& (Figured 1) of qilT-PCR fe^ts-jpr- iibfpsis.nmrke^Cόllaga I. (COI-L I), aljshiH -.smwtfs- Biϋs&k: setm |MM% p^felst derived grøwtlx mtύϊ mcφiϋr-feøfe p ^J D(iFB% traBsfcrπώg: .gmwtlb , iacl&r reeepξφr lkta^l CTGPB^ .mήim

Inhibitor of metaU^protsiaas^ 2.(T1K# I!) after 4& ks -as .ooβtø], and m -the prgsøβ∞ <sf I

nocrπali^ion to C)AFl)B «;X|i^-$slDi.) levels ps:.a house ^' -keepiag- ge?.πi ^' .

In wait, tfr eotjfesh. th ^' at ths rφservβd..redύρ! ^: ].f)τ.ιs ιτv fφrøgeβie nmrlørs are. noi accøutifel :fb? .by decreasing eel! vkliillty.,. mi i.mύή MTT mil -viability assay -w^

perforated 6ft t-!is ^: LX2 cell MQ assayed m. tfe -esbαrøg Figm. ϊkvτaiirø&fe feehthd a^sing vfabilitv 1$ to (feøpsώfeitliat ihe cόpφϋimds. §m iM simply toxic- m. ® nύn~ &P&B& .manner,, b&t mϋm: thaHhsy exerted, ^antitativ^ .mδaέiύra1>!e ^; effects m \h§ ty&\Qgy$k&pkte:M&l&& cells: tbrougli speciϊic .m'όk^ular pterakiQ.^,.

Cell ViaδilltYi TreMmeαts Vs, Goπtroϊ Untr^iteci

Obtøi$e<j r*jsαi^ shoved that tf>e-. -.slgBilkantiy .«l«?crease4 <«pr§ssia.o of fibrosis jiύarkefs wϊiδ m>t dae- to <le6rfei%ed,.oeli Viability- (Hgύrø # ^:; 2) but. rather cius lό ak actual effect pδ BiRNA e.x|3.r^sioivn# Hgu^ Shows .T^ults- of iN MTT viabillly test for human hepfilc striate cøiL{0SQ.-iin&]J^ i« the ahseneerof .poiysaccbadiles.aS' csmtfd,

5?

mή in the presence .-of Dpmmt (:1; rag/tnj), Csl^SOO ami øR~S00 (OJ jng/ml) { -.OB sight)- ltm -am. shew thai mm$ 0fihe .t|iree:|3oJy^cd^s4das..slgfiiiep% ^|£fecfe BB vkbiii ty ^■ withis αrdr maggot (t jpkally ^: 2?4 to 1014 \ιi fbese <5xφέrihiesits)<.

Similarly, these was no _; signMk^it dT«sc-t of aaj' αf Che three -poiy^ebarides on coil 1X2 |>?olifeπttlø«, ine&sufesd X\$hg W-Thynύβn% hVvQϊpQX&ifan ass^y .{Figar©

Flgtire No S FmlrfeMioή Resalis Of 3 GaIfetθ-Foi^>-iCύ|iaιϊdejs (}.n hopatic stellate -.cdi | ^: LX2 CdI liae) ysϊωg _. 'I-I-Tiϊyrai^w^ _. teyϊpQ-ratioo Assay

The ^{: '} .results (Figim? t 33 shows huraaiv hepatic stdiste sell . (HSC) .iiae LXI |jffiIiimtk>B tet jisϊttg 3H-Tlχp»<iin« infcorpαptps a$s^- in th» sbseϋόe of polysaccharide as ©$ntbl*aiκl in :t!xvprest;.«ce oil mφύ DXV&N&IV -tϋR-200 o:r GR- 3&0. The cals &h$w tMi now όf the th:r^e pt>lygacϋharit!es sigrafeaotb -effects ihc; cell proUfeαtbn ^ifcin error mssgia(typka!l;y- 1:0% to 30% in these experimeflte).

0.6.6- «δn- s«e ϊhM thfe p!acto^havm«)gέ1a$tøβBate fe>m. apjϊio showed ^' dftwmt-gakstioit of a .fϊili p&pύϊ of activat.ie!.fi»relagd i«RNAs; mdk#feg- a global silfct m\ Bhm ^' mg dows. sϊgltøjj: celi spedftq afetfvsuo^, TMs. iis.s .maπilbsiatjeα αf SE>rotie _: |ϊrfjgess

.Bxampk 3-

JΏ- vivo Stodies Qi the _. admixiiire of the- -present ^' røwβϊϊόπ in Model amm;ϊ!s

Palhogeπ-lrεe m ^' ale WMar rats were hάwsed -at si cbhstøit: iempεmure- mύ supplied wifi .teboralpry ;ckfw mκ\ \\>&i%t ^: .a$ Hfrtiwk.. _. 'fhhβc&WMk- (TAA ₅ , fmtή Wάko Fpre. Cherøicat Ga, Osaka, Japan) >^ss used to <m«iucfe _: ^' th:er ] ^? {br«sis- Hbclel-. The imiiKtlόs regiffios mύ d.o ^' s«ge- ^' ivss 50 rø#<$% iM^p^iionedly adm^iiϋ ^: feted twfee a week ktp mts- {.π ^ss 8-1.5) _. for iφ-to -S weeks. Tksa4mk|ιir$ \vss. gdminifiered tiβφr mtmpemφMϊύbϊ ^' ox. orøljy evgi'y ^' day. &r ^: ?5 wseks. For testing .deversibiJity of disease- tte a(lm?k'fufe wg.s adniinifiέtfed ήtlw i.n!.rapentonea!Iy o.r orally ύuήύg lte last 34- weeks- of i^ jiκtetku> .period, .2448: .a%t .the ^{' '} ϋnsϊ l^fiχjtϊτ4 _> -\fe,P^is--v^t«/aϊ^.fcϊi∞d _s ^' rød Che.pmtøneai amity wύ$-ctp&\φ The Ih-^ was _: |.>i|r&ss(! wltIi pli.04phate4«i!fere:Cl saline, via.- the porta! tø remove φ blood from (he whole livsr lobules. Subsequently, part of the- liver W8S trøated with l ^' ø% ^' _: fonnakiobyd^ and \&zά .for Mstofogk- e>?sadriatioi>, T!.t<? s ^: emai«mg $mt \v&$ ϊk&ii m ϊxqmύ miirags« &ήά Stored at -M ^& C ^' for fibrosis tiiartes ^'

In other imiϊBsi . model fibrosis and cirrhosis were induced by Bik Duct.Ligatioru- Mi$ mm kψatϋiβnwmά .©cl ^' jfoe common bϊk άm$ wert-Jig&ted at Mo •dϋJb^xit ^' sjfe,

The pis :v?&e advr):bls|erfcl witli tfe aφτ:vktøe- In this exMφk including- eorticosterϋid (1.0 mg/body}-- iMrAperitoaealJy. every d ^' a¥ _. .tb.r 2 wesks. The ϊ€tguϊis werό ϊkmϊ tresimenl with- -the ^• admixtyye ^' -etjhesr- mtraperitofteally or ottάlγ xvppt&sstά ibxosfc of the: liver by at bast 50%- γmø a)Mτ<ύ \&κmMd immvds,