CA2042569A1 - Stabilized fgf composition - Google Patents

Abstract

ABSTRACT

Methods for stabilizing fibroblast growth factor (FGF) in heat, acid and/or proteolytic environments are provided. Such methods comprise combining or otherwise complexing FGF with a salt of sucrose octasulfate (SOS) and in particular with the aluminum (sucralfate) or potassium salts thereof. The present invention also provides FGF-stable compositions comprising SOS and FGF. Such compositions can be used in the treatment of FGF-responsive diseases including the treatment of wounds and ulcerative conditions of the gastrointestinal tract. The present invention also provides various diagnostic protocols employing SOS as well as diagnostic kits with SOS as a component thereof. Finally, the present invention provides methods of purifying FGF with SOS.

Description

Docket No.: 39,5g5 Stabill2ed FGF com~ositlon In accor~ance wlth the present ;nventlon, tl~ere is provided a method for stabillzing fibroblast growtl factor (FG~ in heat, acl~ an~/or proteolytic envlron~ents by comblnlng or otherwise complexing FGF
witll ~ s~lt of sucrose octasulfate (SoS) and in part~cular with th~ alumlnum (~ucralfate) or potas~lum ~lts ther~of. 'rhe present ~nvention al~o relates to lo FGF-stable compositions comprising SoS and FGF. Thls composltlon can be used in tl)e treatment of FGF-responsive diseases ~ncluding the treatment of wounds and ulcerat~ve cond!~tlons of the gastrointestinal tract. 'lhe present invelltion also ]5 relates to tlle use of SOS ln varlous dlagllostic protocol~ and as a component of diagnostic kits.
Flnally, the presen~ lnvet)tlon relate~ to the use of SOS to puri Ey FGF . :

Backqround of the Invention Ulcerating disea~es of tlle gastrointestil-al tract, commonly referred to a~ peptic ulcers, are dlseases ln whlcll there is a defect in the eplthelium o~ the ~5 gastrolntestinal tract. 'rhis type o~ ~eEect usually occurs through the comblned actioll of hydrocllloric acid an~ pepsin. By definition, peptlc ulcers penetrate to at least the ~ubmuaosa5 more superficial lesions are referred to ag eroslons. Peptic ulcers may occur ln many locations oE the gastrointestlllal tract illcludlng the stomach, duodenum or esophayus, ln Meckel's dlvartlculum, at ~he ~igll~ o~ a sur5~ically created ~na~tomoEIle, alld~ rnr~-ly, ln tl~n upt-er ~e~anum.

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'Iwenty years ago, treatm~llt Or peptic ulceration consisted of bedre~t, a bland diet, ~n~acl~s, and~or surgical removal of the ar~ected area. More recently, -rec~ptor ~ntagoni~ts have been used in tl~e tr~at~ent o~ peptla ulcers. Tlle two most commollly used ~l2-receptor antagonists are ranitidine aod clmetidine, both of wll~cl~ act tllerapeutically by inl-ibitlng gastr~c acid secretloll. The effectiveness ]~) and unwanted e~ect~ of th~se two antagolliBts has been extens~voly studied, e.g. by 'Ihomas et al., in Clin~cs 5~gllL_tel~21Q~Y~ Volume 13, Number 2, at page~
501-529.
Whlle treatment with tllese antagollists has been ~5 widespread and relatively successful, many peptic ulcers do not respond to ll2-receptor antagonist t}lerapy. For example, while the reasons are not clearly understood, some 20 to 30~ of duodenal ulcer~
do not heal a~ter four to six weeks o~ tl~er~py with eitl~er cimetidins or ranitidine. Moreover, recurrence or relapse o~ the ulceratlng condition is not uncommon it}l ll2-receptor antagonists.
Sucralfate has also been used as an ulcer curative agent with minimal side efect~. Sucralfate is belleved to inrluence multiple mecllal~isms o~ gastroil)test~nal protection and repairing/llealillg includlng i) adsorption of pep3in and bile acids, il) cytoprotective actlvi~y Or the ga~tro stimulation of mucus secretion and l~lcarbonate secretioll by tlle gastrlc mucosa, ili) 1~) protection again~t damage to tlle prollfactive zolle~ al-d lv~ prot~ctlon o~ v~aulnr integr.lty. ~.ltt~ougl~ ma~or adv~llcq~ ve beell made in tllq undQr~tal~llng of l~ow ~ucral~te prevellt~ ero~ion~ nnd ula~r~, anC; l10W 1~
~caeloratq~ tile ha~ g Or ulc~r~, a mn~or gap exl~t lrl t11n eluaidation ~ cllronic tllerapeutl~ Actlons o~ thi~
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Fit~rob~ast yrowth fac~or (FG~ a8 been sl-own to be a po~ent anyiogenlc ~actor wlllch, lnter respons~ble rOr neovasculariæatlon in wound healing~
T~3ere are two types of ~GF, acid~c Fibroblast growth factor (aFGF) and basic ~ibroblast growth factor (b~GF~. al~GF and bFGF are, however, acid and/or heat labile. Tllus, the u~e of FGF in acid and/or lleat environments such a~ in tlle treatment of peptic ulcers ;~ requires tll~t it be ~tablllzed to tll~t envlrollment ln order to maxi~ize lts t71erapeutlc vo~
Recently, how~ver, ln ~CT applicatlo~l serial no.:
PC'r/USU9~3~67, publl~71ed March 8, 1990, tlle disclo~ure ; whlch i~ hereby incorporated by reerence, t11ere i~
disclo3ed certain acld-resistant ~GF compositions lnoludlllg recombinant FGF ill combinatioll with i) ~tab~llzin~ aqents such as ~lycosamlnoglycan, glucan ~ulfates an~ sulfated cyclodextrills, ii) ~ntisecretory ~gents BUCh a~ ii2-receptor antagonists, i~i~
cytoprotective agents, and iv~ ~ntacld~. These novel composltions are disclosed a~ being extremely effective ln t~le treatment of ailments in wl~ich FGF woul~ be potent medlcament but forltlle ~cld and/or lleat envlronment to which it is bub~ected.
~5 There continue~ to be a need, however, ror new ways of furtller stabili~lng FGF so th~t its diaqnostic potentlal and therapeut~c potential in the treatment olF
gastrointe6tlnal conditlons, wounds, and the llke can be realized.
~() ~h~ ~Qn ~ a~rd~ Wlt~ pre~ v~ o~ er~
pr~v.l~J~d a m~l;hod rOr 3t:ablll~.lng FGF, whlcll comprlse~
comblnlng or otherwl~e compl~x.lllg FGF wltll a E:alt o~

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, sucrose octasulEate (SoS) alld in partlcular with the ` alum~num (sueralfc~te) or potasslum salts thereo~. As : Used herein, "SoS" lneludes salts oE sucrose octasul~ate ineluding, but not li~ited to tlle alumlnum and pota~sium salts therof. The stabilized FGF
eompo~it30n oE the present invention ean be used to treat mammals having virtually any disease ~hic}l iS
FG~-responsive, and part3cularly in dlseases wllere there ~s an acicl or heat envlronmellt, or in an envlronment where protelytle factor~ are presen~.
Speclflcally~ the eomposltion o~ the present lnventlon : can be used in the treatment of uleera~lng diseases oF
the gastroin~estlnal tract by adm.Lnlsterillc3 to t11e patlent an effective amoul~t of ~G~ in combinatlon ~ith SoS together with a pharmaceutically acceptable earrler, vehicle or d~luent. Tl~e compo~itlon is al50 seful in treating wounds where proteolyt;e agent~
would otherwise interfere with FGF'~ therapeutie value.
~o In accordanee with anc3ther aspeet o~ the pre~ent invention, it has been Eound that SOS has a hlgller ~ffinlty for PGF, thus making lt useful in l)urifyillg FGF as well as useful in cliagnostie applicationE~ where tlle pre~ence and/or amount oE F~F 1~ important.

Brief De~eriptlon o~ the ~rawinqs Flgure 1 illu~tr~teE.~ the eEfeet oE hlgh temper~ture on bFGF with and without SOS.
Figure ~ illustratQE tlle effeet o aeld on bFGF
Wltll A~ Wi~ )Ut 80S.
Flgur~ 3 .illu~trake~ tll~ ef~ecl: of pe~s.Ln on rtlbFG~
mu~ln CS~3 wi~h and wll:l~o~ uc~r~llr~te~
Flgur~ ~1 illu~t:rnte~ rfl"lty oE SoS Eor brGp by 1~E1 ubility to ~n~ouple bPGF rrOm llepar.l c~ompar~:l Wit~l 2 M NaCl.

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' F~gure 5 illustrates th~ purlrlcatioll of bFGF ~rom a fluid sample with ~ Kuoralfate-Sepharose column.

Deta~led Descriptlon oE the Inventi The prcsent ~nventlon provides novel stable F5 co~positions for the treatment ~nd/or prevention o~
dlsea~e~ in mam~als whlcll are responsive to FGF
tllerapy. The present invention also prov~des ~or methods o~ purifyln9 FGF as well as certain diagnostic appl~cations wl)ere the presellce and~or amoullt Oe FGF
must be determined.
llle mcthod of treatlng FGF re~ponsive diseases~ in its simplest form, comprises admlnistering to t21e mammal an efFectivQ amount of a ~tabllized FGF
composition or a ph~rmaceutically accept~ble salt thereof, wherein th~ stab~llzed composition c~mpr~ses SOS ln combination wlth FGF.
~) For example, v~r~ou~ ulcerating dlseases of the gastrointestinal trac~ may be treated by adminlsterlng to t)le mammal ~n efectiv~ amount of the stabill~ed ~GF
composition. Such ulcertalng d~seases include reg~on~l ilelti~, ulcerative colltis and peptlc uloer (elther ~5 duodenal or gastrla).
llle stabiliæed FGF composition o the present inventlon can al~o be used to treat otl~er conditlons ln mammals which would be responslve to FGF therapy but for t~le existence of an acidlc, heat, and/or 3~) proteolytic environment. For exam~le, ln radlotherapy or ctlemotllerapy o~ bl~d~er cancer, tllere often res~lt~:
ulceratloll~ oE that orqan's tl~ue wlllcll could be kr~atq~ Wi~ll FG~ 1~ the ~GF w~r~ ~t~blllz~d to acld eilv1ronme11t ~ound il~ ~he ~ e~. Wound~ c~n ~l~o produ¢e an acld and~or proteol~t:lc environment whlch ., . ~; .

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would respond to ~tabllize~ FGF composi~ion oE thls Inventlon. Other conditiolls in whlcll ther~ is an ao~d, heat, or proteolytic environmellt alld w71ich would ~j otllerwise b~ re~pon~lve to FGF therapy wlll be apparent to the 6killed artisan, an~ include-i) tiss~ injuries, b~irns, wound~, postoperative tissues, thrombosis, arteriosclerosis:
ii) musculo skel'etal conditions such as bone fractures, ligamellt an~ tendoll repair, tendonitis and bursitis, skin conditlons sucll as minor burns, cut~, lacerations, bed sore~, slow-heallllg an~ chrotlic ulcers 8uch as those seen ln dlabet:ics, alld in tiss~e repalr during ischaemia and myocardial ~nEarction: and iii) ret~nopathies oE tlle eye inclu~lng diabetic re~inopathy and neovascular glaucoma, skin disorders including psoriasis and retrolental fibroplazia, chronic inflammati~n, rheumato~d arthritls, and certa~n neoplasms that are lligl-ly ang~ogenlc such aB growtll oE certain benign and malignant tumors nuch a~ hemag~oma~ and angiofibromas, and solid tumors, rhe sta~ilized FGF composlt1on oE tlle pre~ent lnvention may be a compo~ition of SOS and e~ther ~GF
~5 or bPG~. aFGF and bFGF u~eEul ~n pra~ticing the present inven~ion may be derived Erom a~ number o~
source~ including mammals such a~ human, bovlne, monkey, ~wine and ~quine.
The preEerred stabllized ~GF composition i~ one 3() wh~ch include~ a modlEled FGF ~uch as a purifled ; recomb~nallt human ba~lc I~F ~rhbPGP) prot~ io whl~h a mu~A~lo~ ; induced ("mu~eln") by cl~ gltl~ ol~e or mora o~ l:he ~our c~y~elnes pre~erl~ ak amlno acid resldue~
2~, G9, ~7, ~nd ~2 o~ tll~ mature prot:eln to se~ltle. In numberlrl~ th~ human bFG~-con~ ellk almlno ~cl~l~, the . : , ~

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N-termlnal ~ro co~prises ~he fir~t amino ~cid. Th~
most prererred FGP ~9 t31~ rll~GF muteln cS23, the ~tructure of which ls mo~e fully ~lescrlbed in Senoo ~t al., Uloc~lemical and ~iot~l~y61cal l~esearcll communlcations, Vol. 151, No. 2, 7~1-708 (19~8) an ~n U.S. Serial No. 161,123, filed ~ebruary 1~, 1988, ~i~icl c~orresponds to EP-2~ 22 ~2~ tlle dis~losures of whic a~e hereby incorporate~ ~y reference llere~n. Other mutein~ wllich can be usecl in pr~ctlc~ng the present invention and which are also de~cribed in these re~erences include mutelns ill wl~lch amlno acid(s) have been ~dded, and where constltuent amino ~cid(s) have been deleted or substituted.
Stabilized FGP compositions in accorclance with the present invention llave b ~ founcl to be l~lgl~ly stable in ~cid, heat and proteolytic envirollments. ~ativ~
mammallan ~GF and ~GF wl~lcl~ i~ recomblnantly modif~ecl as ~bove to be acid-resistant are very low in toxicity.
'lhe two preferred form~ of sucrose octasulfate W}liCIl may 1~ usetl to st~bllize FGF are potass~um SoS
and alum~num S0S (sucralrate)~ Wltich form of sucrose octasulfate is used in preparing tl)e preferred ~tabilizec~ FGF compositloll will depend on a ~umber of factor~ lncluding route o~ admillistration, requ3rement for solub~lization, and t11e llke. For treatment of g~trolntestinal diGeases, tlle pre~erred SoS i~
aluminum SOS (suaraifate) becausQ lt wlll~not absorb to the blootl stream.
e preferred route Or a(3millistratlo~ of tl~e ~tabillx~tl FGF compo~itloll of tlle pre9ent illVellt1011 wlll al~o depend on ~ num~er o~ ~ctor~ lllcludlllc) tlle con~lltlorl beintJ trt~lated ~ntl patiellt conveniellce. ~or e~ampl~, whqn u~t-3d to treat ulceratillc3 woulld~ of th~
bladd~r wlliah are lndu~t~3d, fo. r example, by rad~atlon :, , , .. ~ . . . . ..

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treatment or c11emotllerapy, t11en the stabilized FGF
composition may be admlnistered by urethral catlleter.
In treating ulceratlng woullds of the gastrointestinal tract, the preferred route oE adminlstration i5 oral, e.g. by tablet, cap~ule, lozenge or chewable gum.
~ther routes of admini~tratlon for dlseases o the gastrolntestinal traat ~nclude rectal, by enema and parel7teral .
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~ reparation of the stabillzed FGF compo~it~on for adminlstration i~ accompllslled by conventiol-al teclllllques. For example, tablets and capsule~ are prepared by employlng additive6 5uch a~
pl~armaceutically acceptable carriers ~e.g. lactose~
corn starch, light silicic anllydrl~e, mlcrocrystalllne cellulose, sucrose3, binders (e.g. alpha-form starch, met~lylcellulose, carboxymetllylcellulose, hydroxypropylcellulose, hydroxy-propylmetllylcellulose, polyvinylpyrrol~done~, disintegrating agents ~e.g.
carboxymethylcellulo~e calcium, starch, low subst~tuted hydroxypropylcellulose), surfactants ~e.g. Tween ~o (Kao-~tlas), Pluronic F6D (As~hi Denka, J~pan):
polyoxyethylene-polyoxyprc)pylene copolymer, antloxldan~s (e.g. L-cysteine, ~o~ium sulite, sodium ascorbate), lubr~cants te.g. magnesium stearate, talc), and the llke.
I~ectal prep~ration~ are also prepare(l by conventional techniques, for example, hy e~ploy~ng an oleaglnous base ~uch a~ a hlgher fntty acld glycerlde (e.g, cacao butter of the natural orlgln, Wltep601s (a ~Bm i ~y n~he~lc ba ~H~ ~yllami~e ~Ic~bal, Federal nepubllc of Germnrly)), a ~dlum ~ cid glycericla (e.g~
Mlglyol~ (~yllnmite Nobal)) or a vegetable oil ~e.~.
ma ~iil, ~oybl3an o~l, aorn oll, cottonseed oil, ~llv~ oll).

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Wllen tllc composition ;5 formulated illtO an 1J-j~Ctab1e aqueou~ solutldn, the solutlo~ prepared by conventional method~ uslrlg a solvent such as an aqueous solvent (e.gL, distllled water, pl~ysiological saline, ~inger's solution), or v~ly ~olvent (e.g.;
sesame oll, olive o~ de~lred, on or ~re ad~l~ives may be employed. Such addltives lllclude a di~solution aid (e.g. sodium salicylate, sodium acetate), burfer (e.g., sodium citrate, glycerine), iso~olllzing agent (e.g., glucose, invert sugar), ~tablllzers (e.g., human serum albumin, polyetllylene glycol), preservatlve (e.g., benzyl alcol~ol, pllenol) vr analgesics (e.g., benzalkonium chlorlde, procaine Ij hydroctllori~e). SOS in its potass~um salt is pr~erred when it ~s to be usecl intravenously because oF
~olubility.
When tlle composition is formulated into a solld preparation, the preparatioll can be produced by routine methods using, for example, a dlluent (e.g., distilled water, physiological saline, glucose), excip~ellt ~e.~., carboxymetllylcellulo~e (CMC~, sodium arginate), preservative (e.g., henzyl alcohol, benzal~onium cl~lori~e, phenol), or analege61cs (e.g., glucose, calclum gluconate, procaille hydroclllor~de). Tl~e preferred form to be u~ecl is sucralfate.
The amount of the ~GF componellt o~ the composltlon i6 remarkably small when compared ~o otller ; pllarmaceutical agents such a~ tlle 1l2-blockers, and )tJ ~epellcls on a numbar o~ ~actors lltcludlrlg tho condltlon belncJ tr~a~ad, whetller or nok 1~ 1~ u~ecl alone or 11l aonjurlctloll wlth ankl~ec~retory n~en~, cykoprot~ctlve acJ~Il k~ nncl antacidtJ, ~n~l tlla nmoullt o~ ~ood lnkAkn by 'Cl1~ P~ ~1QI1~ .

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For example, wllell u3e~ to tre~t Illceratlrlg d~seases Or tlle ga~trointestinal tract in ~umall ~dult patients, t~le amount of the FGF protein component of tlle !j compo~ition to be administered orally is generally between about ~ g and 30 mg per day, preferably between about ~ g and 10 mg, more preEerably between about l.O~y and 3 mg per d~y, and mo~t prefer~ly from about lO~g to 300~g per d~y. For oral ~dmlnl~trat~on, 10 llg to 150 llg oE the rllbFGF mutein C5~3 or its salt may be formulated witll sllcr~l fate (generaly ~etween al~out 10 m~ to 10~0 mg of 6ucral~te) a~ a tablet or a c.lpsule tocJether with ~ pllarm~ceutically accepta~le ca~rler, diluent or otller 6uitable velllole. Such a lormulation is bene~icially administered one to four times daily to bring the dosage with~n the preEerred range.
Por certain dl~eases oE the lower gastroilltestln~l tract such as peptic ulcers and ulcerated colit~s, it ~) i8 preE~rred tllat the stabili~ed FGF composit~on be co~ted with an enteric copolymer such as hy~roxypropylmethylcellulose phti~alate, cellulose acetat~ phtllalate or metllacryllc ~cid copolymer to further protect tlle stabilized FGF from acld and ~5 diyested enzymes SUCIl as pepsin. Tlli~ coated composition thus passes into the gastrointestillal tract sucll ~s tlle diyestive tract and alimentary c~ l where it tllerapeuti value is opti~lzed.
Tlle amount Oe S05 used to ~tab~lize the FG~
J~) componellt o~ the FG~ sta~ilized compos~tion depends on a number o actor~ ln~ludilly ~olubllity. Tlle ~mount of ~ucral~al:e ylv~n to h~nlalla ls il~ catlve of tll~
amo-l~lt yanerMlly u~2d tlll a~cordalloe witll t11~ pre~ t lnvelltlorl. Fo~ ~xample, ~or orQl admllllstr~t.Lorl, batwa~n ~bout U. 1 ~o 6 q/~ay (pre~rably ln dlvid~d ~11--doses) may be used in c~ombination with tlle FGF
component o~ the stabilized co~positi~n.
T~le SOS component of tl~e present lnvention may also be itl the ~orm o~ other salts. Otller s~lts which may be used include pharmaceutlcally acceptable c~tions, e.g., sodium, ammonium, trimetllyl ammonium, ancl the like.
Wllen FGF protein componellt is brougllt into cont~ct lo Witll the sul~atecl dissacharide in an aqueous ~edium, the ratio oE SoS to the FGF protein compollent ranges ~rom about 1 to 1 by weight, up to between lO,OOo to 1 by weight.
The concentration o~ SOS in an aqueous medium ranges preferably from about 0.4 myJ~l to ~0~ mg/ml, more preferably from about o.~ mg/~l to 400 mg/ml, more prefer~bly from about 4 mg/ml to 40 mg/ml. The concentration o~ PGF in anaqueous medium ranges preferably from about 1 ng/ml to 5000 ng~ml, more preferably ~rom about 10 ng/ml to 500 nc3/ml.
For bringing the FGF protein component into contact with SOS in an aqueous medium, mere m~xing o~ th0se materials in the aqueous medium accomplishes the purpose. ~s the aqueous medlum, use is preferably made ~5 of distilled water, physiological saline, glucose solution, bu~fers such as phosphate bu~Eer and Tri-hydroxym~thyl-aminometllane-llCl buEfer. The ~tabllized FGF composition can be used as it is, without ~eparation or recovery 3() ny the processas de~cribe~.Qbove, a hic3ll1y nt~bilized c,ompo3itic1n o~ FGF is obtaillecl, whicll CompoHi~iOn can b~ safely u~ed ~o ~reat mammal~ æuch as hunlana, ~a~s, guin~a pig~, dogs, mice, ancl the llk~.

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,, ~, Wit~lout wislling to be boulld ~y tlleory, it is believed that the therap~utic effect ~E suc~alEate ( the aluminum salt of SOS) o~ gastric alld duodenalulcers may depend on its ability to act as a ~ustained-release carrier and stabilizer oE ~ndogenou~ ~FGF. In accordance with the presen~ invention, i~ is believed that sucralEate extract6 bFGF from norm~l mucosa and extracellular matrix and carries it in a biologically 1() active form t~ the ulcer be~ or other injured areas of tllQ gastrointestinal tract. It has previously ~een ~hown that large quantities o~ bFGF are stored in the Qxtracellul~r m~trSX (seQ Vlodavsky et al., P.N.A.S.
~USA) 6~:2292-2296 (1987) and Folkman et al., Amer. J.
PatholO 130:~93-~00 (19~83). Tlli~ is consistent wltl tlle earli~r report that administration o~ exogenous acld-~table bFG~ facilitates rapid healing of induced chronic duodenal ulcar6 (see Sza~o et al.~ Dig. Dis.
Sci.:34:1323 (19~9) and the above-referenced PCT
Application Serial No. PCT/US/03~67). Thus in accor~ance with the present invention, it is believed tllat SOS stabilized FGF is efEective in prolonging the biological activity of FGF in healing wounds, burns, ulcers, and the li~e.
In accordance with anotl)er embodiment, of tlle present invention therQ is provided methods for 6eparatiny, purifying and ident.i~ying compoullds SUCIl as FG~ which have an a~finity Eor sucralEate from a sample.
In general, the metllod comurises contactillg a ~mplc aon~:ainin~ ~GIi ~or o~her colnpolln~ls wllicll bintl or otllqrwi~a complax Wi~ ucral~at¢) witll a col~mn aomprl~lng SOS a~ a component ~hereoE. ~ny FGF present in ~ha ~;ampl-3 wlll bincl to l:h~ cral~al:e compon~lll; c-~th~ aolumn. FGF can 'chereoe be elut:e(l ~rom tlle co:lumn : :.

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w~th an appropriate hlgl~ concelltrat~on of ~alt, or acldic acid.
In a variation of tll~s em~odiment, F~F may be contacted with a bl-aLfinity column of the type described in PCT ~pplic~tion No. PCTUS~/01660 International Publication No. wocls/o~l4~, tlle di6closure of which ls lncorpor~ted by reference3 w~lere sucralfate is substltuted for tlle heparln component o~
lo tlle bi-afEinity column.
In one preferred embo~iment, a sucralfate ~epllarose column is m~de by mixing ~ucralfate ~nd Sep)larose beads ~PIn~rmacia Fine Cllemicals, Swe~en) in a ratio of a~out 1 to 10 by welgl~t.
Tlle eluates whicl) mc~y be used ln practlcilly the pre~ent in~ention will depen~ upon wllether a standar~
affinity column is use~ or whetl~er a bl-aff~nity colu is used. In yeneral, a 1119h concentr~tion salt ~olution such as sod~um cl~loride is used to elute rGF
from a ~tandard affinity column. Otller eluat~s inclu~e acetlc acid. Preferred eluates will normally be aqueous solutions containlng physiological salt and suitable buffering materials to maintain the pll at around 7. Tha preferred bu~fering composltion is Tris, at a concentration of a~out 10 mm. For a bl-affinity ~ column ~uch as tllat discussed in the above-referenced ; PCT application, the eluate~ referred t.o thereln mc~y ~e u~e~.
In accordance w~th yet anotller embo~lment of tl~e pre~ent inven~ion there 1~ provlded tlle use o~
~ucralfate and FGF in varlo~ lagno~tlc npplication~.
For example, ~ ount o~ llep~rln-l)ln-ling an~k~ eli~ll growth actlvi~ ln urllle l~ lncreA~ed wlth blad~er aanaer ~Cho~lak~ a. W. e~t al. cn!~c~ar ne~. ~
45:~90-6~,1985~ and ~6:5507-5510/19~6). ~rl~u~ u~e Oe : .
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2 ~Y ~,t SOS in a speclmen ~ottle woul~ st~ilize 5uch activity present in the sample, thus ~llowlng an incre~se in ~ensitivity and relia~ility o~ FGF as a cancer lndlcator.
Similarly, FGF in 6pinal fluid has been correl~ted wlth incidents of braln cancer. As above, SOS coul~ ~c use~ to increase the sensitlvity and reli~bility of tl~is cancer assay by stabilizing the FGF present in tl)e 6plnal fluid.
In lts ~impli6t form such ~ss~y~ compr~e contact~1lg the sample flul~ suspected of contailling rG1 ; witll SOS and analyzing the flui~ for complexes o~ SoS
and FGFo This could ~e done ~y measurin~ tl)e incorporation Oe [ll31 thymidlne into the DN~ of BAL13/c mouse 3T3 cells ~Klagsbrun et al., PII~S
82;B05-1309, 1985), the disclosure of which is incorpcrated ~y reEerence herein.
SOS is al~o useful in 6tabilizlng FGF found itl various tissue culture, and llas been f~und to be non-toxic to cells in vitro upto concentratlons o 400~/ml. Thus SOS ~an be added as a component of tissue culture in order to stab~lize any FGF endogenolls to cells of the ti~sue!culture.
The recombinant human basic FGr (rhbFGF) used in the ~ollowiny Examples I and II was produce~ in t~e mannsr described in Example l, 3, 6 or ~ of EP-23~,966 employing a transformant E~cherichla coll K12 MM2g4/p'rDG69 (IFP 14532, FE~M 13~-12131).
rhbFGF mutein CS23 used in t~le followlng Example I
wa~ produced by the manner descrlbed in the nt)ove-re~erenc~d ~locllemlaal ~nd 111oplly~lcal lle~earcl Communlaatlon8 vol. 151, pates 701-70~ ~19~13), an-l neferena~ ~xample~ 1 and 2 a~ xamp.les 1, 6, 7 and 2 o~ U.S. Pa~en~ ~pp~ kloll Ser. No. 1~1,1?.3 whlch ' 2 ~

correE;E)ollds to EE''-2~ 2~ A2 employing a trallsformallt ~scherichia co1i MM29~/pTB 762 (IF0 14613, ~E~M
E~P-1645) .
The invention wlll be urtl1er illustrate~ by reference to the following examples wllich will ai~
the understanding of tlle present lnvel-tioll, but wl~ioll are not to be construed a6 a 1imitation thereof.

EX~MPLE I
.,' I
Stabil~ lon of I~GI~ bY Sucralfate A. Stabillzation aq3illst lleat A solution contalnlllg FGY was prepared by mixinc3 0.25 ~g of human bFGF i!n 50 ~ll of l'ris buf~er ~- contain~ng 20 ~g of ~SA. This solution was incubatecl : at 60C for 10 minutes in the presence an~ absence c)f 2 mg of pota~sium SOS. ~t the end of the lncubatlon perlod, bFGF samp1es were di1uted 20-Eol~ with TrLs buf~er containing ~S~ (1 mgJml) and analyzetl ~or cJrow~l factor activity by measuring tl~e locorporation of Ell3lthymldine into the DN~ of U~LD/c mouse 3T3 cells 6ubstantial1y as describec~ in Klagsburn et al., Z5 P.N.A.S. ~USA) ~2:805-80g~19~5), t~e disclosure of wllich is hereby incorporated by reference herein.
Ag can be seen Prom Figure 1, potassium SOS
~tabilizes FGF against high temperature. Tlle activ.ities ;:
r~aov~red ak tlle control temperature of ~C are show ~n ~ open bars.

D- ~bi~ Q~ c~id ~ n a~ld ~o1u~ion contai~ cJ F`GF wa~ ~reparec~ by mixltlg 0.5 l~g Or F~F in 60 l~l Or 1.5 M aaetla aclcl oonkalnlng 20 ~g o~ B5~. 'l'hl~ ~olution, wl~lch had a pll .

~: . , . . , , , :
.....

of a~out 2.2 was incubated at 3~C for 30 minlltes in the presence and absellce G~ 2 mg of potassium SOS.
th~ end of the incubation period, bFGF samples were dilutQd and neutrallzedlwitll l~ri.s buffer containil)c3 BSA (1 mg~m1) an~ ar1a1yzed fGr growtll factor activity as above. Ilowever, wllen the ~FGF samples were aci~lfled, they became insoluble and were tl~ere~ore clarified by centrifugatio~ otll ttle ~upernatant al~d pellet wilicll were ~ubsequel~tly solubillzed in 'l'ris bu~fer containing ~SA (1 mg/ml) were then analyze~ for.
growth factor activlty.
~s can be saen ~rom ~igure 2, potasslum SOS
stabllizes FGF ayalllst ~cld ellvlrollment such as those found in the gastrolntestinal tract. The actlvities : recovered at a control pll (pll 7) are shown as open bars.

C. Stabilization ~qainst Proteolytic Factors ~ 20 A rhbFGF mutein CS23 solution ~200/1g/ml) was ; 1ncubated at pll 2.3 and at 37C in the presence of pepsin (4~y/ml) for 20 hours witll sucralfate.
Sucral~ate was added at ~ ~olar 1:10. The residual mitogenic activity was determine~ with bovine fetal heart endothe1ial ce11s ~TCC CnL 13~5.
~ s can be seen from Figure 3, sucralfate protects rhbFGF mutein CS23 Erom the proteolyti~ effect of pepsin.

~MPLE II

~.1~.,!,~-/_o~ IrG~.crQ~

A Pharma~la l~t per~ormanae llqul~l chrom~tograplly .: , ; , 2~

(F~'LC) system was used to demonstrate the affillity of FGF for sucralfate a~ follows: 2.5 llg of bFGF in 0.1 ml Oe saline containlng 0.1 mg of ~S~ was loaded onto E
TSK-CEL lleparin 5PW column (3.5 cm x 8 mm inner diameter, obtained from Tosohaas, Phll.~ P~). 'l`he column was pre-ecluili~ria~ed wil:ll 0.6 M tlE~C1 ~ 10 mM
Trls-llcl~ pll 7 ~Tri~ buffer~. ~fter loE1~lnc3~ tlle column was rinsed Witll 10 ml of Tris buffer witl~ 0.6 M
NaCl. l'he bound activities were elE~ted witll ~ ml o Trls buEfer containlncJ elther 2 M NaCl or 78 mM
potas~lum SOS at a ~low rate of 0.5 ml/~ln. rractlons of 1 ml were collected and analyzed for growtl~ ~actor activlty as described in Example I.
~s shown in Figure 4 potassium SOS uncouples bFGF
from a heparin-Sepharo~e column at 78 mM. It further shows that the mitogenlc activity recovered by SOS was at least comparable to tllat recovered by 2 M llaCl (Figure 4, upper panel). Since prev~ous studies demonstrated tllat bFGF adheres tlgl)tly to ~mTnobillze~
he~arln (see Shing et al., Scie1lce 223:129~-12~ (19~) and Klagsburn et al., su~.) an~ could be eluted witl hiyh concentrations of NaCl (i.e., 1.5 M), it a~peE~rs that bFGF has equal or greater a~inity for sucralT-ate a~ compared with heparin.

D. Sucral~ate SephE~rose Cl~romatoqraplly A sucralfate-Sepharose c~lumn was prepared by mlxlng 0.1 y o~ ~ucralfa~e and O.U ml (bed volulne) o~
~epllaro~e CL-6~ armac~a) in 'l`rl~ bu~rer wltll O.G
N~laCl. 2 llg o~ ~FGF' 1ll 0.2 ml Oe salln~ contalnll-lcJ 0.l mg o~ DSA were lon~ed to tlle ~ucral~a~e columll. 'l'l)o ~olumn wa~ rln~ed with 10 ml ~e 'rris bu~eer wltll 0.6 NaCl and elu~ad con~acu~lvely al: a 10w rate o~ 15 , ' -lB-ml/hr with (1) a gradient of IJaCl (~0 ml) from 0.6 to 3 ~; (li) 20 ml of 3 M ~JaC~; and (iiij 30 ml of 1.5 M
acetie acid (pll 2.2). Fract~ons o~ 2 ml were eolleeted. Aliquots from eac~l were dilute~, neutralized with Tris bufEer, and analyzed for growtl faetor activity as in Example I.
~ 8 ean be seen from Figura 5, when sucralfate W~5 mixed with Sepharose bea~ to form a column, a gradiellt of l~aCl up to 3M was unable to elute bFGF wllich llad prevlously been loaded onto the co].umn. ~iologlcally active bFGF was reeovered from t~1e eolumn witi~ 1..5 ~1 acetia aeid (p11 2.2) and it~ aetivity analyzed by tl~e growth faetor aetivity assay ~seribed in Exampla I.
Tllis further demonstrates the strong affin~ty of ~G~
for sueralfate as well as the usefulnes~ of sucralfate - in purifying FGF fro~ a fluid sample.
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Claims (24)

1. A method of treating mammals having a disease which is FGF-responsive, which comprises administering an effective amount of an SOS-stabilized FGF

composition to the mammal.

2. A method of treating mammals having an ulcerating disease of the gastrointestinal tract which comprises administering an effective! amount of an SOS-stabilized FGF composition to the mammal.

3. The method according to claim 1 or 2, wherein the FGF component of the composition is selected from the group of native FGF or recombinant FGF.

4. The method according to claim 3, wherein the recombinant FGF comprises the rhbFGF mutein CS23.

5. The method according to claim 2, wherein the ulcerating disease of the gastrointestinal tract comprises regional ileitis.

6. The method according to claim 2, wherein the ulcerating disease of the gastrointestinal tract comprises ulcerated colitis.

7. The method according to claim 2, wherein the ulaerating disease of the gastrointestinal tract comprises a peptic ulcer.

8. The method according to claim 7, wherein the peptic ulcer is duodenal.

9. The method according to claim 7, wherein peptic ulcer is gastric.

10. The method according to claim 3, wherein the mammal is a human, the composition is administered orally and the amount of the FGF protein component of the composition is between about 0.1 µg and 30 mg per day.

11. The method according to claim 10, wherein the amount of the FGF protein component is between about 1 µg and 3 mg per day.

12. The method according to claim 10, wherein the amount of the FGF protein component is between about 10 µg and 300 mg per day.

13. The method according to claim 3, wherein the mammal is a human, the composition is administered orally and the amount of the SOS component of the composition is between about 0.1 g and 6 g/day in divided doses.

14. A pharmaceutical composition which comprises FGF and SOS together with a pharmaceutically acceptable carrier, vehicle or diluent.

15. The pharmaceutical composition of claim 14, wherein the FGF is selected from the group of native FGF or recombinant FGF.

16. The pharmaceutical composition of claim 15, wherein the recombinant FGF is the rhbFGF mutein CS23.

17. The pharmaceutical composition of claim 16 wherein the ratio of SOS to FGF is about 1:1 by weight upto between 10,000:1 by weight.

18. A method for determining the presence of FGF
in a fluid sample comprising:
a) contacting the fluid sample with SOS; and b) analyzing the fluid sample for the presence of stabilized FGF.

19. The method of claim 18, wherein the fluid sample is urine.

20. The method of claim 18, wherein the fluid sample is cerebrospinal fluid.

21. A method for separating FGF from a fluid sample comprising:
a) contacting the fluid sample with a column comprising SOS; and b) contacting the column with an eluate which elutes FGF.

22. The method of claim 21, wherein the column further comprises Sepharose beads.

23. The method of claim 21, wherein the eluate is selected from the group of NaC1 and acetic acid.

24 . A method of stabilizing FGF in tissue culture comprising combining SOS as a component of the tissue cuture.