AU2013231037A1 - Myostatin antagonists - Google Patents

Abstract

Abstract The present invention is directed to novel, recombinantly produced peptides having myostatin antagonist activity. The polypetides may be used to form fusion proteins with one or more further polypeptides that enhance one or more functions. The invention further provides a method to prevent, treat or reduce the severity of a myostatin related condition by administration of at least one polypeptide (or fusion protein) of the invention. Furthermore, the invention provides a pharmaceutical composition or medicament comprising a least one isolated polypeptide (or fusion protein).

Description

AUSTRALIA PATENNSACT. 1990 COMPLETE SPECIFICATION FOR . STANDARD PATENT DIVISION A-L IP Australia 16SEP ?013 ECEIVED FEP FAX P.pc of Aplcrt \ot1f~rpuisPry Ltd DE B MOUR-fA, Moniz.ca; BFEERY, CSok J ME'fL, Victc.:I; BOWY~ER, F okert Synrdc roixbi; NICJ'iOIA S, Gina Dtmf! .A dte~s, f'or serve~ in A PAR K, el I J~i. 610Ninmus Ci"akc 3tri-et. Cner' C 'Thc- ibvtowi::g ~t ,~ sa fxi.tcj1i2i o3716 tis iwcitiot induding the best tm-thod c-f jxr~ ~ it knuwni to 1J." MYOST8-1ATIN ANTAGA(PNISTS FIL OF TkW INVENT IO'N TI~e pr~t v~tiou eates to novel otirs wvit. rnesan tagoit A tiy. Th pr(S'oet inv~tolfd. "'.ithor: relateJ, l.sc 'the tvmvl proweins in the tr~eain. of mycttti 5 artfer] disorecrs .M-ostatim (Lir iYD*,-.,) is a .g Tve" eg Ttor oF nuscle growt-h and is suvuctuiallyreazed -,c the rat bnrl ffit tctoy f3rThf3 rrhfivQvciexner a] 97~ o paifia vy.osiati j.,: a poten" i negative re:~trof skeetal Muscle ri~ d-rlpt~ 10 4W in O-du~t ifte. iy~an s is :unci a -sie Tanga of spukie froyn, fi~jL to. trmmunials id the m -o-sratin pTy!kml is Jni ibly cosi'-Ouved kru'd 1"omrogx.s axs species (.7N .herzon asri. Ujtee, J 997a). N.yostatin exe'ts~ its bi ooical le, -~og m~i-mon vvith th.,ei ~ric sm'eptor a~.vttype 1T (Leo t L', 2 'X N . yostatita J. ahi'o J'~nito rgdaeits own. ewpovessiovt Via a ln'wh in!i!4 thath inctnplt;e-.y uu'so:' tp Ser.(piie;: eA al., 2,X02. 1.5 fkzebbapragEada el -~,2003'.. It hs been demou'srauud tl.ha myos~atf.zn inhibits jin:.yoblas. prulifm-oM7011 i and ifhb!rlnidtW:,R without indruing apopteos-is or stirrnitinp twsce protein br.~akdov;;n C'Thotmis at ul., 2000; Larigley (Atal: 2002Q; RMos to 0, 2001, Taylor fft all, 2001). Krc.Vchou't micce frrayostatin havie geatly inmrased. -nhlsJ'J mass overthe enI.te body, Nlyosati.-null adc have 203 approximately 0%geae bodty Iegtthan., normal takoe, and Oeribiit a 2-3 f!O".d invrease ill n i*tu muscle wiht dae T'o faiscie fibre h r ai and hype-i-t-ophy. NIM:sral m-utatioils in muvostatin have been identifiied as being rzsonsiblle for the"obemsld phenot'-ype, sueb, a.- 'he Bfegian Blue and Piodmoritese cattle Nbreed;s iehret a'1, 199Th-r; Kaurbnidur et Qi .l997; W~OWe dt al' 97). A rri.nu hntp beenm obse:rv'ed in a watht htvj a 25 detofmi.vc rryustatilu gane (ScI2ek ie al' 2.00-:). The inlte.;:.premrsnion wfthe tole ofnayo-stalin int varcl- biological prcs~svia swudie& cf niyosiatin r.Ldl nimah hvs been confouuded L-j inabihiy tov.) rgusi between p-atIdevelopmental ceecs ind. effects Uhat Teltek toth ,aoi rno,'mtn ;ThIr eum-lig JInvaenl 1-d adultit. Jioweve.; .uyota has bec-Is iliCaed it. a rumbter of' disorda; s associalted with rsuJ -0 vessiv.g or mu- iscle atrophy, 'mach as ha seenm 4inindiVidvai"S aifeCtd [Y IV, ae, prolonged bed rest, muscular dystrophy or in age related sMrcopenia (Gonzmkz-Cadavid et a!, 1998; Langley et al 2004; Zachwieja at a] 1999; f3ogdmnovichs et al, 2002; W02006/083183), It was demonstated that in vivo administration of myostatin induces cachexia, a severe fbnn of muscle wasting associated with cancer and sepsis (Zimers at a. 2002) and that may akso 5 occur as a result of extended bed rest. Furthenuo.-t., up-reg tion of rmycsratin i. glucocortcoxd-induced muscle drophy has been obs;eved (Ma e al, 2003). C 2 inge5 in rnyostatin expression have been shown In oier condtons, for example, upr-regulated in cardionyocytes set heart damage, asd dow regulated in regenerating muscle (Sharna e aL 1999). 1G Myostai has also been linked. with n.any other icAogicai processes. For example, knockout transgenic mice have altered corcldes bone structure indicatng a role in osteogenesis (Harnrick. 2003), Fsthennore, myostalin has been shown to be involved in xegiiating glucose and fat mw-tabolisn thus it mny be i.mplicated i typo 2 diabeFes and obesi ly (MoPherron and Lee, 2002), Myostalin. has also been shuwn to be ;nvoilved in the inthunmato response during 15 wound healing (W02006/08.3!82), The key role played by myostatin in the regulation ofnmuscle growth a-ind differentiation and tle pathology of many diseases m disorders has led to the search for antoanist f myostatin!. Whilst many myostatin antagirSts have been developed, such as anti-myotaLiu antibodies (US 6096506 and US 6468530; a iuncated activin type UB receptor, myostutin 20 pro-d ain and follistatin (WO 02/085306); myostatin. inhibitors released into culture fro; cells overexpressng mycstatin (WO 00/43721); domnnt negatives of myostatij (WO /3350); and small peptides including the -WMCPP domain which binds to and inhibits myostinj (US 200'1/0181033); there are cureno' my osiatn antagonists in JIJJclti Thus, there sill exists a need to develop more poem; myostatin antagonists fur use as 25 therapeut 1 agents. Accordingly, it is at object of the invention to provide proteins with myosatin angonst activity for the treatment of msyostatin rented disorder , and/or to provide the public with a usaxnldicho. SU IMMARY OF THE INtNTION 30 The present invention is directed to novel, recomxbinanly produced peptides having myostatin antagonist activity, In one aspect the present invenion provides for an isolated recombinaut polypeptide having mrsyostatin antagonsst activity, conprising a C - a ,tmucated matur nyostaan peptide, wherein the C-temdnal truncation is at a position at or between amino acids 281 and 329, or a fragment, variant or derivative thereof. 5 The isolated recombinant polypeptide may "e selected from the group cojsristing of a C tenuinally truncated nature myostatin peptide wherenth C-tenninl at cation is ti ancno acid position 21,22,283.284, 285, 26,2, 288. 289. 290, 291, 292, 293, 294, 295, 296, 297, 28 299.V 300 3, 302, 303,, 304, 30-5, 306, 307, 308, 309, 31311 , 3 12, 313 314, 3 1,, 316. 317, 318, 319, 320, 321, 322, 323, 324, 325. 326, 327, 328 or 329, or a ftagment variant 10 or deriv atie trereoE.. Prefirably the isolated zecombinant polypeptide of the invention is selected from ite group consisting of a C-tci-minaliy ncated mature myoatatin poyopeptide, wtherein the C-termldnal tnmration is at anr! o acid position '29. 320 310., 300. 295, 289, 284, 282 or 281 (SEQ ID NOS3: 3.-11), or a fragment, vaiant or derivative thereof, or a polypeptide havius substantial 15 sequence homology thereto. More preferably the isolated recombiUant poypeptidn of the invention s sectt.ed from the group consisting of a C-terminally truncated mature nyostarin polype-pride, wherein the C tenninal truncaion is a at amino acid position 320. 310 o;: 300 (SEQ 1D N0S: 4-6) or a fIagment. variant or derivative th or a polypeptide having substanial seqence 20 homology thettD Tl'he invention also provides for an violated polvnciectide comprising a uclend sequence that encodes a polypeptide or rte invention, or a complenwntary sequence thereto. Variants of the polypeptide and poiyanucletide sequences oC the invention may be desirable as a way to produce a Myostatin antagonist having selectively altered binding characteristics or 2.5 having imaprc'ved biodisfributiou or ialf life in vivo or on the shelf Preferaly the mnyosttian. antagonist p olypeptide of the invention is pat of a. fusion protein including, in addition to the autagonist, one or more polypeprides that enhance one or more fulctiOns3 setlectd foiom the group insisting of purification, formation of ptstein complexes, tissue localization or ditrihutiorn, uptke/administation, in vivo stahitiv r id/ar in vivo half 30 life. For example, the fUsion protein can include an imunoglobuLin Fe domain such as an .lgGl Fe Rramn. 17Ix, XfiJfl p:',-ottLrany jacude a pu' rjibon subjxuene, rtKch as anl etope --n& a FL AG tag, apn hd sequence, ar fl Q -Tfusion. Pre.Femblyr dieta seiqwnces cor-nmie SEQ, 113 N0: 13 and. 14. The Iyostatif agodds piiypliAdn . the ivni rnay~ srird oie or- more rodf .5 anndno acid residues, such as a gQyos1atec ixrrinoc acid, a: ?1X~jdazel --xanik acid, a ;ameyiaed ~noacid, an ac y1!ed amino aci a; x~oiylated amioaid nrnixioaci conijugated to a iipwd mnoet a 13 amwio "16d a o'an., amino arrid ojgtetoan ia. dcl 'at~nga gt.t T11h inveion -also provide; for ap.mcti coirspwocn We=rs~ga k one Lolated 10 poy-epd ofte nvenltionl together ait apbna s tuticaiy a epabe cr-er iz. entvfcniion also provides a rMtod cf rgulating nmscle growth .promoting adipogeuc te ano ad/or proonxoting bone growth or innexalizatioa. in an imnnai Com1prisin-g admunlst'erng~ tk) -,d ia an akl~ective amount of at leas,' o lyprePfide offthe invenuoi Vie~ai, dkhe .1.tbdmy be -- used to produce intae~m scl iass, decr-mase~d -)' f'at dQePoniticn amid/or imnproved bone growth in- a sheep, c-at-lev deer, pouhtry. turkey. ig horse. nmure, nn, at dog Ori, human, 110 animal rupri have wrinal or almorm; J .levels ot rysa iu..n aninn'Js 'ill nanulal e.r of ruyostatin, treCa~tmnt w')ith te atgns oF the invelltion tvdj ir"Ot inicracdrcsi inass.. mIn naL With nunnalrjse na.4s, suct. treatment ild resultlt i.. anllccnei 20 mus~cle rness and mnay be particularly usenn in tUS Teo -wdcinTd tr.I mm~s\ith rcd-uced m.k l mass, due to0 muscle damaige or trainw, -. asting d=e nod 1..er st: Crc, trcatn-int trjfE- tagoni it of the .tnveaiuon. illJ mesoe W- te muscle rrsassn to iorynri'. In) fnnmru With abn'onn.aj mysat ere.S, the ;;.sci mas will in-.viriawbly be reduced, antre en with rnyostahn antagonists f the; inveyonun MI 7VAStr the muscle :mass back "-wards nonnal Tein;.abon dso avie awrnEtod "o pwrevenft, tzoat or ted'.uw a he severity of a iriy.:.srati. -re,ated pathologic ?Ljo& 'iki hratrzd t least in. part, by a! a-btwaiA 11cr-oltntn deveiopmeit or: metabolic aactiVity &.4 1utUSCie- or naipose tissue in. a patjtenz., sidr.-Isai methodCK Colrj -riseai adalinister.iuag &Cn etivv a;Ylflt a( least onze polyneptide. of ile 30 -iinvention to a paTirml ty incorsd tc~ 111C p.thologic cxmifion rfhYV" klclu&e srdr re.later!i( .1o u'1cle hyparu~oll-y; xla~cle atrophy and nrus.Iie -wa"I"ing a atdwivb infl-ialfajry it.yropft-hivi, mu ~scular dystrophief , nnotor nenon cliseases. disenses (if the n o suw'intodiseases of the peri ora Sd-ue to O'Doeririe ahnenahiten nr tabolic sy 'dronme, FGVI cecer, c~ormi. tcexainwctivity or rprojonil bodurst aud fiffier vvasti.g onditiow-- cardiia" firl, o storosis, renalir or disease; liver faiure or dio~~anom-cia; Ab~fv diabetes; arv wound healing. 'As anoth-er aiierynativo a poop-IklrtiA of Ih-is vrd ni may bro (oonjug-:li to- ccadh-ef Paicuia~ vcbe cornpourid to enh;rw the 1-hferapautic eft-ect, on 1the target ruell Cr ir. 10 bydelivringa jecoind compound in arte-fifort tc.ra h haiso teaetci~ain stated above. h-n. thn!se cumfiinions. the niyogstatiu atiofis -the invention. mi-ay be i-ndepeadently aad qetuyaenzhiister:e~l or c-diitrd Tfo pirsent inv e.... on also arJ~e ~mto of refgiflating ve growth of vaz animal ct) ing at& ts-ng to said animal anf ,,r.r amit otf at Ivast, one polyp eot~ iS nvntin.Preferably, the method ry bfo lisrd if)onc n:aedmsl raws inm a catle, deer, porJ-Y. turkey, pipg, hor1se. ryiwmse". par., Cfat dog or Ilnunari. ).MLEF TiESCRIP'iON OF TRiE FIG13RES -T: ini~o.M1u -iviiJJb f~ I- 6rwez (ICSC.no-ed vvih fikrrne o~ .0iI.?~ n f iJgures in -wbioh. FIG. i sows theeff'et f i's tatao 300, 3410 vwnd 320 (at I awlr r."ni o 20 C2?I 2 uwbatprheain 'emo~s~'eecli.e o ~ -"' lhots 2 <..2.ms ."'LI inhibitfry eEffM'. off myostain:im prcdC 2.. .~ iferation arid recovery sigm -iiocfin wntagordis z 300, .310 aisd] 320 at three conlce:n.tration 1, 5 and 10 1,10. I ho~ rho number of PC14M p--ositive nuc,;eif otn i-%Afqedfibres fromn young (I rnonth o1d) -wilId-type m. Isotted fibrea; vvere incubated wth no) Wagoiohst (control) or with 5 zg 25 of nryctatih antago~xst 3 00 f br 24 ot, 48 ow FI.4 SO satellites ce,,fLl tiwitio!n dan friou yoiug (). inonth old) ui-tte ice. Isolated fi Lres -were inc4ubated wi,1th .o W... (conrtrol or 5 ~gx'sfAtajaoit300. &.;: 24 or 48 hours. AfAh'.vatecd satellitr7 cells wver detoctod by IC'NA labeling thrfon-gli ICC. PCNA pormti;vo r~uC.eiwi couned Pe~r 100 rryonni~ei &Ad dm-v a converted to erntg ~ces~which ise EuorrealiZed tf- thC OCntroS. kP"~.U FIG. sho S atelIteC c-f1l acivpdou daa trona Old (2 yearoid wiki.-typc inc. soh~tud iiMmes Were eubad vvith no an-Ngoiisz (co;AVoI) or 5 Lg of r1y o5atin go st3031)or 320 Ab 24 or 48 hamr& Activatd satellite cetis vvznve detzfted by PCNA lael1gtrough 5 ICC PCNA poii v clei were cr:fIjrfxIe pes, a 1 ynccid raw' data convve~Te"d to pfrcentage increSe;6 whfic-h wereo n-cimaiizr to the- control, '-<.0.05., FIG 6 ~hws he i iniitov ectof yoi~P o primary inyobia!sts fionr '.u' ',9- yczar cl I.) m"Cek Fid rcoverig Myosj-tutn an~ ~s300, 310 or 320; 'HO-, "? slif"?c ;'rf Th chentoI;A1iifi tofy effect of nvstizon prhixiary ranyoblas*a 13rom younpr (I I L ;xothold) mieand recovery usivg nysti tao;t 300~ ian4 :10; PIG. 9~ow the sateilitc C=-ll activatn data f~r mjirc rc vrng sahi (co )rol o umyostain autagonis,' 300 (S pgo; body weidg hrec tiro.es per wekfb, six %xekn; PIC. 9 idbow t " he caioapsiCity of *noiSts rIe'rivred fl'oirn mice redeving salUn-- (control) or n-wos tat n wa gonsr 30 (6 p~t L, Ad) weight) tirn tim es pe cl Annk for six wee'.k s; !5 FW.101 !shows the migration capacity cTf bo, irro eie rarpae ftomn nmico .rLceiving SAline (Ccntoi) or rnycstutir3 L togonist 300)( (6 4i&/g body wegt tretne e wirek for six ek; 'FIG. 'isf1Gwf. 111e aweage penvcot cflmigf in grip strength itnice rc ii slne (control) .)r 1myo.Stutfin. ffltzlgoniis 3.10 (6 gig body 'weight) thrre times Por wecic fobr Six weeks; 2 0 FI.12 shovvs the aveage gtip stengthi of -fir co-itrfl and tvfate nilc of Iig;ire 11 a," day 0 andJ day 42; PIC. 13 .Alows thice fffcct of 11yostat-in antagonists 30C., 31 0, and '320 (at 1" 5 L.I:d I C0 pg~n'l) o.. bhuian prcllat~olil-kiratiom. Te vo~t wxef 1cittd ibr 1~44 ihsurs'5; FE G. 14 show-vs the effef-cf of myontati-r autagouaisi 300, 3 10, arlnd.320 (at 1 " 5 and 10 rgn~)on F1(1. 1 5 shovr- 'd ;&t -MyoD anid Pax.7 arc --. jprr eto in liesfm~~ ~ ~ id gastiroc -- -1nil uIs Cle S followQing 'my 0Statin 3:. f0a tagonist t: e atient i n M-d "4 ic C'; FIQ 16 shbrws that antagonism of rmyoswrtrn with 300 enhances muscle regeneration and reduces necrosis in mdx mice; F1Q V/ shows that antagonism of myostatin with 300 decreases oatine kinase activity in. nadx mice; 5 FIG. 18 shows that antagonism of iyostaLi with 300 increases gri.p trnh in mxmce; FIG. 19 shows the increase ino grip strength aeftr long term administration of 300 in wild-type mice; FiC 20 shows ara incmased wound healing in mice afer skin biopsy when treated with ryoStati antagonist 300; 10 FI 22 shows the collagen. depoSition on the wound aea i. mice afttr skinx bun with and without treatment with 300; FIG 22 shows increased number of myogenric markers (total number of centrally framed nuclei (CFN') after nuscle burn when treated with 300 as compared to saline; FIG. 23 shows increased number of mnyogenie markers (number of fibres with cents.aley 15 fonued nuclei (C$N)) a ie muscde burn when treated with 300 as tomparedto sane; FIR 24 shows that antagonism of myostatin with 300 rescues the expression of p:$ox01 afer inibition with myostatin -FIG, 2.5 shows the average % body weight change over 22 dlays a.et treatment with Dexamethasone ox Dexame thasone in combination wit3 00; 20 FIG. 26 shows the average retroperitoneal fat-pad weights afer treatment with Dexamethasone or Dexamnothasone in combination with 300; WFI 27 shows the average Myo) expressioni aiter treatment with Dexamethasone or Dexamnethseone n corbintion with 300; FIG. 28 shows the average Pux7 exp-ssion after reaunent wit Dexamernasone or 25 Dexameliasone s cormbination with 300; 'N2 shows t-e rayevbia st proi.) rti~ Isa meus &tn'e~s20 n 2 tti cora~on~(1 5ald V) pig/mI) in.j addition to 'T0 10 ~.~1a oiiec rol afier F, .isand 2 hu; and. FG, 30 A (control) Fnd 11c300 treat&A) show van Giasrn stang of nhe mscl 21 dy fe Jv ur jury, n whic-h thii wvfrrws d~att co'hgenj depos its oin munscle, c e. oraftn flhi-otio ku o ce Saline zloat'ed rr.,Sd h (DCntrol) shws a Wign''er mT to olg doposi t W"71-bU computd to .)00 Sntai -2 W lrO4atod tI)S ig i- '~~530 C mid D shuovw FIA&E nn-gof the LusclAe. Saline iiecntsEd lpusce (C) shcowvs nmrrutic.. dognxing filbrfs 30.0 onioust treated muscle- (D) shr-ws darker-; tamecd fibres, many with cenimally former nu&1 ci. 1 0 -Uldicating 3cn anI regen--ang - -bres. FI i I -11 shos the- high ~euc.ehology of thezaosat seue C 0:18 species. Unesdefined. ec-ise., all technical ardsinifcre;;S ed here.-in havfe -he save -mean1ig as 1ceu uciy ondrstood by onfe of ortdiinary -i~ll i1n the wt ro ~vvjRIoh this inyonti on 1 5 belonigs. Although -ny -,tj hcd-s tvnw cmoiions smaror aiov~ otoedescbbeS h.-A-rAit cal, beI isew in i'he practice, or te sting of the" pr0eent -ivention-, '!."e o-ifrerred fnie-tod ad compositions ae doeibied heroin, For prs-so hep" sn nvnin, tefojlowinig teirms areI definedlow "Is uIsed dir ougkwiou the, spcwcainad claims means awny icrese i Ce=l 20 s;ize. .iyp.erplafia7 as used drnja espcilinandi cl Aims ;mean any ncas cel "nceatropby" 0 --i f-oug'ho-u- the~e~icto and cdahuu iuc'ans any wastin'0 or ls f isitiss-up resulting fr-onn flhe iac. C& u~se. 2.5 "Saco~7penia' as u..sed throughout the scicainanti clai.nsl means' a declineI ill muscle mss and errrmaCe Caused byr old a~g- cm; well' as re na.ea or other aP.? lte usl disorders haatrIsed b'-nusc-.1c jitophy ,md a deciasc, in the abi'Ay Of satellitew cls to n111tbUtov' ox "aritrgonist" Of MYG9sta'ifl as -used Iion:ut tJe eiicto nd Cl'aimns mcusay pnd.r~a at t eease either i3 wAhole C," in part, the: actihy of Aucegiv-owthk' is to bcc unestod as wasdug Quos divisRiot a;.d/orr diftexerdtiTIon W0; mcle 5 Wels and includes th-ke di viswin and/or difr i~nrA' ao J-erFIsc. , fU.sion of suclh cellsh vvi-d, each otlse ancLVor -,ith existingj inusde fbres. and it als.o- inmc-s ;orae protein sythss Irl nyofibres jea~iing to higher protein ient and greatem-r. sc fibre vohanime (imscle fibre by-pe.Arophiy). Thef terml "p(.'o!;rl~ceotice" is to bne uinde!rstood as meaning a tpiyir oif' dwcoribonucleir: 0 acids or ribonuclei. W&id anl inaludes both sigl s~re n obesr~ d povlymers' including, IN -, MINIA, eDNA, genomie DNA, reconibin~"t UNA, nuclIeic acid niolecukes prepred fmrn natural or aitifiia wucleosides or nuclvot-des, andA all other 'noin Eonas -of polyinicieftidtes. The povm~o~emay be -- solated faom a n-ataraily (mx; jug ur, p roduced u,.sing recou-idnant or -molculrz_-.aT bicoocal techniquems, or produced synthetically. A .Y pyuceolide imay include a, Wol gene or amy pa- thereof, and does not hae to include oper! vrzading frameo. 7'1-! 'he f, MsEo all poiynuc~f!ieodes according to .m- present in-ven'ion inc.ludtes- any and all opei leading fraymes. OCe re-ading frames can be established -using Irnown techniques in- thet art. Thesechnqe in dleth analysis of pynU ioide seq fences t identify Hnow stAt ad stocc -om. MIWAy computer so.Rware programmes 20 thal zaut -erfomr __ n ~.ton ar'oite ill-li A proteine" nui~le orj0]y",ie' to be undL-rstooc as 1 iw.aYJin a p(Ayrier ofi naturally ocs1iurping %nd/or arfiial alainro acids ovalenitly Jioljced via pertide b-onds, A p.)ypptido inchides a -po!.ype~idO that Imns be(n isolated i-om a iitriyocrrn lto rproduce uLsinig recomo'inant technijjques. It is toC beI; appreciaNled that a pAvype-ride tha.tincludces a leadker 25 orroseun, or a poiyp bide that erosa post trnionlrmo.liaix2i ntne to fall w ib th definitions of a polypqepta. Whs termi doies not Wocude a polypepide ta h1as bensyntiietical[Iy produced, as s~rn1-jeic polypeptides canj b.e ro- aIcin pIrticuiar, synthetically rrjado polypopt' J&E may 1otIid Correvctly, arAd xuri -may ne the bLologica]1 av.-tivity assouiated with a maturally o:.mmurink or rcibnty rduxed ?IlYPep ti.rc 30 Teterm, (iaget r vaat" Ist I'eO uders to me1-an ;.Uny pc yrmcelide or CI)-polyptptidc or pral qec h iss been nodiffied by sab.stitui&on, insertion or delecatio of 10 one- or lnorer -f, tie or i~ ~ oearSoarjdR, bImr that has -nibstantially the sianp acivity or Jfuiction au the nnmcoJified. se quench o_.. paril e uu Prefierably. a vatikt contain-s conservative siubstitntions. A "conservative si-stituzion' is one W), whic an. 1hic co~srbttd r another amonl adid that hs Amfrrs propaei sucll .S t-sat 0oe skled in the art of perptide chemistry wudeypact i-2 socondarTY ~sb re- AnDd hydrpadhc~r namm.e of ben pob~ypptide to be svb.sia~uiaIjy unchlanpge. Amrinio acid substitirmos may; geunly be made on the bAds of Animity Wn polaxjy. charge, ;olubffity. hydr5Aioiciy. hukobiiiityand/or the tvmpbipatuhic naTiurr of the r-esiduen. .1For ecmi negativffly charged Pmino PcIdas include aspardu acid and gutanibi acid; positively changed !0 wn '~ino Pci&ds imc-lurdk lysine -;nd argirtine and ammio acddns with uncfnn-g'd Ipola '.. head Vr ouIpr.S h ,in sinia hydrophilicity vifiuezi include JQ7eud -e: ,21ucnan rin; ychnai Canine; awparagin.Id glutaiue; ndserinw, tllre Onine, W ~ll~rad tyrosifi.r% se groUPs of aMino.k acmidf -,t __ay r Present Cons--ervative Changes nc, d (15rTpo, guIy, gin1, asghi asn, ?:er, ib:(2) cys, aer.yrth;()a! iiioe aa:p; (4) -lyq, aig, .his-- ard 1 5 (5') pho,. tyr. tp, Is. Aminoc a;:.k.cs uay be casnifj.;d sncoraing to 11he mauvvte of their §de Sroup . Amino aids wr-th aop larakyi s..de grouvs ilweglyc:imn laninve, valine., leuc1ine, and i.50101cine. Srn and tlireonr rTe have hydto.'xyi groups on their- Side chains, and because. iydroxy1 grovupE are polar and capable off hydrogen 1xinding& thcse amino ficid-5 mr hvropI) lic Su.1tur groupz 'ed Soly kirtid I itam a~moacdr. are s.tot cisly prolr but Can become, negatively charge 7oair% Giutamine an aprgn avm's sjn..1I'ia to gi au-C; acid Lind anpm 1t acd except the, side chains contain, aulide *n-sLyr-Ame, tvrgini an 711 hst"CHa nw mve onu. or s~nhov. amm gino, p-"' n side chi-us 2,5 which cautccp protons, and thus' anumtno acids act as bases. Aromiatic groups- -ray hev found on the Side cha--ins orpanileie yo ine ad tttph".a. 'Fyro-siu ,, polar because of its hyciro)xyl gmou:p, bult t- 3top .; an pher -ylairsej a~rf o-pl A variar. njriy lo. a E1tceanati'vely, C43ial ictCn-r A plyvpuptide variant atxcor1ding to thle inwmof vesirnf ma avu at least one s"Jsbat.'ition, 14 dkion, .30 insetrtion, or dito.an6 mrfay be made a-coing to ra-Ltagergsesis znmethods -cK.norwi in. the rIrt, Sui.- moiiain a be maxde- to a polynucdDide sv!-enc ihat encodes a oyetd vrnuof dtrivu.tive Of nie' Invention -- r34 may be ivttoduca-d. using staurdak . rutager,-esi..s w.d~eoa partictilpr Joi by synthesiiin =V tig c Iecte id.es coritahniig ;ah meatquence-. ~ta~rc b r~1ktirj~;te-s enabling h~gwkxiu to fragrrimnts of iheti'. wqune. ligation, th ; r. i ojtrt' pe en.code) a 8:.walag ha.Nig the-- dwosred atio 5 r~id i~erton.a~b~itwiiior dcee.So-u. O!gonucleolide--4ireczted st~ocfcniiei P-r-cedure s c.an -31W bc crnploye.l to provide all aieroa'd e.in I 'rulrirew -In prudeterrmined G :.donfs can be adfitered by li~ttto~dltoo nernby maethIods wveR. known iii the art. !dteimati've-V, amrictv of rxiupu-ar onal ii thods C-.11 be- usmo t( g atie vananin Y!avoslan It a ,ollist pootidw of LIhe1 pinion, ir-cuding seqieenco2 based artosid ,"itotr- b--ased. methodifl snoli as prote~i design auernitire. (P.DA) as decscribved Im US 64033-12. 1A s"bould nlso, ben Opiei J'tai :OR"ivae. ip availabhe to -very aucuirately pukhir t1fe hre dimesl oal Trncture of, a nptikk- se.jlrie rIadwey. AIM5) Thaeref ure ilt li epoasible For scn Ion killed ,n the arftto listi sno 0.fwr o~e~cttee~c fvrttost h 15 uena o cwe of the pfqAtide Pnd therdocre any like-ly e-ft -an the activity of ,t vep-ltide.. Such varniitions mre also iecrprnd within vibe scope of' tile preseRnt inven.;tion A "deri-vatv& plyepk of 'lievn~n;en that r, anmino acid -xpcu rs hais been altrec- in soiu way to p-od-ace a PolYpetd aig rtyin'sesibit.FrxmP~ ae.-ino ackds canm be replnced by U.he sami ammoI acijd of difrn cirlt.or non.-naluafiy 20 oocunic LEsAI-E.- 1LC,'Is C-an bae iserted. or j mstuted in th polyvpeptidf. kpetn ely, th polype-ptide. -. ay chomiw~cally modified to innovc crus4~i~ig wilyci I)Y1 GI Lw az; polyethylene. glycGI(U 4(40,S35). 3 uch o.-IFjL rvate ~ h-ave iimreas-ed se-rum hal ives ini Vivo, -ia.aiaii~,fjsca~nrates i.uid Other poete that .-ale chetn Very in frntaig rhmnmace'r icai. C-oinositi ons. 2-5 A polypepidjc of theivnin or afragrnt, vwzaa or drvtv t:eefa ther bioloalca fiunction Of antagonmising siyoctat1 in ativitv. 'T170nxe whether a polrpeptide of tfxe ~nvntoto a :iam u.vrato eiaieteef sable to ainagonLEse n-,yo.1tain af~ivi ty, puch acti-vity an be tested by going ipybiasts -. n 4the jrveselic' ojr asne(otr)ofa c.aKdidatz polypqeptide of the invventin, Ani Ir,crear in r.1 growth (of m~iyobla-sts, -Which 30 nrodu cc m utin na tlicrefre limit ilhoir own -rals, of proliferation, owt-er o'o m5,yn flb~st!5 rmat 'i-id n1) ot receve h canididate Vile~ieinia e..ha tfic pteptiduo has yostatin Einiagonisr." owlcivity. A suitable cr~ll fine could hiv n-airm 2 jzmyts(TCNO T I~ ~~~' VVIVI hoofr.i-wi b app ec'.0ated Ifhat aany s3-IUlblo mvbitcl raCould C ubdt s~c .as -primary oine, bo vine, porclie -or humyfan tmyolla'Is, The- tm" iae as tvied hterein rdefers to iy) a of a j-no:e sUa as i5 polyp; p-tife r 5 ncodrjn olyrmcleotidc from ito rnatuni source. r.rimen' or -nlideu (ig. Ce.,a~o a r;i -from Fun inftact cell s.::'uro,"). and the- t-m 'cpun iidt' aS toJ- \Cre jy caStt: that tfxe Prwteifl fOT pollype-136a (A fh e inen ion, (-r its encoding -_peynucl eottie Js -zs:;ont-a.fy frcx- of kls so lattkon vwnju. folifr oyui ide protetrns or popet be, r examnple, as ,. pwificatbon pod-uc. of h<~ost vell cn~rof as ?, purifirgd prorlud( firor a o-eonai .0 sol-.tCc. l t'solated'l pfolyov- de thwoetorce is One -Lust 'i rerwivcd -fionau its original n runn.Plefebrably, -sich -xolypeptideni arm at least al-owt 7 0 %.t" 75%/-. S%, or -S'O pure, at least alout 95A purc, fr at leat abIx-.t 99%A vpor. for fexWample, where sau ;3 egyc of pur)jji~y rofers io Ifir- percertago of deecais mystI ngkuat polypTptide ur its encodiig po lyrleo thti rei n ;eartuirlative to olter do.ut'able plr3te 15aridor polypelcidena. Tbew !term "rmbs.wrxtiaiiy pulrirke-" fir ",;siu 'taill isolated" as uszed Ihereill mearns a. mrixture: djs -. kon Ai a '5etlo-iich as a ryoarmiagaitpoj ypoeptide or' ts mooling 'oyule Th~e cat ini atni~iJlv ftee of w&,ioclat"'or with 'ocverpovuloids Proins or -polypopfidte. but fo-r 4h pesne 4 rj- pproteins Let rn be, rem-'oved using conViUf. -- m~ WJAS suth a affini-ty oz nar.ograp..v with atip if av iboly ojr lig-and, 20 ni~ whch ubsantall p~niiie orsubtaniaiy ioli~e tuostun agoist iepieor erncod tu-g it retierta' t,io arielcacea s a5d sr~edhrein or retajiis at lastofo>'s~oi; aci~a ilgclatvte. "'.,le eyofresi n to~x be -uidev~rto'od as 4enngte! initiationo tasriton trans11cription of ascto of UNA mnt nIN-A,, ad "he tnaiof o ue rrjR.NA into a 25 :-ajolypeptido "A ijio -Iiatov of 'ete xprex.WnSI" is cfjyjfed as any rnond)ati al toj ca-use, ir. a staiisticafiy 'Zignificanlt il'sikm, an inraeor ex s in geno exreionc, and va actI at any poin -a m th gen eprsso pathway. Tbhecu ien 'cxLmn-~~'a sdi~t~. neSWi~Jai ais meaus 4cow-ssing at huanti port o.thai is to Say whenI inedetngine cetcaimus intdnfta entejatures 3 - nrcfaood by that, term in Geach O'Etimn ail nee'd t, b" prn' qt bLut other faoatwous CFIp also be Th eas s~~ttaJ" rvecp. t1ds to.", -submezariaily boracitogo-as." or "subtaria. identity," as u, m kloiAjes a characteriAtic of a* nucleiC add0W Oha. P.Mino Wil ~zte ."erem sin Xt.d' rincteic aLcd or amiino adid ecluence has ati-as abow, 70 a bouzt 7 percent secqutmca identity uscanared toaslce eeec uli cdo m cdd it~quuce. More typically, ;a ~.ected svtquc.,rice and.J ftli-- refrence "Squeficoe wiJi. i-ave in least abouji" 76, 77, 7. ,C, 81 , 1U, F.3, V4 or even 85 pecrtsequeucr ideraijr.v, and wr p~reAaby a ias~ 9ow .~7 ,90, 91, 9:2 3, r 95 petcent ieq n . iduty .orc pr rrably srIIii, hihyhoaigosqeces ofleri sh;5re g:reatfer than at inst about 96, 917. 98, or 99 ptercenft sequence idntity teeu t-he selected ! oquence and the ene 10squneto 1-vt~ it was: i:orripared. Taue percentage of sequ--ence identvty ma;- -. i calcu" ted oWer the enie Ieigth (if tihe -;qii.-.neru, 'o be co-npared, o r iy lbe onicu.ta-dd by exchding ,madl deletions or ariins-which to)tl ss ta,,n bout 25 percent or so of tht chosen reicre-ncte sequence. Jibe preference ,equeric- -may he a sub::et of a la2rer seunm udh m~ a pc~ol! of a geno or fk&igequience, or -a tePetiuive poricn cf a cbxomosLone. 1-o-wever, i 15 the case of sequence homology off two or in~epoiynucleo-ida se~ences ; h xeern seq uence wril typically conprise P.' leas, about. 18-25 nucieotide,-, -mor.Ie ty-picrifly at lerast aboutLY .26 to 35 nuclecotidea, and oven-- moFe typic-ally at 4es bu 0, ),0 70f SO. 90, or even 1(09 or so nclectides. Desir:Lably, which. highly homologolvs ffagicaents a:ce desired, tae e~etof percent identit-y betwiemn the tw;o seqruce-s vwU, be at icast abont 80%, prefdibhy at 94)le4 aou 85%", andj., j-yiure prfrb about 90% or 95%vi ox higher, as rea;dily detemindb oine orT more Of !:he -,ueo Conrsnagrths.lu o those, :.f skill iz the art. such a.s,, eag., the PATAprogrm anialyis de-scribed by Pearson rmid-- Lipi - i (1988) ,k,~ usd ifrenn. "'X identity" rTefers Mo I'hepr~t of ijdentic;a amino) acids s2itu-ated at corm-spoinding ;wimno atnid residluf Ponilios inu a. sequwrico wien ,wo or more polyp~etide are 25 alin d their anue Oe'dila~~ using a ,,gje~ -3A.S a'l-orithmr.) (e. gU Ali5O'lSe at , .cAcic.hz- Res. :25:3389 1997)), wijchwig~~S equance gapsadseec imacs acc-ording to Uh default maif,-infigs prvddby the on 1Institc.,e offfakdalh NCTI data base (Bethesda. D Fze xeet>wnci.J;igo/g-n"MMiph 30 The toes-rm: "!subst-antially conlte~ay"We sdto defirjfe eithicr ami acid ornucleic acidse~Ience. nenn.- thn- a wrular ,suabj oct q~cfor 0 enple. ;n olio-scot seci-ence, SsubStantially. con-iplen acutary to afl of, a 'portion of, the ,.el:cted s~ec.adtu ,4 WIIspe.fCficalily 1b.no to a potioni of Si niR"TA -looing the. s e'kr-,J seq jr.f; lypicall the sequen.ces will be hbi ,hly emlnftryt hc rIRNA. "target" sequienc<, anid wti) hiive no more thian about .1, 2, 3, 4, 5, 6, 7, 3, 9, or '-"0 bamism-alohos througphcmt the corinry~oi'ionof the qno.In many insr~tnct&z it nmay be dwirr4bMe for the 5 smuccs to be xo rx!.! ta, i.e~, bID c aipetfy comuplemuracy "o tile seque .Qf to Awhi;R~h o~eoigouc~otC prcifieaiiy bind s, wtn thewei.bre hfj-.vo :?ejo rnismwhes 4. on~g the u~emntav-tth. -. ,, uhaly complimflthy se quenccs 'will tyqpicafly bind quite speifcalyto thn target sqec region of f t- iTCYA-a wid ill thece-fore be highly reflfcietil !I) tCf and/Or evena iiiihibiit. ttike trnsaio f the target irP~ e nceuto 1t, poiypqptki:e product. Sub.-iantiaull cornnlernentv.y oigo.w'ee quvzu;c- pI -~er than avout 80 Jperrant __cigcnu +.ioide spec i-fically binds, a 'mi wil moxe prferdlv be, grfeater (hhan about 8 percent complementary to the crre :Cuig iv uKLNA t4argetr s-,quienme i,- which h 15 otagon-uclotidz .fe~ita~binds. III we~il asec as ~ecbdabove, it will be desirable to .havs - evrm more substraltaily corPlementary oligoauelectideces in n;* i acfic;e of the ineiUcr nd if) such intnetecgncetd eune ~iibe greaiteri. than- - ou~t v(J percent rflne~ay~ h creonm nN arget seunce to wviKwh1 dith ignclftd specificaI~y bbnds, andjc mray in rmtaiim bi ' pf.tl iar about 2 0 95 perwc.l n~met to ffhe comrespoIfding rsRNA target seque-nur to whvfich tre oii~gnucleriri ( s~cificaJ~y b and evo'ri. vp to ancl including 96'1.,,97%1 93 . 99f , and efve~n 10.3% e;Xact -ac oeemarycf to all or a Ir"J~on1 o'f J:ir. target iriRNA to vhc tkie pt'rcit sinrilaity, or mper!2nhlou pe anf f Ay of the scloseMi sieqwinc- , may be 25 detoxnnined., ;6z example. by colipsming saeluenicc Iitormnri don Csu h AP conqpwutei ~:ogar. er~ir 10. a;'ai~ah fom Jthe tUni vercst' of Wic n( ~~isCompu'~ter O3r-)u (UWOCO. TheGAP~ progxrni. tutilizezs thep a~gs~rt elto. of -wdlmai d Wu!-sch (90.Briefly, thec GAII pogrant deins iitanity asC the- uibr o~f aligned symbols , 1nucle1otides Or amino acids). that air,: similar, 4--Iividtd by the toWa number of' vy-buls ir tr 3'0 shlorter of the tw o qu.~J ess. 'Iue frfereddfiult aamtr for 4he (SA proga neue 2.; a unay c1pais. ~alrix (ntinga valu f I for identites and 0fAx waon h entiis ~ ructetdesaud the eigt cipariion.umatri-x of ( iln4-,(v and 3urgcsi '1986i) (2) a poenafty of :30for 0eC.L gap f'nd -- a add-iioalC 1 pcrd o ic yibo nechsp (3) -no p naly f br end gaps. D EJTUEDJ1 DES~CRlITION (W T*r-i1- I'V,Nt. NTWN Tersetivnmis direcbted -- o now; nrci ,M myostatin antagoas activity 5 for uisen the U treattuenit o~y!itnrelartd disorders. ~3pec~iikaiy, 1ti pre n7tat invmnte~. --- E dhected to novel dr.iaLnegatives c" riyaqtatin campnpnsing mnnr'Yw Yyostfi'tm re cibinant .- eptjd,:s hav-i--g a fC-t .niai. -rancatifon rt a Position either- at or betwilemn a-nn~ aoid posins 2'?i. to 329 ;f SJ.iX ID NO . ra fagulntu, v-arint or- deiatv hereof I 0 he myorctafiln proton is initially tran'Sla t edC as a '375 amino ac-id prmm-~scr molecu-f: having v scrtr inisn~ tthecmruaoyic csin signal (RS.k cR~f fh ;.nri o~udprot 11=0 nilic cn rr cysteima resid-ucs in thoe C-cen-vinl tcgOan 4o faciltate tilt f6orman;AQ- Of a "stio t 4nur.Myoistafli is adtiva,.ted by furin edpoes clAmvag- ai- Arg 26A '14-~ri reN rr;:aOF i~nvnscaedp~:ie L andte 1. 5 iuazwre. C-tcx,-nt0a docmain, which- diinerisezQs to forj 4-hr: activfe n-:1iYost.atJn~ mcleoule. AfiRreoi~c!ng. ahrdinrf h A ptd aarinonoaedburito the0 hmm&od ie of t~'emvstdnin an igacltive' ColjipiefX (LCet tci aL 00)"te rtflS. fr exan'lr, folistti, Wn ap.(3~IT I. fflitatn elaedgene andl !-I"LT are alsoknow to bind to and ~ei~aethe sretion and tiaiof thie lwatent~ .myostaiin voMYp"ex ije andj- INoPherron, 20 2001; Nicc.as eft a], 22;hill ot al., 24,102; 1-111 C-t aL. F.03 Wnge al. 2003). The aino a.cid seqaenrc of fthe- htiuan myota6tin p-recursor proter-in rnoleci-e i.% sown inl SEQ L0i N-102. The cormesqpoudifig rncieoon - e qWmhcz Cflsxh.dm tlie ri-yoivta p-m, cursor, protein !5is v s Iw in S, Q ID "40:1. Prf.Mlovs aivo,,:atin a ooIsr s -Cnrsn n~emotn eit iswihae(.ri~~ 25 U.ri ta, at wvzino- r 6d Pos.i~u.n .30 ol, 3.5 are kioown (WO :1201)1/53:350J). Thsen;tagoniStz are trancated t, a. position so that lae-V tme art- -etained that are lieyto play anfi impotlntrole- in. de-enmniahlng thcThree dimensional struc.-im and ass-ociated nercins ti othe-r moteculfes, Losjs 017he koy vystoiune mesid-ces wou)!ld reoerieetd to negcativoly rnp-ac t on t -eir ab1ivto -bnction. For simple, -a C31 3Y ---- ubsl-tution in te rrm 5,(ti gene 3) G 11 car Jti( zu !ISe a C', -, fhnetOn rc-suuu ir th e P 1 r,, oioese p~i; tn O..r et ol 06) Suq~is.', it lia:; rbetmSO~ fo~r thm. trsl t."111 that naiijre myostalin peptids 0--tenminally tsi~ccd at pr .itons chose zo or ~cnigcystiine residues xne bIoiogica-Av active. The;e 2aovel penil dfs have myostatin anhIgowust ar;Uvify and are usoisjj irn the tredmer.. of m-~ostati.[j reAMe disor3ors It has been dotfrrninid that; a vosa peptide tfat w~js C-tv.-rninalty 5 lnaca ted at 2sosto 6-w tht criticaLly removd PE but one cystejc~a Ihrn the~r mysotatin vetde as Uut bicoxgicall ctv. h h avelor lm ow 2for tll- fi~t tizne thatA 1:4 least ,tWo Cyrtoineoese atv, requiven .na pqn~ae an riolrr ~ptidc in aider to re~tain 'biolog-ica acivity. Wihitbeing botind by theory, Wt. is cori.!;idre-ti llrat a peptd t ai only a singic uYstaVfle moiety will ulo' 'be. al Tr; fQJ.1-nn diiiatethree 'isronal 10 strkfushe xrquired friolgi acti-vity. Rt is thought that n obnarv rodsc C temmiatly unneare0 mycat'.iv ok cp ' R-Jr iw~to ii into a xibrodfhru acti-ve auxo. imacjive oforaioa fns The extaot fo~oraiia brof.T C-11 r i~icicsi E7ctivc p~eptidies is not known, Atempis by ihe nertr to ipukf hti verajons of'th recnombin~ant iperjtide cif thce inveution. px(rr xde only inactive ao;:m sfrult oiVr).. Rt is tScxnsidered thaz, for biological1 activi tombin, . tly produced poeptie ar; ntec~ssary, 1711)s, Ttle p.resenit inv1"enticon provide", fbifa iso-ated utec""binant poJvypoj.t64 I - ; - iravn mytai ati~nitaciitcols'prising a rxer~nlytucte auemottnpptide" wvhetei t-:c C-tria truncation is at a pciinat or bct-wcen a mino S 8 a~d 329 of SM 11) WkNO 2, or a ,r,7tvnat~ d,v w-e ijwreof 20 The isolated recorib in-Antp~ppie of the inmaamo. niay In, sreiecti Irain the group constng of a C-terrrinvallv -uuucte natcre mvraostatin pkip~firdn wMiacrrejinte C-~rnra irancation is ait amaino acid position 28L 282, 283, 284, 285, 27F6, 207, 28?8, 2P, 2)0, .291, 2R2.2913 294, 29 5, 296,n297, 29, 29, 3043 :301.302,303,34 05 0,30,38 39 3 311, 312, 311,~ rH , 3-3 3i6, :311, 310, 319, 320, 32. :322. 323r 3,32!;, J26, 327,328 or z.5 .129 of SEr- Q TD) NIO 2 oa ragpmnt, variant or dotivative the-reoCA. Prfrably thte isolated. reo-n bhma.t O po'-,ptidc, of w.e uvention is, seee--ted fr-om th g ir conssng of a C -emially tummed iatre rnyostatm P'l eWI) e whelrein the G-terminai tfvnfcatiotn is at am11ino aci,'d poiin329, 320, 310,30029-5, 289, 2S4, 262 ()r .281 (Si50i NOS: 34i 11 or a frgenvari~rjt or Wcivalve tcirot~ oa a. nolypfcptid~ -b~ 1.1tnta 303 fequencc. homiology t-hereto.

M 7efeer"aly tChe Isl - exrimhjrjatit polypeptide of tJ.- e ivr.. is s-,eed ftomita -- roil, covsb-tir!g ofaC ? tninally t cete marine *'iuos!ttn pIolypo'-ntide, where in( the C tennal tru mai is al amico acid position $,3 .0 oT 3 00 N-S I -Os.-, or 6) r1he polype-ptides of thbis Invention can be altmred in many -ways to- ptod ace variants or 5 deriveivea hevng V.o"Immoved rjai~kntcas wudbe rrspprcciated by m. Akiled -. ,orker eorzmxple, fuinctoal groups" my be taddeta ~ polnAiy and/cor the. ability. tol fR)IM bcd ingn boJ.d amd wilUl althe ii bhi of t~Opippie.Sni n functions. group may alter testabiiity 1by changing fl-i srrn ralf-]Ifi3 cW"'i 01 , 2006) or by rini. firef r-clea:se of thae polvvp(:Vidf i frm a Tnioctlk. at the Fw~tSt. Prther a;- n--cto.Al gro-i may 10 Ll.T bicornapatibility, for iexpump.ie by mrinimijz;Ing 1he side cffect-5 of Che polypeptide to the patient- Addi-tioD oF . filnecns group cp c.", 01 Llindkigf to uiar:I cellso Or tissues- or Eac1itaticg -lie trnm tiznto the target cxfs wil-.l enhanpuce. delivery and tigetr Of t"he polyppt-4a'. It Is also understood that. the -pipiidej may' be tvaneated fromr thi-e NJ -Itenrinal to. improveperc~~tS 1-1 tim group T0 a poiypeprici Lti.Iinv1 J. tln.X ny t a b-olgicl 110enl ta bin--d-- to a Specific Niologica-1 l ~b~itnce of :lite, . 'hc biological J sac rst is Lb itede -get of he. dolive:Y nd" ta-rg I n'o moicule, tliat bInuds lo At, miabiiig tIme dlivrY of 017heJ VfcrnnrQUrid. toV) tissue o; elsOJl inicrest. Ligand mnay -function as a biologeil targets g --violecue by selctvely binding Or having F. 2 ;0 s~cific affinit-y for another- oubstane, A liadis -!e'opgnized an-d bound.l by a speuific -'iding body or binding p ater, Or receptor. EXamIPle s Of ligKandO c'tal targetingg ur arn~juenis, 'Imptensr" v, bioti'rivtvs etis -ail-acosammiro.e and f~oyanremlns -cpos stSrates, oezmsarnd oftosaniong othetrs-. COther s-ubsta.nces that cm. fiviction as Hpganda' for delI:erY anld tageting art eti tris mot~~ 25 curbohy.drates. ipo.Certain viroteiris Or proteVin fragments (e.hon nrls. ItOxins), ad with sclec-cive aifinty ibi ligator int are r- du,~ through recombinant DINA, gf-netic -nrt moulecul~axr enneerngp. Ane tPe of target'Ing sneeeis en antibody, hichtem is 'Used herein toD Ilud2Ce all 30 classeS % of antibodies, rsoicoa ibodien, C-hilmned atibodies, Fab.' 4inrs, fragmmet enrzymes such as neunainidatlses, plasma protei, avidins, streptavidins, chalones, cavitsand s, trgobr, inrni facter, globuiliusihv ros surfactants, orgaomtlls'ubstanzcs staphylococcal prmteit A, protein G, cyrochromes, kltins, certain resins, and organic polymers. Targethig molecules may include peptides, including proteins, protein fragments 5 or polypeptides wlich may be produced synthetcaliy or through recombinant techniques known in he air. Examples of jpeptides mclude membnse transfer proteins which could iacilitare the transfer of the compound to a target cell interia or for nuceear translocation (see: WO) 01/15511). Otr ioditcadons to the poypepudes of' the invention include conjugates tc, a biologically 0 comnpaible polymer such as poIlyedlyne glycol (PEG) and related polyr derivatives, Drug-PEG connjgates have been. described as proving the circulation time (prolong smum half-life) before hydrolytic breakdown of the conjugte and subsequent retlasc of the bound ;:;mokecuie thus increasing the udgs efOicacy. For example, US 6214966 describes ihe use of PEG and. related polymer derivatives to conjuigate to drugs such as rotei , enzynesi and 15 'small molecurles to tiprove the soiuoty and to facilitate control release of he drug. Altesmiely, EP 1082105 (WO 99/59548) describes the use of biodegradaie polyester polymrJWs as a drug drlivexy system to facilitate cntoled release of the conjugated drug. The novel polypeptides of the present invention may also be modified in a way to trm 0himerIo zmoleculos compris~ing the novel polypeptide fused to another, heterogoi 20 polypeptice or amino acid secuence. It one emhodiment, such a chimeric molecule comprises a fusion of' a polypeptide of the invention with a tag poypeptide which provides an epitope to which an anti-tag anybody can selectively bind. The epitope tag is generally placed at the ain-te-or caroxy-tenuinus of tie novel polypeptide. Abo, provision of the epitope ttag enAbles the tagged polypeptide to be readily pirilled by affinity purification using 25 an ari-tag antibody or another type o' affuity matrix that binds to the epitope tag. in an alhermative embWJodUIme-Il, the chumeric Imolecule may comprise a fusion of a novel polypeptide of the invention with aF hmmmogiobulin or a particular 0iAo of an inunnglobuli. For a bivalent forn of the chim.eric molecule a fusion could be to the Fe region of an IgG mtolecule, 30 V lious tag polypept'ides and their respective antibodi; are well kow ini the art. Examples include poy-Thstidine (poly-his) or poly-hisidine-glyrdne (:poyhi-giy) tags; the flu HA tag polypeptide-and its antitbody 12CA5 (Field <t al, '988); the C-myc tag andt 8F9, 3Cy, 39 6E10, G4, B7 and 910 antibodies thereto (Even et aL. 1985); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., 1990). Other tag poiypepsidcs include the Fligpepide (Hopp et at, 1988); the KT3 pitope pepcde (Marlin et i,, 1992); tabulin epitope peptide (Skinner CI at, 1991); and the T7 gene 10 protein peptide tag (Lutz 5 Freyemut et ai., 1990). A another atrnativa a polypeptide of this iventon may be conjugated to another phannaceutically active cempouim to enhance the therapeutic effect on the target cell or tissue by delivering a second compound with a similar mycttatin antagonistic effect or a diiferent aursvity altogether. For exarriple, US 6,051,576 decribes the use of co-dmg fomuwlations by 10 conjugating two or more agents via a ie -nkag to improve itE pharmaceutical and phannacciogical properties of pharmacologically actve compounds. For example, a second myostain antagonst may be selected frm any one or more known myostatin inhibitors. For examp 3 US 6096506 and US 6461535 disclose anti-myostatin antibodies. US 6369201 and WO 01/05820 reach ;myostatn peptide iauunogens, muyostatunmuitcners and myrastatin 15 imunoconugates LcOpabl of eltinug a. imune response and blocking m5yostardut1 afTAfity. Protein inhbiotors of mycstai are disclosed in VO 02/085306, 'whicf include the truncated Actn ivin type: receptor, the mnyostatin pro-domrini, and folistaiu, Other myostatinhibitors derived kLomr the iynostatin pepti de aenw and in4lude for example myostatin inhibitors tht are rclefed into culture from cells overexpressing myostatiu (WO 00/43781); dominant 20 negatives of myostainu (WO 01/53350), which include the Piedlmontese allle (cysteine at position 313 is replaced with a tyrosine) and matJue myostatin pepides having a truncation at a pos11n either at or between amino acd position 330 to 375, US2004/0281033 also teaches small peptides coup;ising die amino acid se..'nce WN4CPP, and which are capable of binding to and inhibiting myostatin. 25 A second pharmnacologically active compound having dernt activity to the myostatin antagonist polypcptide of the invention may be used conjointly with the polypeptid- of the nvention to treat the myostatin related dhoniers, For example, the polypeptde ray be vsministed. 1i conjuncion with polypeptide gro wvh factors, NSAIDs or CC)X-2 inhibitors, alpha and bet blockers, ACE inhibitors, bisphosphonatcs, oetrogcn receptor modulators 30 antibhypertensve agems, ghtamnate antagonists, insIN, atibiotics, protein kinase C inhibitors or various over the counter substances as would be aporeciated by a skilled worker.

20 Other modiications to improve stability and half life include the identjfication of susceptible amino acid protesse cleavage sites wiofn the polypeptides of the invention, and replacement of such amine acids with alternative mino acids to prevent protease degradation of the polypeptide in plasma, in vivo. A person skilled in the art will arpreciate what type of 5 fictional. groups might be added to achieve the desired result in rd-inister the polypeptide to the patient and thereby improving thje overall therapeutic index. The present njventio n. iurther directed to anriJogs, derivation and vrwhints of the polypeptides of the invention having rmyostars n uetics activity. .As;ngs, derivatives or variants of the pepdes of the invention may include sequence 10 rmordjificaLions or non-sequence modifications. Non-sequce mo1difications can include acetylation. methylation, phosphomnethyation, carboxilation or glycosylation as described abov~e. TEe specitc recombinantly produced 4erinaly4tcated polypeptidcs exemplified in the present invendtson are shown i r.eiaionl to :Jidr plosiionX onl the1 C-erniat portion of 15 myostatin of SEQ ID NO: 2. Preferred aalrogs include peptides whose sequence differs from those of the invention by one or more conserative amino acid substitutions, deletions or inserinons which do not aflet the biological activity of the peptide. Conservative substitutins typically include the s;ustitution of one amino acid for another vntsiilar chag.'teristic, e substituflons within the 20 following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartici acid, glutamic acid; asparagine, giutamine; shrine., threonine; lysine axgine; and phenylalaue. tyrosine, examples of conservative substitutions can be takn from Table 1 below. TABLE 1 CONSERVATIVE AMINO ACD RPPLACEMENTS 25 - - -------------- -------------- --- -- - -------- - ---- ---- For Amino Acid Code Replace with Any of --------------- - ------------- ------- --------- ------ ----- - -- Alanine A D Ala Gly, ltA, IL.-Cys. D-Cys - Arg - nR Do----------- D---------------------------------- Argnin R l~w Ls, D--Lys, hol-o Axg, D-.homr. Mee D-1 ------------~------ --------- ----- --------- I---------- - ---------------------------- For Amutititi Acid Cute t -. ja.e wib an~y (if ioragic~ N j sD-AUT3, (Itl, D-40Ghi. (--n, f)-GhI Aspattic) Acd D-Asp.D-Asn-,Asn. Gbf, D-OlGu., (~li, D-.Gln e ct~r D-Cys. TN~-Ci, Met, D-Nlet. -!k, A-h G-11Ii!.-tine Q. EJ-Gix-'T11 A.,,n, D,.A.&n. O. D-0113, Asp, D-.Asp G-I~tmF Ici D-Giu. il-Asp, A-sp, Asn3,u, D-Asn, G-ii, D-Glm Glyini: G A-Lyt, D. A.xDjP, bet.ax, - anoAg Met I -- - - -- - - -- A t -- ,-----------.Or M~rthisyiric PA D- 11e. Val,- ~cy,, Lok, D-ILu D-le'. al -'a 01 el5WJ I Fiue I A-be Tyr D-*Tv LDpfiD-Hs f-.D L D.-So, V,,'.' D-Vh.aIJ-i, Me.D.-MP -- -- J) - 4 e ( ) -- ----- --- ------- --- ------- -------- ---- --- ----- ----------- ---- I---- - ---- S r ~ r~ , Y ct - A t.D I~( } Ty~osiri D-Tr brI-hLDp.1i.D-i O~Jr jJic~ icldepei'i~ nh m~ifcaiins hih sxh '.c pptde tJr.ty -t i~ g a ------ n -fr -------- -~k -- ------ -- -- --- ------- -n n ----- ---------------' -e la e h~ e t b~~.rid in t- ---- i-- PVT~ce Ils D-Mcte S-NkCy iuligs that D Incluc D-esues al er th ;- -- - c u ----- -----~ ------ .cid., -------- ---- ----- n- -c d ---- -o - a u a l -c a -n ~ ~ t e ~ am'.i acd~,~ bea ~'r ~m-a antnoacis ad cylicanrogs 'ThP T11esen3t iWI0IntionF fLrther conT ja~sNtri truncationrs of polie fy~i des o't th in:Itkon. 9116h vasiant.. -%-"11 comphSce lthe -. tnial uvc ted volypeptides of the pren tnviin mrbv tJhe arloacifd, ftc'r theiv~ ni elnd mce seq ufmtiaLiyr:Loe ane, wher-eby such.' an N-terminally trnonated petd e~u ysai naoitcactiviy. .T 'f h pvfsefa irivenu 'oll aso nencpae~r.uziei- c rJ steews encAm th noe Recent sr'uies s.ugges't that myostatriu is I pogeri re.' of feli cycle -pwogresision and tucin npart by -eegiting 'both 'fhe p-'rolifetaticun and su-ferentiatic'n ste ps Of rnyugenesis (Lanugley v, L-1, 2.002; Thomas rd. al., 2.000),, Severl studies hawe dteon-!trated C. role for 1.0V myo--;M-fi) njot o~nly during emibryonic rnyogc--nfsis. but also i -po-stnatal rusaile cwh il sby Weh-iug et al (V'Veli:h.g f~t 91., 2000) and Carl son. et ?at ('CarI10on et al., 1999) indicated that at-Iop-etd 1nsl los d-ur tc. hind limb suspension. in. mice. was socae withinceasd mystain e~ll JinJ P14mWKSri., huureastd In-yostiuv kneves were as asseiaet dhse'~e uele astngsee'ni i:1 131Y yptients-i (Gnalz2dai t al.,i9I) -450 Oexpianwimo for-z the e'tj evel-s off 11kyosta-ia obsere. during nuscle dirnse is iliar 1ryt tirn ay lfinctiori as an intbtrof.satellIite cell activation. indeed this !i supported ty reetstudies : WWichT. ";how "tta Jack of myos tatn. tesuhs h6 an increased pooi of acti-vated teflte cin v~ivr) vad enhifanced f.fmenw - f s--atelite cel-ls Qnk-Croskery m. al., 2003). Mysain hibitucts n-ave also beenx shocwn to r--c1rma~:e thie act"iaton of' stv-i-:te cells, iswel ~20 as tv iucrease-, tfli migration of macrophages -Lurl -A-ycjblvsts durng ulee.'.o (W0060i3 t 2) and in -woun-d heflin-zW20103I~3 Methods smAnle fAr asayngJr ywosfa n antagonists auti viMy of the polypeptideo of the present invenion my he b~awd oan any of a variety oj. f linmdooogn nldn knomvn in vivo Tafifal.moels r'r ip" vitromdes For example, £xrint tat n^xyrc-statin atagonrists cfflhe mvvt.u'J( non may first be tes-ted vusinug ran in vitro finlfbre sat lute c~11 activation agsas. 1r;:yobIP-sts proliferation assays! bioassay (WVO 2093/'00120i) or mycblas.'t Tnrk---h,-jrg ni gration says, as de 1ibdbelowk". Myc.--adti antagon'ist picyly pepfides of --he inzionwat ase able to bucrease F'ad a ca-I activate ci, Yn'obcat" profil'bration SAMdo myobiast or rracopag igatxm. nvi~r rc.JFI tayn, be t-ersted for -Jmir ability ;o treat lxxost-:t-in related 301 disodets in armi-ial modelIs in VIVO. Sun m--ociels inclidv anK aged muemocdel of sarepei (K'irek. 200): a mouse model o-F musculr dlystrophry ")Mdx) (ranabe et fi, 1 9&-'';. - mu moael of ibetes (IMket 1ia, -976); a mn uew mddl of ( besity (.0rididharan it fal I 95Sa) 23 n~oex~ moel jfr~sk~ njuy Khk? 20W), a model ryf sprfIaor dkep skin woi-ands U.,btz e al, 20101) )i auOdel of bumns, (Yang et aJl, 2005)j a uvnous-- *2chrz.la 7mocteI wheme m~~eis illjected into mnice -. o incluc mmuscie 1wastdig (M/a ct al, 200-3) fraous crc- mo1del- in whic&i co ton amoacfOh (2)clis Or L ~ug oircit;ionaya (LL,-C) 5 CCUS SI nto MicC tW induce mumcic w~asting assuciatwd withl culner. The oajdpciypeptklesu of the i-nventionm prdetably bind to lirtal-re wit'a a Kd. ulf I jiMl Or ie~ r~~iIo-e ef~ibvwith - 1 o 1001) a, 10 r;im 01r evfun _. uld or less. Tho pzcesent in-veonmi also dj~tdto a pharmaceautiical co~mposition comfpri-mg at least oxne novel pulypepidh of' the inyuntion haing MIYOSr~an anaoi~eactiviy moethi vdirha a I ~ liar'.aetiicii orphyioioical~y tsb.('auier. A ~ ~~(J pv~~~e fti nention~ o-- salt the;;o mnay be included in ri~ oefi~ul snccetahk carcicr o~r dienimt, ideailly, in anj fmcr±onnt suqffcioert to ddiiveyi to a -patien~r a the.1uteniy l.ve arriourit without c,±sim.-ug srirous lkoxth; fects min the atient tefd. Tl on uraio of wAtive pobypept ie ill tile compositions W.I'l depend or b rr ~. 15dj'ibution, iatvtc.and cxcr-,et.ionf rates of4 the~ polypqritid as well 8.5 other .factors 1,nown to ruo.se of Akillink tlhe art, It is to bet noted thatw. dosage valuors wLito also vxry with the sov(erity of ~~recondition toj ber aulevied. ft is to be furtiie. underto-.od that for OnN. particular subject, spouific dosage 3ein'nS.ould 'eadjustc-d ovrtime accordingr to te -irndividiual nee~d WAd the 1 oi~siordjudgcnniicixt of the person. acmnsen rsimcarisinrg the: adminis-tPrion of, 2.0 tie coios.;tions. Fxipesof'the, technI:-1ucs and protocois mnentiouced above :can be ibunri in In ri nq:o L . 0k 'ha.ttcei1.jC-t Ic S .1 ,cines i6-5 e diti on1, 0 slIo,. A. (--d) 19 SO ,Solutions or spenosions used for uxtnintsadetmml, sub u-cn-qcous, Or topical. Lp.PliCaion muay VncuS NOe following eompnucnts: atei diflealt .nmch1 as fotr t:ieto. ~n sohuimn, flxed oiN~, polyethylene glycok , lycerine, prCor.0-ene gI ycol of o-t-her -miex 5 -xjlvents; atbcrilagents such as benmyl 61lcho. or ine.'j pa 41ribes "'atoiatsi.-cika -- swrbir; acid or soin iuft~chrelating a genii- suchi ashmdrneer~r acid; buffers. sufch as aeas.citrates: r pohar and nigents for tli-ic a(djus-tment of toxicit.y such. as somn1 ci~o'de oz etioe Stable pIarnceutiuAily ficptal catres fr -r ian-wral. application, such az intravenou-5 :30 uctjjos ritr sl a injection, Include szeril-w water. Iphy silogical, sadije, bacter~o-jtan 'fije slieC011Itum1ing 0.9 Lu bd en.-Y1 PAichol) wrlf.vropiiehfec 24 saline. If administered lntravenously, referred carriers are physiological saline or hospohate bmffrede sanna. Methods for preparing r fo;.adations ;ncuding topical frnulations or transdrmai delivery devices such as patches are known to those skilled in the ai For eampile, see 5 Brown L., and Langer R., Trannra Delivey of Drugs (1988) Annual Review of Medicine, 39:22l1-229. Polypeptides of this invention may be prepared with carriers that will protect thi polypeptide agaist raid elirnmaton fram the body, such as through controlled release formuations, including implant> and~ microericapsulated dejivey ysry systems. Biodegradable, biocomptibie 10 polymers can be used, suc as ethylene vil acetate, poiyanhydrdes, plygiycolic acid, collagen, polyorthcesters. aw polylactic asai Linosomal suspensions are also suitable carriers fo the polypeptides of this invention. The polypeptides may bo (cotnjugated to a lipid by known methods for incorporation into a liposonm. envelopf or the~ compounds may te encapsulated inftc the Jiposome. Liposomes .5 ay be prepared according to metods known to those skilled i te art, such as is described in E3. 4,522L . Fr ez ople, Jpusome SauIOnS may -be prepared. by dissolving appropriate lipid(s) (such as steIryl phosphatidyl othanoihuiue staroyl phosphatidy choline, arachadoyl phosphaidiy chorine, and caokesterol in n. inorganic solvent that is then evapmorated leaving behind a thin film of dried lipid on the surface of the container. An 20 aqueous solution of the active polypeptde of the irvention or its monopuosphate, andnr triphosphate denvamves are then introduced into the contained. The container is Thmen swrld. ib aud to t3:ee the lipid aggregatres, thereby forming the liposomal suspension. Ffor nasal or pulmonary administration, the active ingredients will be in the fonn of a fine powder or a solution or suspension sitaJl for inhalation. Alternatively, the active 25 ilngredievts may be ;n a LOiXn suitable kfrdirect appication to the nasal nucosa such as an oitinent or cream, nasal spray, nasai drops or an aeroso:. Oral copositionf s may include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or coimpresed into tablet. For the pupose of oral herapeulic adiministation, the active compod cn be inoporate with excipi ud in te fon 30 of tablets, troches, or capsules. Phannaceuatically compatible binding agents, and/or adjurmt materisis cwn be included as part of the compiion. Methods tot encapsulating ~ostius(mchas in, coating rjE hwxl gelatin.) for oral adn-istration are well ,ujo;wn~ in the art Baer Riohar--d, f-'ovrolkd Rekase of!higci 4t gent&, John 'Wiloy ;n.d Sops. '1 986), Tecnniqc-.es to ove.tcvume vfir.iou u.' hiers Ch n1uei s-layer barrier, the enymatic brier and the awm~orn barrier Pare el .irwn in the ':0. (JBernikc,..ch6rurch e IS, ei.ps Caps-ades, 'asoc ±es kaid the like can-! coutair. andy of aie "lo I Crng ingxedi.enr.st. or.......... eorioin~~sOf a simrjiar rjrluxe: a biun'din ;uch srcrcvtai cellulose, gem-, tragpoanth or gelati.n; anl excipienit r-ud a.- -3tarih or lactose,, a ij -Integrafln.g agent -tiiau w. igin-io. acid, o crjr.u starch; a inubpicant such ais zua~grizsi'USj1 Acaratf;; a "giudant fSUcJI m&~ COJIoi(aj siho. 10 - d-de" a agn!wc a scrs or smecha.rii ,!r a fla-orn agent sixt as rnmethyl saheifYlate, ox i-nge tVhvouing. N~hizu the dosage unit fw rrii a wpsride, it fcaf couitain. ii,. addition 't) m-ratertial of tU;c above t~rype a lquid ciryrior suhas a. iatrry oI n acdditjon duoag imit i'onns eari. oot~xVa.iOL us other lwwatanar .hc .ndt .. ... ...... . Worf tIJO dosage unit for exanple, cotvoagls of sl hellac, o-. mother enteic agents. 5 AJw.terati ;'ly. pcJlypqwtides or this havention cwfld N:' a xnaiFdasaonun of ari vlix'ir, fruspeIsion. yr wafers Che'1-Vy.n guml or the like-. A S'm p may crrtain. in addition to 0".w active co pu~s ucrose as a rsweetelin hgeor. andl",i presex vntlve-S., dye and The jp!resen, invention. is ditecteod to a ietho~d of treafling, a YK'cs',t-. reflad.pdo'go 20 Condition in a. 'rnamnad. whereinl dhe m-ethoid grcaycrssat leasr thte stop of a.l-rfin..- t mllg to a rna'-uuvaj. ii ileedo thereoft an efffcri ye wr-.untw" of at least one potyp aptide of the invention having myc"'L9.61. arirtagonisti ctiiy krx a time .fietto ]pre;'ent. treat or '7rliorate 'he 55yMptomsL- of said inyostatin redated pathological ecknrli'don. hI prfi Le .... odiin'erit the' mammal is a himaz thet has, is usetdof havi1, or has been diagnosed 25T .vith one or more mycstatin. related pathol ogicai con-LRIons. Th~e- Pathol--ogic C-Oditiov is chrceieat least mn part, by atai omlamu devel(ien ortabt!'-- actiit of muscle or adino tissue ia a mnaUMna arid in.2y Inludo disorders related to moci ijfxn7ropM-; ', muscl atxoohpy tvl-d rnuilsoe AN.risting soitdwt infhumr..tory myoAtie s mata dy; 5troplies,motor neuro;2 cdxi ease ac of ne 30G eroas a junetio-n, disea:,s of 11he ]DWipheraI nerlVE, rvoatisdue to endocrine abnomlio.tlie's' riae!tboflcO .sndrone M!T ,Canwer, saricLpeni.- cacfhexixa anidotr wasting 26 conditions; cardiac faibuNe; osteoporosis: renal faihwe or disease; liver failure or disease; anorexia; obessty; diabetes; and wound healing. hIlammatory myopatdics that can he treated include: Dermatomynsitis (PM.t'DM), inchin Body Myositis (IMM) and Poiymyositis (PMJDM). 5 Muscular dystrophes that cass be treate-d include: Becker Muscular Dytrphy (BMD) Coungenital Muscular Dystrophy (CMDP), Distal Muscular Dystrophy (DD), Duchenne Muscular Dystrophy (DMD), Emery-Dreifuss Muscdar Dystrophy (EDMD), Limb-Girdle Muscular Fariloscap'uilhuerrii MgsculariDyL'trophy (FSHi or FSKD), Dystrophy (LGMD), Myotonie Dystrophy (MMD) and Oculopharyjgeal Muscular Dystrophy (OPMD) 10 Motor neuron diseases that can be trealedA includr: Adult Sphina Muscular Atrophy (SMA), Amyotrophic Lateral Sclerosis (PLS), Infantile Progressive Spinal Muscular Atrophy (SM SMAl or WH), iatermediate Spinal Musculhr Atrophy (SMA or SMA.2), Juveinile Spinal Muscular Atrophy (SMA, SMAJ or KW) and Spinal Buibar Muscular Atrophy (SBMA). Diseases of the neuromuscular juncion that can be treated include: Congenita. Myasthesic 1.5 Syndrome (CMS), Laber-tnaon Syndrome (LES) and Myacthenia Gravis (MG). Diseases of peripheral nerve that can be treaedinluce: Charcot-Maienoc Disease (CMT), Dejerine--Sottas Disease (1S), and Priedreich's Ataia (FA). Mypth- duea tndbieties that can be treated include: Hyprthyr.oid MyNopathy (f T M)7 , and Hypothyroid Myopaty (H YP'OTM). 20 Other yrptIhJes that can be treated include: Central Core Disease (CCD), Myotonia C ongenita (MC),. Nemalin Myopathy (NM). Myntu MyopaMyopathy (MITM or MM), Pauyotnia Congenita (PC and Periodic oaraysis (PP Metaboli: diseasers of muscle that can be treated include: Acid Maltase Defiiency (AMD), Carnitine Deicierncy (CD), Camitine Palmityl 'Transferase Defiiency (CPT). Debrancher 25 E;xyme Defhiicincy (DD)), diabetes, Laciate Db ydrogenaae Danciency (LDI-), Myoadenylate Deaminase Deficiency (MAD) Mitochondrial Myopatby (MITO), obesity JPhospshotylase D)eficiency (MPi) or PYGM), Phosphofructoinase Deficiency (PFKM), Phosphogiyccrate Kinase Defici eny (PGK) and Phosphoglycerate Matase Dtfncienac y orPGAMM), 27 The polypeplide of the irrvention can also be used Rit treatig or preventing congestive heart failure; tor reducing frailty associated wth aging; for increasIng bore density (such as for maing osteoporosis) or acceletraing bone fracture repair; for treating growth retardation, for the treatment of physiological short stature, for attenating protein catabock response such a 5 after a major operation; for reducing protein loss due to chrorfe illness; ibr accelerating wound healing; for acceleraing the recovery of b'um patients or patiems having uidergone major surgerv; for maintenance of skin thickness; for maintaining metabolic hoaeostasis, fbi treating renal iaihue/dsease and liver ilure/disease; for treating growth hormone deicien adul-s and for preventing catabolic side effects of glucocorti coids; and for treating a number 10 of neuroi.al system disease conditions, including CNS injuries/'disease such as Spinal cord injury ant stroke, and PNS inj uries/diseases. These disorders canrbe treated by administering a therapeutic amount of one or more ;miyostatin antagoist to a subject ins need thereof. ,1n a further embodsment, the. invention contemplates the use of one or more rnuscle groww. } factors wich may be co- inistered with the pharmaceutical compositions of the present invention to g Ve ani additive or synergistic effect to the treatment regime. Such growth factors may be selected from the grdup consisting of HGF. FGF, I, MOF. growth hormone etc, Such growth actors may be administered either separately, sequentialy or simu~taneosly with the pharmazeuticai compositions ccmnpris'ing at least one polypeptide of 20 the invenion having myostalin antagonist activity, In a further embodiment, the invention contenrates the use of a second pharmacologically active compound having different activity to the myostau antagonist polypepti of -th inventIOn to be used consjori ny -with the polypeptide of the invention to treat the nmyostatin related disorde.s. For exarumpile, the polypeptide may be adcdiistered in conjunCtion with 25 active compounds selecred fiom the group consisting of polypeptide growth factors (as mentioned above), NSAIDs or COX-2 inhibitors, alpha arid beta blockers, ACE inhibitors, b'isphosphonates, oestrogena receptor modulatoxs, .undhpertensivie agents, gitamnate antagonists, insulin, antibiotis, protein kinase C inhibitors or various over the counter substances as would be appreciate. by a skilled worker. Such active compounds may be 30 administered either separttely, sequentially or simultaneously with the at. least one poiypeptidc of the invention having myostatin antagonist activity.

28 The present invemion also direted to the use of one or mornsmostatin inhibitors in manufacture of a merdicament fo treating yosaWn. reJhted pathological conditions in a. patient in need thereof. Thie one or more rayestatin antagonists may be selected .fiom the group of myostatin antagonists described above. 5 'Th medicament may be formulated for loc or systemc ainita. Far example, the mdicamn-tmay be torrmulated iRb eetion directly into a muscle, or may be fbnmuated for oral adrunistration ior systemic delivery to the nms'cle for the treatment of muscle wasting condition. The mIedicarnent may be forrulated for topical adnniiijstration fo. the tmatmeni.t of wound healing, and may be formula R for oral adniurstr aron or the treatment of obesity 1.0 anid diabetes. The mediCam-ent may' fthtier comprise one or. more additional umAcle gruwvh promoting corvpounds to give an additive or synergistic effect to the treatment of muscle wasting ucOUditionls or Ior Jucreasitng muscle massV. 'Tc medicamet may we tormulated tor seurate, sequenial or siutaneois administnadon. of the one or m:iore myostatin antagonists and the 15 Ocnet or mvie muscle growth promoting compounds. Without being bound by theory, it is thought that tie novel poiypeptidcs of the present invention, which have myostatn antagonistic activity, are effective at preventing Or rmyostatin retired disorders n part via three mechanisms. Firstly by inducing satellite cell activ'ation, proliferation and dirfferntiation Satellite cell are the muscle stemn cels and are 20 thus involved n. muscle tissue regeneration. Secondly, by enhancing myoblat proliferation and migration to the site of regeneration, and thirdly by enhancing macrophage recmitment. It is known that nacroph ages act tO attract myoblasts and thus increase miyogenesis. The effect on macrophnge recruitment has previously been observed with other myostatin artagonists to result in improved wound healing (WO2006/083182). Thus, the present 25 invention should ad so be effective at improving wound healing. We show for the first time that novel myostaiIn inhibitors, comprising a Ctt'ninally truncated mature myostatin peptide, wherein the C-termnal IrnMcation is pror to amine acid 329, are useful. in treating Tyostarj related disorders. In aged-related ;odels of sarcopenia, the myostatin antagonists of te invention were effeecve riot ny at improving muscle mass 30 and strengtI but also at redm'ing sarcopeMa related fat depositions. These trmcajlts were surpnsing given that the C-ternmxinal trincation in these peptides was close to or excluded a 29 cys-eirne resting in the dingpdpurn ol the three duirc~nsioziai striuture;, a kcaturf 1rholight -,c be esserT.tijaj for biologierl aetivity (jearaplorg et -- ml, 201 ) This lravention umaiy also be sid Wma11 U3 wnAMs~ in Wh pas.iS, eikmeutcls andii -Fokare3 -refrxred to or i die atefd ij the spedhf~coo of tbe n)Pyie ation. i-ndIviually cc. Wavel~~y, am! any QT 5 l b to; o." -ILY two or re-c sid pars, events or &utread whero spt!cific, -ce3are. mcnriconed. here-i hx have kno,-wn erquivallints ian at to, which this invention relates, rjch known nquillalert,& art- drecmeri. to bo incorporated hecrcin as if ind,ix'dnily,. 1 0 Th tbh wn xanipi.s w-te itucluded t. ondaeri'e bohrtso ICivro;, I' sluouki be 1p~prqcjflved by thoset, of' skifl ini thle art thlat -e!te ,slsd t ~amres h~c folowreprsen t ruiue~discovered lby the Inlventor to hrc .v' ell ill die practice of th -- i-nVention, adx- th ai be cffnsidereull to institute, -re6ed moe rji r 13hactive. 1-jov'ever, -. --;e of sj-ili bn Wie ar , should, Tlit light of the present 15 appr-ciate that inamy changer, c:= be- made if) ru suc 'Lloz~is~ r isclosedI and stil, obtain a like or smiaesuit without ep Rtig~om the spirit wnd scoper ofi the Ex~nip~e E ~yotain tgo ists creme myokt proiferatio. in tr 20 EiXprescss. a ad Plwif eatfo Cf myosUALin antogonists A. 6DN c ore3-)n rd ing to t Te 267 -3 29; 2 6 7-0;, 2,.5-J- 310; 2-57 - 3 00; a vd 2 (f7- 2,as a ni. o ac~dvi of bovine myostatir. fequenece, lira~~ eerdto asrrottnacaot32.17, 320, 3D)0, 300, ad -l80 1%):espeotivelv.wsyuiyP! anhfe adc-loue into a pE'r 6.1 YvTo u~4rmg Etuxlaic.Fpress3ion and purificationt ofn-qostatin -:ntea~onist:- 329.. 320, 25 310, 300, ard 280 wats done according to Temwanv turr's Qgeprtcluerniv fconditions. yieldiyig pegtides With On .N, terilinlal ft m, H HE 1. 1 H:~HE E3 I; Of H I k~ am 3, li 3 ) kull C teurinal ag (E~ D P A A 11 K A RiRJ-i A . , AA A T A E ~ 30, Myohlast assay Bovine C2C12 myoblasts were grown in Dulbecco's modified eagle mediurm. accord to e standard techniques (Thomas et at, 2000). The myoblast proliferation assay was carried out in unicoated 96--well microtitre plates. C2C12 cultures were seeded at 1000 cells/w=ll. 5 Following a i6-hour attachment period, test media containing either reomiblnant wild type myostatin or different myosttin antagonists were added and cois were incubrxed for a further 48 or 72 hour period. For competluon assay, wild type myostatn w;as Comrpted with dirrent consetrxadnvs o;fmyost4in antaoiSts prior to the addition into te meOia. After the incubation period, proliferaton was assessed uisng ehylene blue photometric endpoint 10 assay as previoJsly described (Thomas e. a], 2000). Huran skeletal muscle mnyohasts (Cambrex, USA) or prouay ovine myoblasta were grown in Dulibeccs Modified Eagie Medium containing 20% FTS, 10% Horse scmm and 0.5% -hicken. entyo exrsact accordinlgto the stAudard techniques. The rmyoblast proliferation assay was canied out in uucoated 96-well ricrrtitre plates. Cell cumes we.e seeded at 2000 15 ceiisAe1. Fc1llowing a 24.-hour attachment perod, test media supplerented with 5% FCS contaning either buffer as a control or different uyosttin antagonists (300, 310 or 320) were added at 1, 5 and 10 'pg/m] and cells were incubated for a father 144 hour period (fot human cotHq) or 4tR haor (toR ovine ceUs). Atter the incubation pened, prohieration was assessed using methylene b photometric endpoint assay as previously described (Thoars et aL, 20 2000). Results The resuls show tait myostatin antagonists 300, 3 -0, 320 and 329 signiicantly enhanced the proliferation capacity of, muyobirsts compared to control when present at I ptg/nl or 5 pg/ml over 48 or 72 bours (FiG. I and IG 29). The highest myobiast proliferation rate was 25 served it the 48 hour time poit fb each of the 300, 310 and 320 antagonsts when present at a concentration of 5 sg/w and 10 g/wi for 329. Myostatin antagonist 280 did not show activity above the corirol This is likely due to the fact that this peptide contains only J cys'teine residue. Addifon of recomibinan myoistatn M 2.25 pg/mi to control medIm led to a 25% inhibition 30 of m yoblnst p;:oliferation after 48 hours. 'This figue was reduced to about 10% after 72 hours (FIG. 2). Vhen myostatin antagonists 300, 310 and 320 were added at three different .31 co~tv~~o~ (1 5and1(1~g/1).0~p ofit-inib n~g effcts Gf .m'yostatin on rn.i'obh-rs w~a~f .res'oued to kvfeIs iilrto hisor'd~nJ; o~~v.oW ............ deanstmatod -Chat mycv~tatin antagoms4ai such~ as 313 310 mmd 320 -- an effectively i:'cce'mte muscle re gonetaLon by Mmticirg myo~asl'~feto (P !C. 2). 5To detrmie wheth,~ die rrivos t a~agopas ase effectCive in dfifferen specie-: We Conflacted haum-an andl o inryoblat~q :roiferatio. assays. Whn uysttiua gonis-s 300, 3~t 10 nd 320 were wiled at lie dibeni oct (3,t2 (. 5 or 1,0 p -v/mfl over 48 or 144 wnoras) tlv PYCEii-exa-ion capacity of huniam and o in.- ruyotbhtl sas .Siificaritly e jh~ammic vvhit:n Qfjjj?:armd to crntzol (1NfCffl 11 ad F10C.1 4). 1.0 'Fhee results indicaffe tat ainiisraio ofmotti rmonss jai i benficial. eft;ects on pabodns Wkfeing frrm Lm £.i3.C d eard dIsodas ()j tviy other coreswhe-r -4x reafsed tote-. rof iferatiol of mvolssis benelwto-al., surh as in ilit. lreatmoent of mu&6,-e Wa.,5ii 3;Exa-.nple 2. ITvoai~in rgulakes svsrelib-e cell activation -Si ibe eeisltda prviosl deribeci Ros .1aft Qt al, 1095). Briefly, I and '24 monthVc:.ld vAd-typC, n-ice qcr-, cuthazid by, CO;, gas followed by crndal d~~cto.TA VwewXC disted ut and digesed in 0:.X% (wiv) fto IA cof(>260 CDU(--2610 igma in 20 DWI-becco's moji-fi l] Eaoe, merdNmx- (Y9Mbj.) 6Jii~ogr o 0 minutee, ,I-!7' 2 ~&e kver.e transfterru! to DMv~EM 4 I(Y 1(1% orst servum (uIS) -F ( ticken embryo fextract-I (GEF) adfibres were separaed by r.entle triturat-IoTI. Isolated, fibres were transAfer.-m to~ 4 well chtbrslies (i."ectou racJic coate-d with iO% rastigel (1.ectori Mrckinsw) andi fixed with 100% ce cold inethurinol for it) ullts ~ex chr up t(-. 72 hours at' 5,C in 0 25 C0) 2 . ProWlifai-;6ti Cell 1'hicear As;tigen (NAis veeuin cells Tat l autrxrely ,indei-go:ing cell cycle but not ini .puecenl: u.ells. A large perceantage of Ibe 3ptellite ee h ichlr "0 thle mule fbrs are qiset and hen Qcc do niot ex-ffes's PCNJA. R(YwCvex. up I Cf-vai10n. -- a' ilte cells ,we ac-rtvate4 -,C express PCINA avFd rege jett! the museie by YeTrztnishin~g the 30 rmsce nb lmre. Tiu~PCNA- is a verY TeAMl antigea t~ madcer or thec acti vated satellite C-ells.

Jib ordmr to m~erijore effect of C myoI"ain '2aon~o- tsbug;tc .o15 .. ~u~ ...... nuvrce {ilre3frm TA mkvtw.,,e uf youug (1. mon L-frh) or old (24 nt) idtypc mrice were cultu)red in prese-nce of 5 4ghmi.::fit!t~ antagcmist 320, 310 nr 300-, in. c~udure iedui for 248 a~nd 72 houzq mcd 0,zed with methanol and warihed ir.P133. iflh,,ed fibves ,vem 5 emHie in G.%Tritosix-100 ill WIS. f~jw IG 0 cnuitas, blockod1 in W% nosjgowst wsvrn1 Jr'J 0.35% ctu~Vagommonan mIRId in PBS3 FWo 30 mnAsat rofom J Lopr tuto t. uacubaied w.)ith a 1:100 dilutin Of anii-P:NA antibodv in b_',!kcr Prn-Jt i anlibody waz dozected using gee.- mntid-mouisp-a-da fluor 5,;'S :cid fibr-os xmmm cou:nters':ained with DJAPT. P P4A po;it-ive Ll.:tjvated _,atLeii ccls wcrze s-oumitd vnfer microscope umd 0 xressec! as a perce A of alltotl myonuciei. To .tst 4the Offhct of ,shoft iiurmn u-yostz; n mntagorii-s' tra C1et nsa cdl?"J iaron -- reiir~ wer - -lated from mico frrorn ~ehtreat -,2i gmfoup (V; riesmrbcd i_-i exwarpie 4.. bek.Mw), ane cultured as, described vibove. Satellite ccd! 1q ctition )va-s Lnve:_,Aiga-tei uaing anti.

PCN.A. ntbodie-i as dsrbd LS R Ps Myosat in R.P.O:. gODW~SCO C6O Pea. S iae Mite iS s~no nuc~e tbr~ ro~nYoung ("I month ild.) -wild tylxe mice e j.;, icef with In vs.i naois30,a significat inraein fhe rj n-umer of atelt "e~ acivtd was obimwed (Fi:G. 3). Similar results Snwe sen hen- -lte ;a;be (if xctivaied cisi&r 20 com~ertd to pexlentage racrease, nomalio against controls (FIG, 4). This.,pri-t U.: rpeated using un-yosit~n enaoiss 33.~ 30 arid 320 on Woei nmscie fibrs ISOM~ old (2 peas oid) nie igniticant inceae ,o ic~ perceYNte of am ''-'d sAWN M wee v,,buicrvo r;j taa atrnt S5StA I g-matopt increwvas sfc -~~with mryoslatin ta 33"J.30 afftnv 4S honns (FIG 5) results (corlhi thav. myostatin m tamgonits 300, 2.5310 nd 20a. potvkr activators, of titeCells in ymung cu.:t a ed Wild typ: -miscle anud iun;Ca-e thata~nsxto ol: _xyostafii n tagonists2 -w. I-,- expected to treat r prevent the mnset. i si tz" te wviff a decrmise in ;;&eei te ce!] aciain such LIS suootpeflmaj. FuxthteIorv i-t foan be iexnccsred diat nrys~i otg nis~ ay be 1used "') rec ce sevcerivY of such ccnditios in calses wheye the ppopoprtion of activated s.atellite cells has already y 30 de1134d.Ths data. also so that the atagonist..- are eff-ective inl incre asirng sateffire ccii activaltioni muscleR Volmq above norn"11 Ofel orsaiten"itle cell r1c"ntijon, *Threfor th~e 33 rAnostutin. UA~goldsts cod.d b-e used. as a trivmeii rior to tri:wrua. due to. medical trwtinemis or prior to the rjn:Ret of Conditi~ons that would be- expected cto esult inj decreased muscee S;.rengthi 5""101 pv e forced. Jwatfcti'v,".y or other od.n. Exam~ple 3. T-'yostathi amgisisis lues-ease iJ1mrslair(Wux response "And i emortaxis Fof S xnnprph"44.R and my 3 abh11mts' Primazy myob~s.s wero ctirried from thae 1hid fi-mb ;.aceof young (4 to 6 week old) or old (24 ont.h old) mice, accovding -b te publishedd pzvotoc ols (Alkn el al., 1997, lvaxrtidgrz, 101997), Mrefly. muscles were- mmnhcc. and diese~hn 0,2'% coliagiznasi' -vpe JA for 90 nzu. Cutrswere- e!ridhixd fore myobimits "'vy pv-jplting on mncc-eted plate:: -ffr 3 Ihours. hvobikk% vjtn erp ei'e mmnjilfe n., grovidU. me-dia (MA) supplemiontad MA} 20 5/Q WOt calf. evum ....... ... (P.,CS)l 1&N 1-15 =e 1% CEEl on 1094 Mattgel coated -p'ates, w.at 37C/%Co. The Cextcu of c~uJ~ure ),jjvw assessed by flow cytumaty analysis MyeD exrsinafftr 48 hours in i5' cultiro. CeL15 wore harveste. ~t'tvs sus--pended a a concntration of 106 cells/200 g' amud fxei overnight isi5 ml -dmntiarol al 2(C Staining vnis pvrformed -for 33 rin at room teoeaoeusiwg ralbbit polyc-lia] anTh~yuoD, 1 :200 (Santai Cnr). f"ollowed ".1 Aklexa iluor: 488 aitirabbi: cnua,1 50(Mlvolkx uiar Probes5). snalysis -was carded Cut i dup'licate NYwih 1 04 C61 eva::111S wul :cle0 in ecxch Ossay. D ebi i w~S ecu by gating un forward ar-d X) gide scauter prvofiles.. Cells were anlzdby IFACSca (I13ecluln Dickinorl. NlrMenrpbages were iso--lcd by a 7eritonmeal las'age tudhn~gme anV/o derI'Ved flOF-D Ione maIRro-w ZY11os3all activated oseseruos. (Z.A .'JS was prepared accsyri1nni to Pepuatli -hled protocol (Coldiv, and Movat. 19L<4). For the clic.rnrtaxis assay ofm ~ ia fist assay was carkdc. out ny.~oblasts fromj. 2 y-ear 25 old mnitse- as -follows- DIVEM co.iiig2$- horse ,:ern= (118)e 5', chilckena embr-yo extrsact (CfI"E) piu1diaysi buff- r wArs used as pmrdit-Vrc cou"tvol. eobn syati(25'TI -- ttil) ar.3d yot1naaonts300. 3M0 :Ind 320 (at 5%-tfj.,;s myvosTntir fconcenr.-tion, ion, 12.i qg/rr) wure added -to pisitfivc cwn'tr' -mvrdif. Plin D!MEMI- was used as inegativ-e con'oLOna. 24-wcff 4lte th botmwl:were filled with test orfoto med-Ia. SYencrty 30 five thovsafrvi' cees'v.r addeA u.o the top We."ls coral!'nlir.2 Toytyeeteehhdr P'1) 0,9 urn memubranes C-of-ed with 1%Matrie The plate wac, ir-,nbated Ror 7 b a37T, 34 CC. The top surface of the membranes was washed whh pre-wet swabs to remove cells that did nuct rigr~r, The memrebrane was then fixed, stained in GiPs hmaoxylin and wet mounted on 3z3:s rerar.er3 ceris we;e c mte on :oar srepresemauAve leidaper membrane and the average n-nu-br plotted. The rm-utis are shown in FIG 6 myoblastt mgration from 5 oid ;ice) and FIG 7 (myoblast migration from young mice). in a second myoblast migration assay primary myoblastrs isolated form hind iiib of 5aline treated (coatml) and myostatin antagonist 300 treated mice (as described in exampk. 4 below). Three rhemo-attractant media were used: DM-kfEM containing 2% horso semmni (RS) and 5% chcken enbryo ext;:act (CEE) (orjtima chem.o.-atactant; DMEM... cnining on 105% CE or DMEMSi containing ony 2% 1HS (both suboptimal chemo-attractetsi Pan DMEM was used as negative control. On a 24-wel. plate, the bottom we1.s we::e filed Vith tet or control nedia. Seventy-ive thousand cells were added to the toi wells. The plate was in~cwbaed fo;: 7h a, 3 7 "C % C ) The Iop surface of the swas wshev5 d vth pre we-t swab to remove cells that did ieo migrace. The mrembrr±.e was then fixed, stained in [5 GiiPs hemstorylin and wet mounted on slides, Migated cells wereounted on four ropresentaa.ve fief is per membrane and the average numslber pi.otted, The results are shown in FIG 9. For chemnotais analysis of macrophage:s bone manow was isolated frm saline treated controli) and myostathi anlagonist 300 created rnc (as described in example 4, below), and 20 plated at i, elif/pate in DMEM 10% FBS5 plus 10% L929 cnted medim (containing CF-1) for 5 days to induce mecophage differentialion. The macrophage were then ha-vested nd. used in the assay. Three oentrations of DIMEM contairng Zymosan activated mouse se.m (ZA S) was used, 33% (optinmm chmo-iratan oncratio)........ 22% and 1i1% suboptimall cheno-a-ractant wencentrations),. Plain DMEM was Used as 25 negative control. On a 24-well plate, the bottom wells were filed with. test or control media. Seven-tyfive thousand call Swere added to the top wellF contaban oyethylene erephthaliate (PE.T), pm meirm. r ebrimes. 'The plate was in cu1bated for 411 at. 37 C, 5% CO. The top surtfce of the mmnbranes was washed with pre--wet swabs to remove cells that did not migrate. The nemobrana was then faxed, stained in Gijis hematoxylin and wet mrnonted on slides. Migrated 30 cellIs were counted on fur. representative fields per mn brante and the average nqurobir plotted.

35 When primary myecblasts isolated Pou old (2 year old) mice were incubated with recombinant myostatin at 2.5 pg/m] in control (CE) ixedium, a signifc d creseo approximately 78% was seen in the number of migrated myobiasts. When myostatin 5 antagonists 300, 310 and 320 were added at 5 gg'm], the chem o-hilbiing effects of myostatin on myoblast migration was rescued to levels Similar. to huose observed in the conftro i (FI .s experiment was rpea-te usng myoblulast om young (I mont old) mice. Again, addition of myostatin antagonists 300 and 31 ('to myostatin containing njmxlia rescued the migration of myostatin cells to control levels (FIG 7). These resuls demonstrate 10 that myostatin antagonists such as 300, 310 and 320 can effectively accelerate muscle regeneration by enhancing myobdast migration. As shown above, myostatin has a negative efctr on myoblats accret-ion to the wounded area. Since myoblasts are known to be influenced by chenotactic factors to direct their mnovement (Bischoff 1994; Jones, 2000), the Cec of i vvo admwinismadon of myostatn angonist 311 o. the i o lf 15 sateIlite cc derived mvoblasts was investigate-d using three chemo-attractnnts. Myoblast; Isolated from mice that had beer treated with myostatin antagonist 30) had significantly increased migration rates in each chemo-atctant media compared to macrophages isolated flom saline treated control mice (PIG 9). it is also known that nyostatii has an effect on the inlarnmatory response, in particular. 20 myostrin interferes with the m ration of macrophages to the site of iniuiy drarinu muscle regeneration. To assess whether or not treatment wish myostatin antagonist 300 in vivo affected the igration o uacrophages. an in vi'Tr mrtion assay was carried out on macrophages isolated fom mice treated with salne (contoi) or antagonst 300 for six weeks. The results showed tat antagonist 300 in vivo treatment signifcantly increased the migratory 25 ability of macrophages in three different concentrates of chemo-amtractant redia ZAMS (11%, 22% or 33%) compared to macrophages isolated orom saline treated controls (FIG 10). The ability of in vivo administration of myostatin antagonst 300 to enhance the migration rare of both myoblasts and macrophages indicates that administration of myostatjn antagonists co Ad be laed as an effectiv e treatment in patient sufferg frm conditions were inhibition 30 of imyostatin activity would be clinically beneficial, including muscle easing conditions and inflammatory nyopaties, as weH as being usefl for wound dealing and any other pth ologies where enhanced uigration of miacrophages and myobiasts can ccnfer benefits.

36 'pir~je 4r P vivo trials witJh myoxta~k ti. ni efi 30 Methods A4n --- nia ia wa coneamr to as the dJ.ects of r.yortain aixgnt.30 inm ro i g rawsc-1e fiunetion. ie mkath old mice ie divided into two gimsof te.i, While thi c 5ontriol group received saline iquNmmnmaos (SC) inj:cticn.,, the ofher group received rayustabstagotst 300 SC at 6 ,ig/grnz.rs. of" BW tlhrzee times a wekfot, z*ix weoks. Tfhe fisictioriat improvement of' s ptIc muK'i was, aqwssed int asiirig rip rimigth of Ane atthe end.of tiaL hio h - war nieastnd nNetos 10 The rumults indicate that Wiill thIjt a.y ~io rh.( grip strmlngth ol Ja emooxl I-nic (lo"s C.f -. 5%), Onerse is bighldy sigmni~i.t smam in ime gwi ,,rnqt of agd lmaed with. "Jx.e, 300 antagordst over a *Six WEek pro.(PIG. . D). The same data woo expressed as grip sitrngth at thle begifinirig and end of the triaJ i both gnips C.1G, .121), and rl;.e san.ev 15 trnyosAtin nttois' 300. Thbi! wvaf dee inl niv u) the incrt'ese obsfei1vek! In siliec n r'ainand ;izzwcrophlagt a;,ri n1j5'ohla~t fmgigra m obtaiuoed n.cciis and fibwcs iso] aTod -ftom ttf.ese ff;ri M atIIcni e [ J ,Tllofj per.od .JUJi, 9 Q.). ( An additional is.;,poit obs-e-rvadinw,! ated., Ati H) ~ agod wild-!tYpe nice sl1o-wed si~niirit a i~nnia0iouofbod), ft!. Ini hoth tt-atlne.11t gr.oups, -all Frc'ice kwerc obe dTo have :2.0 4 d:itinct -re dlution i.m body T±coynpared itthhe Faijue treated -m This wars -w-fj notice Oe. Ithuaars aip..hraged I tal titurvstti aV)~ri 1 ~sets h'b've reduced the amount ofsbuae ht infcludingp the I'igui~lfs pF ad in thVe Mice -*rear.ed WithJ 30 he omipwued( wo thew fcontol mice, (result not hw) It has previously been sfown -Wn delerion1 of mryosta.n turinLg cTnlxyo omc. stages afi-c-ts 2 rmsntlbtacmltOx. Eknwevel the surriing ltt her-- is that reaimnI of mice for a 6 Nveek Period ha!; reduced zhe amount oif far, tis;sue. This Gou-l be duo to the osai antagonislts c-ith I e mobilizing, thetrv ycrie stored ic.fh air: s tissue1 intoffering wt fatty Rcdd biosyfntdesis or aohrehisi.R adesthis veein.ifntV clearly eejple -Gh, oont-oi of -fwt tissuem rsize by 6he ,,.yostati nitagonis~s as oppose-d to the letsn bzorty naSs 310 d.-,w to a co.1pl-?ete abseyxce of rrryostatin (myostathi uubl -' t ~~~~U ths oa tatrryotait or ptvrevetlrg tfic r-cducticn .;- muscle rriast and s~oghinOuced by sa~wcoponia but irf also usefall ini reducing th;e iyj.nueased f.rit deposiicao t Jat .- also eusoc iatecd ith oldi ge- fn Ldition, it is ejjvisaged thmjl~e m Ost: fin. atyao"'Lt of lhe presi]1 Lave ilt non.ord ig 5tfio wn'ount of.!W fat e.,ue in. rolinical csito of obe Slty nor m-fexir alnd ib;ts A n in 'vivo i-l was r-TRifn-d in nwhid. 4 weck old mCx icce were injeectcd 3'x per wvokwit 300 (6i~gg or aine (1 0mnice in eac-.h trnatnen) fory 4 wwks'. 1ody wexiglts were reconiod 10 ';egWlly ind blood twuc c-oilecded fo-r creathnie, kinase, activity to ass'eSz the ex-tent of rmlu.dlqJwe damagee, i. ip tests were pe-rfor'Med aVs a mleasure:C of strength. After 4 -iweeks of treatment r;m c were cutdand inldi'viduei kbdelimb muscles collectud -R Tprotein e'xpression analIvSis Protein wa ex~fracted fi'orni ih (" ;un limb mnuscles, biceors acris~ ,ia antei wd 1 At Cas on .en I5 g of protein wevs seoa ated or). 4-1 2% SDS FAGBi, rtuwsj;fered 'to ni~oe;.1.~so ieibyali f- ad imuuoprobtxd vvith Paxj and MoD 'L .1 mi~cron trans-vere Sections w'ere: cu, fkom the midhelly reino ah n a -onomu Mfumlie vetn vwre vv~ne ith. aetogvi and =54si (1 & E2) to is aliea mei46sure ;--I cas -of ncroS-is and. regenerad on, -hse vreas -were analysed P=n aOlyrnpus LBX50 2() mcrvoscopee (Olypq:, To~kyo, Japan), a SPOT-RT 4.01 camera cu.sonde ijgrOn Int-UrEUr tS. StlinU'g NAgiTsM'j. Areas -were inesn-e-od using limage-Pro is(ea Blood wascolctd bN 7ti*beiait 5 %mt hepgrinise;d n-riirohtmatoctit 'xibes. Tubes weRIC CANtii 11-1 Pn Mi-e atoIt centrifg (AC Lvms) '"or i raiuutes andcithe wh piaa 2.5 semo-~ived. The mreoed pia.srna 'has cetarifuged' i-gain at 25010 g, for 5 -iniri-ites and il~zn skored pt * -i u intii analysis. plwwlra w~a3 Trarhe ims, o.h .1 n bo Jeh S~iti 2W 1 o of, 1)Ic f4 colle-. iorn to han~ns henotv5iS. OCr:Ai ne kir .as iaEcftivi (y as determined uisine, C0. *N-aeiy.L-csteae relnt.-ne jdiso Uit (cka.coulter) within 6 Alwek's of irn colleclion.l Muscle Strength -was5 asssd USIJ~- 0i rp5te; ;~ar M-~ 30 Tol'VO )3l~i oky0 Jan) at flt LI'e~~U~ c.d-oi u n. of teiallTe -rncaxnUuxn forced byddlvanouevdeb'~ pLilledb kwr by the waiJ. isrccoxdedi ill 'NO sis Mrzb Men of three gridp tOStR fvar eaehod mvo w as calculated and the -mean. maxima.ior of 10) -micc per gw.-np was3 determined, 5 &Wf~i and Wa7 are vrgmlired ''.~tfX to n'rnet ]Botb PnY7 prni ayf ~v c~'tet)ik ,-a pyotnt mmIker- of nw -,ueis P'ax'i ievcl indicate -h-- !xfft of sakdilite ceii11 poooi" svtlt a's Seatellit~~ce- r~-rea and MN-yoD expressions firles tho. loievJ of myoge.-ests focurmrg ithin a rnascc. To invcstigate - P7 and MvoD :pmteia levels inj ui.errmc e. n z mico, Artstnrn bl t alyses werve erfod. 0 As si own hf FIG -15 Vhr w.as at S2btia' H, egulation of c t. nax and. MyoD P,~ i 0.. S (x] in hor. Lp~ievscui and. ptc-lferating satellite cells. l pvrcaidatior. of Payx7 is imiicat~ve of injcrcased aotivatiou of :;attllite Cells: bigher7 Pne~iecll :aufnbor amd itrcscdr lueco"' 86H., renewL al tillPortant rtlf:' mui gene~l 0 iJixIJrg .~ni linicatie o-f an inc'riasc in yges via uprccglation-- of p2l arid MVyognin. Tve 15 trguliaio.a of both Pax",an MyOD muggest-S thlat 3001 rffectivcly anjfagonixcs niosai at is WO.l eFtabIshed iflat absence of rnrost?.hn leads to: imicrfet Al od-iie l antiveiot nd -self Renueld no eis. T-he pvcr;entatgc ;nera of ragenerafing and necroki aras Ewe nowm ii.FIG, 16, Th-is result h; 20 csss.W.ith tho !h-ypctI~esis ti--at xysa antagonaists inraem~cerege!n-rtion and repair by acdivaingJ Caeiielljs tIfiby' inwrea-ring the.. fcmnatio-n of new nmmc Rhbos ax)d. decrunsing levels of mcrotic tiss3ue as also i.-dickit-d bjy (heenhnce M-YoUE itid PaX7 expression vact rerd ned', CR ale in the, blood (FIG. I '!). Crea~~~~nc, b*4s. mci~ sice bvihs a~vdcl'i myvs ,Mifl 300. 2-5 Crmaine kjpinase (CK) is !n ntracllla en-ynn con .noxily use d asa primary miir-ker to mopsrultor extent of ms.edamage inmdy. -umvce (Bogdanovic. it al,. 2005)i audlmmims, Tlih cfb;nage , in cx entine kin rue activity ftrm the( C-mme ment, toi "he co~znpns."tion ~itetria~l shown--- in FICG. I!?. '1l a-uger decreased i creating ldmzse Rctivilv, Jkt the 300 -trc-tecI lc ~.pasts ies- -ilsocle C1amage. in ne atismJs die to.- a changes nte n::otn of mu11lj cells reg_5eratig versus damaged or necrotic state. Tis corroborates the histol'gical data showing zems necimotic area in 300 treated mice Antagonism rf mvostaui with 300 increases gr ip strength 'The mean of three grip tests for each mouse was calculated and the mean maximal force of 10 5 mice r.er group is shown in Fi. 18. The sigiicavt increase in i , strnter 4 weeks of 300 treatment indlates a. functional improvement in the 300 treated mice. We largely arnribute this to th- arneliorAtion of the pathology of mdx mice muscles through increased regeneatiAv - etctsv, va ntarnism o myostatin resulting in a partial recovery of umeAle fnctio. These fnudings suggest that beneficial effects of myostatin antagonists in mdc mice 10 are likely to be due to effective treatment of .;uscie damage enabling a rescue of grip strength n mdtc back to wild type (Amthor et at, 2007). examplee 6: In vivo skin biopsy trial Nxcthods", C56hl/6 micf at - 10 months of age were anesthetized, the skin at the batk of the neck was 15 shaved, and two wo rnds made on the skin with a 4 mm biopsy punch. he mice were :njected suboutaneously with either sale or 300 myostatin antagonist at 6 pgg of body weight at day 1, 3, 5 and 7 afer injury. The mice were allowed to heal for 5, 7, 14, 21 and 28 days after which they were culled and the sidn. pressed r histology. For the histology, kin samples were fixed i 10% formnalin, etmedded and mounted in 20 paraffin and stained with Masson's Trichrome stain. The sections were aialysed for the size of wound area for each day following the treatents, and the values plotted. Resuts: To evaluate the impact of ray ostatin antagonismr, in wouud healing in skin we neffocrd a skin biopsy study mi mice. Animals areated with 300 consisrently showed smaller wound area 25 when compared with the saline counterpart, particularly at Im later time poins s shown in FIG. 20 indicating that treatment with 300 antagonist can positively influrece the rate of skin healing in miCe following a biopsy37 wound. This result shows that aitagorism of myostatin can confe.rimproved rates of healing in a rnon-tMuscle tissue such as skin and indicates that the 40 use of nyostatin antagonists has applications in improving healing of skin after damage fnom wanes, bums, sugical i.ncisiors and other skin trauma. Example 7: Pn vivo skins burn trsal Mothj;~r flurlsal S 1060/6 mice at ~ 12 months of age were anesthetized, the skin at the back of t-e neck iwas shaved. and a od--ho netal rod (15. rm wide, 7 mu cr was applied t-. the exposed skin of the nince for 5 see. The mhnice were iected sibicutaneously with either saline or 300 myostatin antago~uist at 6pgig of body weight at day i, 3, 5, 7 and 10 after injry. The mic were a .wowed to -seal for 51 '.14. 21, 2 35 and 46 days, after wich they were culled and the skin 10 frozen ri liquid nitrogen f hydroyproline analy'sis. Aier a burn w iry in s-kin a h'ydsnxyprojine assay was performed to detenuine the amount of collagen deposits on the wound area. Hydro ine is present ;i collagen, and gives i. a stable helical conf-ormarn. Thereue, measusing the around of hydrxpoi resent in a 15 given tissue will djetermine rsxe arnont of collagen present in rhe 300 treated groups showed a ten~densy toward lower jevel of collagen On the wound area when compared with the control group as shown in FIG. 21 suggesting dit the myostAtni antagonists could be usal n) he reatment of n s tissue where the desied outcome is dereased fbrosis a.ie; wound hefirnhg. 20 Exanple 8: In vivO muscle burn tridl C56bl/6 mice at ~, 12 months of age were anesthetized ard a red-hot metal rod (1.5 rm wide, 7 mnm long) was applied to TA muscle for 5 sec. The raice were injected rmbcutaneously with saline or 300 mnyostati antagonists at 6 pg/g of body weight at day 1, 3, 5, 7 and 1) after 25 injury. flfie mice were allowed to hel ittr 5, 7, 14, 21, 28, 35 and 46 days, after which they were pulled and the muscle processed fr histology. Histological rmsclr waples were covered in OCT and frozen in isopetren'e cooled in liquid nitrpn, T-e sections wbre cut in a oryostat at 10 prn thickness and stinxd with I&E and Var Gieron. Regeneati;.on -uarJkers nuch an total number of tmy fonned nuei (CFN) and number of fibres with CNF were analysed for each day ibilowing the diffiTrent treatment, ard tfi- values plotted. 5 Mature skeieta muscle fibers have rxelei located at die: periphery of the 'fiber. However, during skeletal muscle reEneration, mychiasts migrate to the site of injury and fuce to pre eisting fibers or fuse to each to torm nascent myofibers. During this proCcSs the nuclei of these cells ar e located in the center of the fiber. We therefbre used the nurnber of cejtraiiy Rmi d r nuclei (CkF) and the nmber of fibers wIAd CNF as motors of muscle regeneration. 1.0 The damage p;eaent in die muscles for the first tme points of the trial was too widespread, and no positive regeneration marker was detected for either the treated or control group (FIG. 2and G. 23). However, by D21 the numbers of CFN inteated groups is significantly ir than that in control groups intdicating increased Trmscle regeneration on the treated animals. This difference is maintained up to day 46 ae;tr injury. Interesdiy.. There is no 15 dufrxenue in the percentage .t danm.ge musCe are between rte tretment aid control groups from D28 onwards (data not shown). This ldicataes that the 'icre in degeneration n the 300 treated groups as shown in the increased numbiers of CNF resulted in. n improvemem in muscle regeneration after burning. Futhennore, v isuay, difrrences it muscle fibre integrity blween the treated and control groups can be seen fomn D21 onwards in the treatment group. 20 lso, vr (ieson staining reveled a higher degree of fibrosis in de control group when comared to the treated groups at D21 after injury (1G 30) sigAifying earlier remodellnrg of the injured area, and therefore an earlier intlu of m yogenic precursors, leading to ;c increase CFN sccn frm D2 onwads between the treated and control groups. Decreased fibrosis per se is also attributed to the effects of the m.yositin antagonist on the extent of 25 fibrosis hat occurs during repair of wounded tissues. Example 9t In vitro Cancer Cachexia Trial Methods C2C12 cells were differentiated for 72 hours in differentiation media.(DMEM - 2% 1 Following this period of differcztition the myotubes were tste with. r binaut myostadin 30 at 6 g/nL or a combination of both rmyosfakin and 300 recombinant protehx at 30. 60. 90 or 120 upg/mL for iiuther :24 hours to detennirne if 300 can rescue myostatiu-mediated muscle '42 wastng i vitro. Gene expression chatiges of interimechate sgnalling molecules criticaJ i myetati--meiateda~heta 'were aiyrsid. Therefore, protein was isolated from cells and Westemi blot analysis was pertomed to mau protein expression levels for p-Fox01. V;ich we have showa to be a critical signai-ngintermediate in Myostatnediated chexoa 5 (McFadene et al 2006). its:tt p-Fox~i is a enitied irnediate sign~alling mnol ecuies in mnyostatin mediated cach~e:xia. In the experiment expression levels of p.-Fox01 were normaised to total levels of Fox0L As shown ~ ~ ~ ~ ~~~- in FI.2,.etetwt gm eobnn Myontatin protein resTults in down 10 regulation of p-Fox0l expressin following a 24 hour treatment. This result is consistent with our viously published data describing the inechnism behind Myostatin-mediated echexmia (McFarlane et 2006). A. maximal recue of p:FoxO I expression is obtained with 90 pghi..300 (FIG. 24). This shows that aplication of the 300 myostatin antagonist can reduc the magnitude of the Fox0i signal that is critical in a propoition of eachexic wasting Examv"p 10: In vivo Cancer Cacelxia tria A study of chronic cachexia was desigied whpiereby C57.B rmice, 4 weeks ofage, were injected with. the gJluocoticcld dru thaone daily at a conceatio of I mg/kg body weight. 2 Tlrc treatment groups were designed: 5 mice recejving da iy ijctionrs of sa5i)e, receiving daily injections of Drxaetuhsone and 5 mice receiving daily ijections of Dexamhasone plu 300 antagonist at a conceniration of 6pg/g body weight, Body weight of the mice was recorded daily and monitored o for signs of cachexiaa Food and water consumption was recorded to ensure that loss in body weight was not due to a Joss of appetite 25 or thirst. Body weights, fat-pad weights, ;ad MyoD and Pax? expression were assessed. Body w.veight Daiy injection of.Dexamethaonie resuted in a reduction in aver-age body weight as ,omprcd wi;t saine injeited controls (FIC, 2 5). Co-treatment with Dexamethascne and 300 antagonisr.

43 resuhed in a smaller average body weght ioss as compared to contros (FG. 25) indicating that the antagonists can prevent canhexia related mleqCJ t es and hence body weight in nice. Tis also incates tLat use of myOstatin utagonists such as 300 has applications in irducimg the negative effects of use of dexamethiaone in human patients. t Fat pad weights One of the clinical features of patients suffering *rnm Cushing's sVynidrome -(over-pro dction of glucoccrticoids) is increased at deposition, therefore fat-pad weights were analysed foiiowing the comnpketion of the trials. Treatment with JDex'amethmsone resumed nl an increase in retrcoerton a l ert 6239%; right, 10 60.4%) fat-pad weights as comared to saline treated mice., with svatisticaiiy significant increases observed in both the left and right rcu'operitoneei and inguinal fat-pads (p<0.05) (FIG. 26). An increase in reiroperi'toneai (latlt .2.3%; right, 39,2%) and inguinal (left 3:3,2%; right, 39%) Tht-padl weights war also observed foxkoi;ng treatment wirt. 300, although to a lesser extent than that observed ihllowintg treatment with Dexamnethasone only. Therefore 1 5 treasstent with 300 partiaiiv rescuedd Dexanmedsnec-uediated izxcrrnse in Tt-pad weight indiicating tr our antagonist cvan not only be useful in trea&ting oachsxirz but may also be used in rkuati on where is would be rsdvantao to redi utefatdeosiio such as in the treatmnt of obesity. My'oD and Pax? expressin 2.0 Protein was isolated ium tho righr. gas'trocnenitis muiwle fromt all mice and Western blots performed ifor MyaD and Pax7. There was a significant down-regulation aof MyoD (p<0,.05) (FI. 2) ad Fx7 p<005)(FIG. 28)l exresion following trealment with D) examethasone. In addition we abserved a significant up-regulation (rescue) of both MyoD (pc0.05) and Fax7 (p<O.0 1) in ile treated with Dexamsthasone and 300 mstin retagonist, Therefore treatment 25 with 300 antagonist ap.pears to regulate the expression of MyoD an~d Pax7 during Dcxarnethasone-induced cachexia. It is well recoguised th at iupregulation: of MyoD and Pax '7 are marzirs oX muLsUCl regenetiton. During muscle regene;:ation in young wild-type and mnyostatin nuli ice, MyoD) has been shove to be expressed earlier and at higher love 2 inth myostatjn null muscle as compared 30 wVith wild type maic (McCiroskery, 2005)., Since MvyoD expression can be used as a marker 44 fox the vf~ry V~v~ de:tjori of n111igi'afdg lu.-obass dus-ig muaci O eei~ailn(r~ COBlS to the itefjury (Ivl0Croskery , 2005) Since Pax', 51s a Y2.arker for -"pteliite ccilf "Owl 0ustaiina, 2004; Z2amrnit: 2-X,4), h 5 higher laevei of 1'axi uhe~aht 300 enhanced Vhis sif-3reva1.pocl Thia- is in ri ~cnrdance wjtia.ar finding hisgeLmc~ttnIhbt aelt elsfaca (c~oakry.20,05.) Taken itogethet; theQ higher Pax7 and ,v 4 yol) l-mee z-esuiti g fiom tu adziuoratin of 300, sruppasrt Uaob---arvation '-hat 300 trcatulunt ad-var-ccil usclk zegeneratioln iin this moelj of 0 mnurcle vastincr tluough -- creasing levje'ls of- ciatd aeftscells and subsequent nryoI~ens S andsaelite O lsl eea FI-xarnle -. :i In vi~ Sarcoveia ra IVt-h pas: Wlild type mice (32 -nife qrpro. 4-j nrh of age.) were ijreted beinosyfrriltl 1o- mnont-ilv with 30 Y,( 6 piggr body weight) or salie fcr a ~.riiOf 10 rrnh rs stenhwu a-asc uIsing -i ;Tip sm;reug<ih pprts(M*3iS.Mrmch;Tnoi~n at rlic! end fif u'hre trial. Thrle rnidiiwny- forced: exert4ed by a mouse whuje be~gpullcd bcwr by the lit .'i mvclrdrJ ciinNcD oe The m A f three grinp tests f r ach -. nousce was calcula~id fine the inean, mroiial frepe.r g oup w7aPs (I tenni;i 2 0 .Res i im Grpstength Is -used an indlication for muscle!; function (WaVzgner ex. Vl 2002. A 0mot ann,,,ual ivial wa.s conclucts-"C to as:!ess tre cfm(Cis of :ytaiatagcnhts -;iurrovig l.ue fncioin jice utri procgre Ssiaon Rion', youung u)o aged ,,tatws. A,5 uhv., jyn FIC. 19 n. il~iicsrit injursas in1 grijp frUgt in. ffice trecated mcnthly Wit. 3,010 over theT 6m sfif C'Cuilmo at. 25 l cth en lf the trial indicames P. ftuj.ctional ijurpr-.overce,.nA -i the 300 created m1-ice ( <GI.W have shmv.wn tht bothly plicatiF rL egm moWtl 10i. 19) as ,velt as tbrin:ight*ly (data not Llln ataqorijt apQV3 aic wt-re equally e-:ffective ini insrjysinjg vnusuc u)ctJon most likely dueto inc'rensed siihecl ciainadsbeun rygnas in addion to the benencr effects of the use of antagonists to increase rnmscle strength in. aged mice, it was noted that during tisi" trial the inital i response after one monh of treatxnt when the me wrre young (4- 5 monit old) showed a sinIficant increase in muscle str ength. This shows that the anltagonists are effective in increasing miIuscle function in. m:uscle 5 with norma kveis of sateflNe cell activ*ton and other determinaumts of uct function. The result indicartes thar the treatment with wmyostatin antagonists prior to trauma due to medical treatments or prior to the oaet of crYnditans that would be expected result in decreased musicle strength sus.n as enforced inactivity or other conditions. )Disicussion 0 Myostatin is a negatie regulator of muscle growth since lack of myosttin leads to growth while hig levels of m-yostatin induces musek waiting. It i- documented that myostatin elicits its biological function, in pt, by controlling the proliferation rate of mVoblasts (Thomas et ai, 2000), Myostan appears to be a potent regulator of ei cycle progression and it has been shown that inre'a'ed levels of' mysatn bloci the e cny of 1.5 Imyoblasts into S phase thereby inhibiting mnycbInst prs feration (Thomas et al, 2000), Myostatin has been shown to'bind to the active tyrpe '1B -exeptor and signal via the smad2/3 pathway. It has been shown thrt the 'TGF beta embevs, includig myostatin. conjIin conserved cysteine residues that are critical for detrenninig th.e t'rce dimensional tcture and are esmental for receptor binding (Lee and McPheiron, 2001). 20 Inhibition of myostaa activity may manifest itself in a number of ways such as an increase in body weight (McPherton et W, 1997) enhanced muscle mriass iduc to hypertrophy and hyperpasia (McPherron.,. 1997 nd Zhu ct aL 200C), a j.uction in body fat content (McPherron et al 2002), an increase in nusce strength (WO 2006/083 I 3) and an inarease in bone quality (HlRnm-Iic, et al 2002 ,. 25 In thds stiy, several re'ombinaut molecules were tested for their ability to antagonise mostatn neon. For the antagonists to be fTncticnal, it would be expected that the conserved cysteine residues would be critical in determiing the three dUnensional structure of the protein. thus tIar. ng biological activity. Given tluu the C-terminal truscations at amino acid positions 300, 310, 320 -nd 329 all resut ini the loss o. essential cysteine residues 30 remn!ting in the disruption of the three dimensional structure, it was very surprising that 300, 310. 320 and 329 were able to be eiTetive at neurasnyostatini activity boh in vitro and in vo- Tlnba dje roMlt ewe unprecedoedx a~nd stirprising~ty idztetha. .x; o.rJ ar,"- these i ingconst - ovel c-o-nrinds, bc:d: fhat their exent three dimensional proteilP -rtrL;Otr. ad thdi meehanirm ~ ~ ~ ~ ~ ~ YMO AM woP~ i~ iptdsiJa se C-tcznninally nnatda posajors .81.282, 2813, St. 85 286, 28'!, 28. 289, 290, 29J. 2.9,2, Z43, 294, 295. -296, 297, 5 28, 99,00.30--.2, 33 304, 305, 36W. 0 30 i 3 1 3131131 3131MS 305, 3,Sin, 319 020 32 321 33, 30, 315, .26, 32, 2 or 329' all Qwai suna rmsnbens, (-.f cystein ies to conf mter %cuv'y :as d.rnjonsurated by, tfhe Itmpe -- oeis expected that till th.-s pqptides viould be 1firjioaL U hc. Sroke of fhce3c cysi":tine residues itn confering fuzwrlon , s SUP.Piorted by itse obsevatND Mht 280, which hv.s an additional cyste-lnc. -esidue A L kcand th~oeon] 7 c r'flein on;e. n rCYSdue apo obe iuanLti' v.cfxarrjrk l) The ur--esent results cloiarv shbow that, :2,w L~oveir' iosraln -aitagwi.,ts are able, 6e--Pite the lIoss of ic c eto:c n usrs and a 'it& nculd stir-ctul oncraio.to aritagonisc raryostatin. This ef~thas bMen shown. in rthe in vitto rind ;-, V-vo triamr dcrc.e v to Provi.flocnefits in; candh~Ons w.~we Ane effe or yahology j., due to !signalhui 'a ficrease d 15 .iryostatn levels, includig W=rou wasting OVnditi(InS afnd IrkrafUaS, 4ha ire ASSOC-Uatd With is keyto drectly cofnfer icencoilkja rsrtwomei; and in. condition--s where the caPaci"y to antgonse aysl esntsin the ter;hnnu-nuient ofpyilgcJprocevs-,es -wmch as; myobla---t :proliftrfixaon seiJ.it fcell acii-vatifon and scfifuwvat, -ingation of my~~ssand 20 matcropbagcs and oih-ets, and hr thte enhanf~cet of these effect coifens be- nefits in ti,-,---ue whr i;'osviaa'ne above Those ccurrin.Ig i healhy t±is$7,xs The pi fv1;.~ . 0 bt-y ftc bfO 5F~nffi An frth 1 rnnnt fI nevnr- of a nTnber11a f discasus a J-i;sorders- that ale characterlsed, a' levl i part 1by an abiuminal anoont. dcvelonmerat or mtabOMi activity of twascle or adipse tisue in P. in-nmi al rnad iwadfe 25 disordeos raedto lnus1~h~ ~oiy uscle- atr'ophy a=d ;ruclc was-tig ascae inflammatr -qy m-yopalacmuiljedstos, , motor n-euron discase!s, disea-?ses oit te rrewio=asua j ulloon:&.sse o the, peridphendr nerve, mor.esdue to end~ocrine qbaoruhtiesnz rit-tauolic syndXOroic, HPVC5ICC*,ca oar oe5 uMnch'$iAL Eic'- wLI otkir~wsi.Jng coditk~us. ~irrZiaothhuo; ost:oplorc'ss; acomna xfrurc or disen.e; livkm 13ihe rdieae 30 a3norexia-, osty, dia).betes; and -Vouud e~.TFrr xwnpJe, it has been shown that caohe-iaz C-cn Ihe induicedL ficc nity the- systematic diitao.o ysak (Z -inmnerx ei al, 2002), "ysttn levels. have, also been how to Ite celvarxed in animals virtl bran in~ni (C'harles- E. Lang, Chnstine Silvis, Gerald Nystrom And Robert A, irost Regulation of mycstadn by gheccotoids atter thenual injury, FA-SEB 15 18071809 2001) and in JIV patients (GonzalezCadaid ei d, 1998) and plasma mryostatn immnioreactive protein was found to be increased after prolonged bedrest (Zachwieja et ad. 1999). We have demonstrated that 5 administration of the antagonlsts of the s results in rescuIng myostatin mediated cachexia signalling molecules (Example 9) and alleviating body weight loss and enhancing muscle regene~ratonin a cachaexta mouse model (Exanpe 10). Thus indicating that our antagonists can be slial for the treannent of individuals suffering from eacheia resulting frm-0 burns irjurics, prolonged bed rest, HIV or cancer.. 10 Treatssent of conditions such as these where increased myostatin levels is believed to bef key signal to induce the deictenous etffets wxill obviously be rssisied by the capacity to antagonize the rnsyostatn signal using antagonists such as are described here. 1, is underood that there will be numNIMrous pathological condions where the role of myostatin in signing and induction of the pathology exists but has no? been discovered as yet 15 Use of the present myostatin antagonists has been shown to corner benefi tst n several conditions where it is not clear whether- increased levels of myo4tatin exist or are responsible for the decline in muscle or other tissue function. It is clear that the inhibition of myostalin can confer benefits in. increasing myoblast prolifbration in wild type myoblasts (xampie .... young wild type satellte cells (Example 2) and in young 5 month old mvcc (Example 11) 20 where there is no indication of abnormal levels of myostarin. Data is also presented showing benefits of the present nyostatin antagonists in two deleterious conditions, sarcopenia and muscular dystrophy, where the beneficial effects are likely to be largely dume to the general effects of enhanced rnyblast proliferation, satellite cell activation and self-renewaL migration of rmyoblasts and Inacrophages due directly to the ehcts of the present antagonists, 25 Sarcopenia or age-related cachexia is characterized by insufficient levels of muscle regeneration and reduced muscle strength due a decrease in. satellite cel. activity aid myogenesis (Conboy et al, 2005; Bockhold et a, 199K). It has been demonstrated that using the antagonists of the invention. muscle rgeneratiorn is increased by to minSJ mechanisms, e. by increang myobiast proliferation (Exataple 1), 30 and by increasing satellite cell activation (Example 2). The increase in muscle regeertion is associated with an increase in muscle strength as shown using both short term. (Example 4) 48 and long term (Exwople I1) adminiration of the antagonists of dhe ;nvention. The results demonstrated herein also provide evidence that the antagonistt of the invention can be used to improve tihe mnus ce mass and strength i a nber of corditi.ors, such as m IuscuJr dystrophy (Eaiuple 3) muscle in1 ury (txampe 8); srropenia (example ii); and cancer acheXia S(Etxamples 9 and 14). As the mechanism of action is similar for olhir conditions, it is predicted that sr.agonists will be useful for the treatmRent of inciiduals suffering from cachexia resuming fron burns nurses, prolonged bed rest, MITV or cancer and sarcopenia. Further, icreasing satellite cell aAdvatiOn Ln muswie Strength in young wild-type muscle (Examples 2 and 11) also indicates -1it the antagonists wi]l be useful for treatment of patients pre-operativly thus prevenung muscle Joss due to post-curgery enforced bed rest In dystrophic-conditions there is constant activation of satellite cesl due to repeated cycles of muscle degeneration and subsequent regeneration The present results demonstrate that the inventive antagonits arc able to increase muscle generation, satellite cell activation and .5 muscle strength in a muscular dystrophy mouse model (Example 5). As this mouse model is aj standard modi fPr husnan dystrophy, it is pnxdicted that individuals suffering fiou dystrophy 'will be able to be sucr;essfrdly treated with de myostatin antagonists of the'present invention. The two examples above show te effects of treatment of the antagonists an grip strength in young and old m1c (Example 4, and in decreasing the sym'poms in mdx. mice (Example 5), 20 where the beneficial effets are Ik-ely to be due to enhanced nyoblast proliferation, satellite cell actvaton and self-renewa, migration ofmyo bW asts and nacim-phages due to the effbets of our antagonists in reducingg myostatin levels below normal wild type, in addition to these mples it is expected that keatment with rthe present myostatin benerficial ef-fests in treaurert of ary miclcal conditn where truscle fincrion will be 25 improved by the effects of enhanced Ayobiast proIferation, satc..te ccl activation and self renewal, migration of rayblasts and macrophages. Such conditions include muscle loss after surgery, growth rtirdation, physiolo gical short stature, muscle loss due to chrnc illness, accelerating rhse covey of b!rnss patients OT otler disorders where improving Imusce function would be beneficia. 30 Skeletal muscle resistance to insulin-stimiated glucose uptake is the earliest known maifastadou of type 2 diabetes Mnellitus CCorregan t al, 199i). It has been shown that the lack of myosttIno partially attenuated the obene and diabetes phenoypes otwvo mo3se models, We agouti lethal yellow (Yei et aL 1 994), and obese. Addidioniliy the introduction of a myLvOS.tain £utafion ;o agOUt jethal yetow ice has- been shown to suppress fat accumulation by five.-fc'd (McPherron er. al, 2002). This, togethr with the present results 5 showing that the antagotists of the present nvtlhion are capable of decreasing fat deposition (Eyxalmplre 4 nd 11), Idicate that individuals suffering from the effects of diabetes, obesity, and hypergiyce.;c corditions will be able to be succesafinly V.reated with a tuerapfutIcaiiy effective dose of one or more myostatin atagonist of the present invertion, The present resuis aso show that the present myostatin ancagronists ane osefuu rj wound 30 healing. Every wound undergoes three distinct ph cases of wound healing. First'y the inflammatory phase for rhe detachment of deterirated tissue and foir wound cemsing; secondly a roliferarve phase for the deveiopmemi of granularion tissue; and thirdly a differentiation' or regeneration pbIse or maturation and s1ar formation (Sediarik 1994). The iilsrnatcry .phase is characterized by intilatraion of inflammatotceJs su.ch as 15 macrophages to ;he darnaged sire. The present results demonstrate tat use of the prser antagoisticrase the mgraoy pa;ty of camaicvrophages (xmple 3), This n with rereased wound size in skin (Exampie 6). decreased tcigen deposition in skin (Exampe 7) and increased musciC regeneraon as Lndicated by increased myogenic makers (Examipi.e 8) suggests rtha the present antagonits will be usef: in the treatment of wound healing. The 20 results show lhat Uhe benefjeial effects can be expected in wounds of v tissuesvhere creased macrophage and myobast migrationdsues w1C' )"elzj cicdtiatflrgeevtiiTofmsce isue a decreased flibro'sis arc usetti outcomes. It is aqo expected that the present myostatin antagonists wi!l be useful in the treatment of damaged cardiac muscle, Mostatin levels are regulated in cardimyocytera flowing 2.5 rmyocrdiwl infarction (Shanna et a, 1999) indicating that ihithdOn of myostatin may Umo% recovery of heart musCe after infarct. lIn addition, myostati ml. mice have increased bone minerd content and density as well as increased muscle mass (Hrimck et a!, 2002). it is therefore expected 1ud reducing myoatatin levels wil improve bone strefngthi and reduce osteoporosis ai other degenerative bone 30 diseases. Rhbabdomysaronas (RM ), one of the most common solid tmnor of childhood, express high levels of myosrali. Uit has been demonstrated . Iat hIibirion of myostatin allows RMSs to progress into the myogenic teniinal differeniation (Ricaud et al, 2003), therefore om anagonists may be useful for treatment of cancer. As discussed, above, it is expected that the atagonists of the present .invention wil be useful 5 for treating hannus. T le present antagonists wil alo e useful for treating other species given that the myostatiu sequence Ls highly homologous across specie:, indeed the huamnr and bovine sequences ase identical (FIG 31) and our aitagordits havc been demonstrated to increase p iMton of ovine, marine and human. cells in vitreo.. The present results also clearly demonstme tha; myostatin antagonists of the inventon can increase p-roliferatiou of 10 myobiasts (Example 1). REFERE~NCES The allowingg retfrences, to the exent that they Provide eemary. procedura or other details supplmentary to those set forth herein, are specifikaly incorpomted heIein by reference. The patent specifications, referro to throughout the text of this specification, are 15 also specifically incorporated herein by reference. Allen, RE, T'mm-Grave, CJ, Sheehan, SM, and Rice, ". (1997). 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NicI13olas G3, Theias M! Laugkv B'. et 61.. (2Fig~ti-carp wrc-hte with, and ~uae svwk on QA ingyustatin, . C:J I Phlv~sio 1 93' 2-31 Pabcersk-y LIR, FendlyEM )Fisher XL' Jwom- 'RMA MTatks Eltf, kte-Cray (In Tate, KMt 'Vehar CIA nd (3orm C-M (I 9) NInumnlian Cell- trarnstfiet exipression ef tisgiue factor for the S od ucdon fi] f wntigen , Ymtelli n. gin'r'r'(lf4~3 Padd-idge IA. (1997). Ussue calttnse of s.eeAW ratvi;c1.e. Me -hod' Bc iol 75, 1l3,1-4.1 Pammon WIL andl uen. Wi (1 98B1) , ye -od E''c- ioo~;a equence coitrpazion. P( .--o Nat I Mcad Sc. US A 8 5(,S): S2;.~ Rebbp~gaa A 13e i~abut., 'My'la -L1, Co sWeAJ, A,"tismw~ L, (2003), "MYo)S~atisigark I 0thow ~irns~.nhnagrovw4h I b .1-c gating pealiway tr; biok); adiji o M CelBiol 23: 7230-42. kjJR S,'' 3M , rU&lOs M, Bejniiirdl A, Thtvos 0, Ciirnac C.; Do.:nieu A. (03 intivoitic'n of. r-totr sections lit my ostaiti enlwices tetrnindditi-1oni haunn 1 io Rt. Camot-tv Ii. Arvo V-\, Dow-sa 1. (2001)~yoai ~'I~tS el Srviv'J ;vgL 2 n~yoenea~llioe-mix .fiophy.s ke~ Connurwn 250: 561-566. .13ore~~at..1. D., Imit, A .Parry. D. J. anid 'Palividp, I.A. (1 995)I. Clthurin"g sateJlize Ceell .Crot living khglte nausde fibe exrialts. in Vitrom Ce-4 Dt-~ ier An 3, 773-9. K~amia 51. I0rbadur Ri, Miie KC,. Snqiet WO1,C) -in C-P, -Coiigen .4- F wke PJ 20 Bassi R, (1999) vsti, raknnggtowzl. factor-bfin SuP~e&fLrjiiv meo-d'ber in expi-e!r5d in ieaxt ni.swlD axid is' tljpregullated ia ca-eiiottixye afe .inlarct. Y elPwil Sclm e 04, VWagnr KR, Stolz LE, ihbne r C, Riebci T, Ko-nen -"V, Bzmwn IThi-S, Le-e S'1. (2'04) MNyost -Lt in mttion -associatAee -,vtb grros sf h)iperftophy' in k ch-ild. 25N I3n~I A3 501,26)-268:2.-. Scdlavik K-M; The {eSof Wownd Vv'iin. Wng P ifn. PublirShed oiu-'1ine: harttinlnhn.14 S~~-~i, l~ackyS, wno~ A-!. Joi -rson NJ, .s S stanma.rs IT,, 'wad! Love 1"N (1991) iThe o (A G-CIft-uhe C-timlia, qitop-, for~ rapid purifi-cation of thu- catabvtic donrnmin ofnorroal rind .'11.1if ras GTa'-wtvtn pocn .. 1fic. Ch~cm. 2.66:14:!63 1'4166 5 MPir VF, Cwainbadut' R. kaplong F, Thonati M, -K. BaR~i JJ, Sharu.-i .M. (20QC) ?hie Imastan goenni is a ucnAemrtga pri of basicb~ xio-ei trL~mscriptiOn factor "A-YOD. 1401 Uefl f3iol 22: 7-364S2. 'Taibc Y. Esnl i K, Nom tira -, f19F. 6;. Skcletttni n.c!e pathokl ogy in -X crmstein I1C Taylor WE, Bislia S,. AM=': j4 et Al (2001) Mot]T isajbiti Qet fjrolii'c raton aad rorrf-in SYMMi~S inclc!-. mtuso;' celLr. Aiu .Y IPhy-sioJ Euo& o tab 28I'0: R22 10228, ~Tlloma- M, 1.pugiey 13. Bevyr CQ et A. (2000)) Myostatiri a negative rgiI.tor cif rcn- scJ e growth, functions by iuiii~mois xlf'air.J iBiol Chcm 27i: 4I0235.4024-3. Narng H., zia-rig Q, Zhn AD (M003) lispf Ovas wVith tha I muan1.rgionr of rnyostar.itv1 t ~P'ci~rj fiopxynPes Crrrmn 3 I '1 & vieh,-ng N), Cai 13, -and Tidball JC (2000);. 'Modulation of nmvos'taffi -xpr ssion during mcidfified n-sckl usc, Faseb 1 _ 4 10,3-11) Aral prooh tii drags. Aaynir.o Acids 30: -35j-367. 20 Y'ang L, Scott 11, Dod-d (, -Mcdia A, 1jia T , Shaakoiwsky 11A., GJiwy A rind .fledgcit EE. (205. dflcaiion of fibrocytes m'~ posttuau hzltr.'- hic. evan WeVtcv epk d. Rcgmun;atioun 13(4), 39 1.-404. >r~ ~r (JlV1 , D..g;r LCFari. OYS anid Wolff CIL (1,994.) Obes..ty.. djabei3,an neo y.-.w Av' /-rfsico e-C !-pic cxriresalu-n of toagou;-i gwjnr , T-*,~F3J .194IS :25 ahwidia ITLS;Ih~. SR' a-Iii pf'osizCdai . 3~ai (-199! lam rtloatn-il~~i.~jiflectve .J*~.i s increased sAft oroqpd bed v-ess wjthl lov-duose- YT3 adinbn~tatao Birvit iwsol 6,:11 rnystti prd~~shypc.-trop~hy wc~ithout hypc-.iplm~a _;l) sd'~ FEF185 Letters -i..eri TA, Davirs MV. KoniLris LCIL Iayns 1, Fsq%.la AF, Toxnn on K,. Mbpb cr. 'i ~3AC, NkVoiJfinan N, leS". (20302) Jndumziou uof cachexsia in inice. by sy-:4ea~uaiJy udvauiislhered niyo.t~l!hr. Sce.-ucf 296: 14-86--A'. INDr$'fRTAi, A'PI(AION The -r~n iJA&Sio p iosovel proteins haiviu.. myol mla-i iragorusoi. autivtity wl~ Lie se' ,t e Irf atinentI o.1 Myo tjmin re, kte d &order s -uU S 1jsorm l-rs ize eharac trwi.Sed, a' 1. east 1.n -part. lby an Ab normal 1en 1"owdel eopau cnt or maet ab ol if a oity of n-n-sele cor ? d ito s tissue -jR7.ina ramal am-n ayO ille tjdo d is r'ri -r & related to nmole hy p trophy; -,rai-ce a L-ophy and inusclfe wasb!ing wa:sorialud 'With ~.I.~doyM-opithirs, n usc-ular dystrophiel-, motor neuron- dismsfeS' diseasee, of the neou.iiirunctio-, diSEases C"' thc periphtral nerve, nvopathies dSuc 'o endokzine abnormdsitiesn, metabolic -- iyndomc.z lTv" . cacer, arorna 15 acheyda and crdhe wastin znii:.s za:.diac. '"ailur; csteoporusi!s; renal fiiilute or disease; liver fail'u.r. Or flisiase; anore'xia;. obcsivy, diayetes; zT.3f wovund healivtg.

Claims (20)

1. 3.11, 3112, 313, 314, 315, 316, 317, 318, 319, 320, 321, 22 323, 324, 3 327, 328 or 10 329. ragmnt varPnt or derivative thereof 3. An isolated reconabinant polypede as cimed in cl.im 2. selected ftom. the group conssting of a C-tem.namly mncatd mature myostalai polyppid, wherein the C uinal tmucation. is at amino acid position 329. 320, 310, 30. 295, 289, 284., 282 or 281 (SEQ ID NOS: 3-11 respectively), or a fragment, variant or dev. iv deof,. or a polypepti de having t5 substantial seq'uenc homology thereto.
2. 4. An i-solated polynuleotide coxnpiising a nuclectide sequence that enc.odes a olyppde of claim 1, or a compkmentary sequence thereto. isolated r a peptide of claim 1, o- polynuclotd o , the amino acid or nucleotide sequence is modified to produce a rnyostatin antagonist having-' 20 :electively altered binding caetic or having improved biodisrsibution or half life a vivo or on die shelf.
3. 6. An isolated recombinamr polyepxde or polynaceotide of clam 5 wherein the one or more modified amino acid residues ne selected from the group consisting of a amino acid, a iEGyLa-d anino acid. a famesyated amino acid, an acetylate. andno acid, a 25 iotiinylated amino acid. an amino acid conjugated to a lipid moiety, a D amino acid or an amino acid coniugated. to an organic erlvatizing agen. . A fusionl protein compiising one or more recoumbian, poypepjdes of claim 1 together: with one or more further polypeptides that enhance one or more 5nmdons selected from rhe group consiting of pmifi cation, formation of protein complexes, tissue localization 3) or distribution, uptake/administration, in vivo stablItV and/or in vivo half life. & A 5io~n Po-4c asi ciainnie in claim 7. hri the one or more tAler :p:oipte ') U :xm~oiobf~mFc cioin~~d as, an Ig(l Fc f 'agment,
4. 9. Afusion pne s cIimed -in clainm '!I ihorein thec one o aCof fii~ther po'.Yedu-. ooxnprisie a pa'ific~tion bsr'c 'eieted fon.I group (ornmorising In f'Pp rag& a
5. 10. A Afisiorr protni m~ climmed in clariIm .9 cuuprisivug a menhrapoyrid o clain 3, a polvbisti dineuQ se.qu--rIoo and a ~gsr~n-e Ui A fusion protein as claitmed in claki 1 0, 4eeb the rJhitdNer-.uuriee 113 lomatd at te N-trInl, of t ecmiar po 'p id Inthe tag. szkjIcj-jw is located atitho 2. Afuisio. protein as cl ill claims 11, fmreytke polyhistidirie seqnenmce consists of SEC! B? >10: 13, 1.3. A fusiou p-r.otein w,, clainud !I. c.Iai-v 10,hei'ebv th C w mrnnitg .scqucn.e conr.)s of -FEQ ID NO: 14, 15 1 4. A pharmaceuticals colmposilion eoirsn tkaust one isolated: povEpde of 61Iail 1. together wvithapacae.t ai ecpaA carrier.
6. 15. A haauica cr ~oitin omriingaticstone fson p~;ote of claim 7. 16, A mmthod of veating musce growth. prolmting ad!'pogenidif -n~ta and/or proluting tkmVcw grovwti or mied~.to.in an in- conising~~~~rfg to said 270 anjimnl au effl1-ctive arn-ownt of at. least onee pubiyprdi of clein I to an anin-Lal m cd thivreof:. 1.A method z,,aied lu claUu 16 comprising adninistration to sa~id- animw on~ f . effc~i~e rncatof at 'east ont! 17-am protein of claim "? to amemnli edteet 3). A method as dained -In claim 16 or 17 for produohijnc: resed mus,.le inan~s, dvcsedfa deostio. indori. nivedin gowh im '. shecep. urflce, dccv, poultry, tn~rk(ey, 25 pig. huxsfe. uon-su rar~, Ca., deo or- ininau
7. 19. .(nc-k o to proenst, cr&zt orr fCe iirit ofInysaiireae ~ioo ronditio7--m, whkhi is chrcei ,at least in part, by an kinurmal an.~ndevellopmn't rx;k smetabolic activity of Inluse j adipo-,,e fis.uu in in patient. wherein sr~id rne.tOwd arjinristnnganl cflfcctivc- kimount i at w lon.,a onec 1poypeptie of clnAmit .~teI in :. A meIuc4. as c.Mirried in claim 19 ;vii-ovein said .rmfthod conmrise .: a 5 effective arflount of mt 1ewa4 one Ii ii- protein of ctaini to a. patient iD reed +hel 2 1, A. -z.lhod ns CiflelmkUl C-laha 0 of 20.,K), ir the pavi:,cik condition is 3clectzo ftom the group conisisting of disorders related to rud5hypei.trophy; m1-ausde atrophy an-d -- uscRi w I-.Ig a~ssocialt-d with jatta=atory mOyopathics. mu-nular d ctrophis, mo:or 11UMr413 diSeas. rdisea e s of th'e niou~ua ucirdimse of ttje pteriphberal nert', 1 0 iyowithic,;, to edrieabnolmalities, Metabolic ~ydon~IRtV. can-cer. srco- enuip cache:xcm and othcr watn er-tim. cric In; sepo:ss;rn L uiue ordi,3ea:&e; Ihver failure or diseaseC: anorexi16a; ob-oty'; dia,"botes; and wNoxid ie=Iing 22, rntb-fAoprve~ veea -- or ameliorat a, v,;iio horeinj thl coludi [in 13 one . wsolnld bhcno.it 1 i ;.;. part, ftern jS,!;rP,,w. -saellite (;ell acutivatilon, ;Iyo-last pzoli ferati k)n, I rn~oph age and uy oblas-- ;-rigratiori. Pnd/cu red icedr tibrosis -wherein spid melbod i*xpihes adins~rjgan cftecdivo asmxI'Junt of at l'east one, pol.3peotirJre of (-la1n). I to al pyfier1t in lrod SA meth:-od zaLz clhainied in clam 22 wh ciii' s aid method compjists adxmiin~.jt'- c; ective amount ofa .laa I n sn protei of cli. 'I to -L. rtent.Li need ftIO. 20 :4, A. method as clai-med inl claimn 22 or 23-, whertirn the Condition is iSellwumd from "he goup consisting, of :1aus d( damlag, due0 to tratumla; =musclc. Manage due to thv radminislTation c,- agien-tssuha ciioery agen!-ta, radiation Lberapyr desarne-hirone- wucl waing due to-0 on bldreet such ',Li- at required aft-er surgery; vwund healing; dsdr related tv ascie hv2ipertrophyr- mus cie atrophy and muscle daniage associated wi-tf. idianuuar.o-v 25 myopathies. usculas dytrorihes, ~n t neu diseases, diea4 es of .. hle eosia LHS-2:m the of i prsrlo nxxve, mnycqiuhi -, due 1&o cendccrine albno-rn-aliie, lyeta Ibolic syneroniet , . H!'/ uccer,,s ,ZcAJcexia are othe~rwaig odir;caia faiure oseoorois:real aijreor disease:C liver failurec or dies;anorexia; o'be"J.ty and 2.5. . nhmakcIncati conpesliou us ciakned in claim, 1.4 or 15~, 8wreui fxe i'- :olattx podypeptie or Muson ;wokax ig Congaed to f4 fliucod pbta~I~ ad ac-, einpoud to tundnc Te fier~'utc Afe on the target ccIJ or fiss.31 ano wha~frein cbe t.iricuia crsitioi frua~dfr eaae na or simultaneously adrainistration of the 5 isolateO polypti'rlo, o-r R1 sif protein arid the second compud
8. 26. A r.~~o frgaIndguce growth of an MWimamp -in dm im-tri -to &aid anMmal an efWctiv a-z-ama of wt least one poiyppTdeof M 0aim I or a frs.ioii tui'otein o~f rciaiin
9. 27. A muethod as claims, claims 26, lo prcdiwce inroso us'cle mwss in. a slacep. caftle, 0 dieqr paoult, turey, pig, hams, cat dog or himan. 8'. A use of at 133!ft ono poly~eptide Af clin 1., in the nutcw o:'ariiam Lfo reguating muscle gcomth, pooigadipcgenic di ei~ aid/lo: r ~onoting bone gvcwth or tuineralizatian in an animal ILI _nenj there of :29. A mse of at bmast ono fusion pirokiu. tf claizu 7, in the mn.!_ufacture of a Intserjca'UnerAt for 15 'gillatIng:rnsl .o~h rm:igaioel difleret',afton mnd/or p-omoti g bone p.Ivwthi ox mlinexaiizat.cns. i awr-wmaJ in nee-d thmreof'
10. 30. A usu off Gcl 28 or 29 fobr p.-kOducing itncreasod. nuriclo m. krreas.: fat doporsit and/cr xrprve bc groth a shep ctle, dexr rioultry, tur~ke y, pig, JA1orse, mouse" rat. Cat, dog or hu1man, 20 3 . A mse of ast oe -piiypeplide of claim I in tlhe -imaiufacture of a Evedicimu to prevent, imal oP reduce e severity of a Imy-ostatin. ratdpathologic coi~m~ia w.hicla is, cilarac;.urisei., al. least.. pwA;: by an abnormal amount. .ovepmVIeA or tnctabolk, ar.iivity o rrjnscic or ndIpHose tissue in a. pfAient in ;,.ied thaeeof.
11. 32. A! ust, of at Ie:;+ on-, fusionl JuoteInJ of claim 7, in th;e inmatfacture of a Tmcdimuerit zo 2.5 Prevent, treat ox_- recfj7Fe .Lly Suverity of a Imyosratia mlelated patholo gic coxudk.in wi0hich .i c~caretiiscoatlea in p airt by an abnormal amount deve~kloen cx.j metabiolic activity o muscle or Bod~ome tjisue in a7 liazient in. neoed thereof 3j".3. A ls.: as clai-ed in C..aiam. 31l of _32. whereyr tie puahnIic;~ co.ndiox, i-3 selkected i*rjm the gronp onissthi of disorders relte d to mucehYPerq'JphY' us an'.opby and mus-avfce diseas,:S, of th o ~ua ~cin iacr olthe peihirinrenoahe due to eriicri-ac abniormalities, metabc~k synrdrrome, HIV, cancer, !3rcopcnma, cae:(ia and (Afitr ;ntn o tii;c~~i~u;o~opr~ s ron fIue' di zasa, ; liver failure or
12. 34. A us~e of at llkaAl onej4 polypeptide of' caim., in ticmaiotr of' a riAkwumit lh , ro-ve.uion. !PwAtneut wrae.o~Jno a voadl-doi vw.r irith ond o is one tha would bcnvtitn In..43 Car.41111 cxce .sd satcffifte cell ti wition, rnyolbi-~ proliferation, nnicrszpage n yol ast riacnand/ior rid uced flbxosis -W ej gaid me-.Jhoc n.pn it 0 adrnirni ag -n c-Blectivc a-mcount" of Ptl~t oe vptid2 ofdcaim 'I 'uo a partial !.-3 72e-ed SIMe7o
13. 35. A im -f fit hea:4 onre futsion xrotchin of ciaim 7. in. the u~rindfhm.tuv'e of a r;~ia.rIP; r~a r ~naU tmmu, us. aml i lwaf..ou ot a1oiitc hr-ith dto is owJ! rhat would btincaxIt., at, w u LeJ Ltlie'! drait, myo1'la"t pxoiif ,atiml, -~;varern-hago and m--~~tuigm-tion amlox' -redhuc ed fibrorAs 'wieuin !4c'ld merkod co'- rjpr.-kw's aduiii5wrIn~g -qjfevv amlolat. of itJoast, one Olypepidet o.fdc-inat I to a pitienmt in ee :36. A, vuae aj claimed in cla,.iii 34 oT 35 whereby the crmitlidri iJ!- s;Ale-teJd ftom the PT-. cossig of rnuscieJ'r d~a7-rvgc to r~nm imso-c datmigc duc thec admd',istxation of 20agent-' squch us cbe.rnothevi--'py agets raito hrp eanhoie njewast-ing due to prolauged bcdxres such as that iequisred alftex surgcxy; wounmd hecaling; :' scrdvrs related to nisck;' hyportsLphy; nmcle atro:phy ~iu~cle da:mage associaed with in~aamat ry rnpathies, ~rclirdsrpemolor neuron diseases, diseases of the- neurn scuj ,ar -juniclion, diseases of tile pr ihem necrve, (flatic u, to en.dkCHiK.bnrmnu-e 25 moaoi .- copcrtiar cadhexia wnid cor war -*in.ogs a~1a taiure o~teno o:j~ e;f .huk-*o disiease; Jivef Pillum. or di: uw.OlCX. obe Sity and diabetes.
14. 37. A rcetbtod of prodncirig an isola te, rr nbirianit poiy-,Ppt~id orci:r ,?dsd mn-eLiod comp rising 'hi: -- ten: 30 a. C.-temli-.ali:, truw~ting ma-,u, ,nFyost.ti ptUm ck a-. pos iou 329, 320n 310, 30. 95, 23~9, 284,22 r8i b, PCR awpiiying said uumatod petid; cloniug said popide i a suitable vector with a C-terminal and N-terminal tag 5, xpressing said pepd and purifying using said tag sequences; optioal ly removing one cr both Of said tag- wequencef.
15. 38. A method as claimed in claim. 37, where by the N-terminal histidine tag is removed by rsrsiction enzyme digestion.
16. 39. A method as claimed in claim 37 or 38, whereby the C-termal tag sequence is £0 movedd by s esirictionj enzyme digersdim. 4 An isolated recombinant prsypeptide produced by the method of any one of claims
17. 41., A method as claimed in claIm 21 or 24, whereby when the condition to be treated or pevented is sarcopenia, n eft'tive ammmt of the at least one polypep tide of claim 1 3r 1 usion protein of claim. 7, is administered.
18. 42. A method as claimed in Caim 35, whereby administrationis daiy.
19. 43. A method as claimed in Clam 35, whermby adaritration is three times a week, 44, A method as claimed in claim 35, whereby administration is once a fortnight
20. 45. A method as claimed in claim 35, wvhueby admiistaton is once a month. 20 4- Any invention described or claimed herein.