Appendix A: U.S. Patent No.

5,716,981

I
United States Patent WI
Hunter et al.
1541 ANTI-ANGIOGENIC COMPO!+ITIONS METHODS OF USE DS] AND sg80~99
5283,928 5,403,858 5,407,683 5,415.869 5.419.760 5,439,446 5.439.686 5.441.745 5;44i;724 5.449513 5,457;113 5$60,817 5.462.866 5&x,450 5,464,650 5,473,oss

USOO5716981A

1111 Patent Number: 1451 Date of Patent:

5,716,981
Feb. 10, 1998

lhP95 Fearnot et al. ...._.......a............ 604d-265
111995 Scott a al. .................................. 623/l 4/1995 Bastard et al. _.. ..... ..-.....- .... 514449 40995 Shivdy ._-.. .. ._-....... ............. 424439 50995 Saanbia$er et d. . ........... . .-. 424450 50995 Narciso, Jr .. . ............................. f&V8 WPPS Bany ....... ..” .- . ..” ............ ...... S/J%‘ Ikai CI al. L..._......._.............. 424/451 % resanr et al. . I.............” ........ 424450 iln9Ps l’ 911995 Helmus et al. ............. .._........ 424I426 911995 Yokoyama et al. . . ............... 424n8.08 1011995 Cul&xu~ el al. ............I .......... 514019 KY1995 Lpngley et al. .....“. ................. 424Mo8 l(Y1995 Wang .............. . ......“. ............. 435074 1111995 Busoemi et al. .. .._.................. m.. 623/6 .. 11/1995 3ag et al. . . ......................... 427R.3 w1995 Mongdli et al. ... . ...... ..“. ....... 53w329

Inventors: Wllhm L. Bunter; Lindmy S. Machan. both of Vancouver; A. Lmy Arsenault. Paris. all of Canada
Assignee:

179

Anglogenesis Technobgies, Inc. vancouver. Canada

[21]
[22]

AppL No.: 47833 Filed:
Jun. 7,1!w5

Related U.S. Application Data
[60] Divisim of Scz No. 417,160, Apr. 3, 1995, abaadooed, which is a continw&on-in-part of Ser. No. 94,536, Jul. 19, 1993, abaadoaed. Foreign ApplicPtion wm Priority Data

FORE?iGN PATEhT DOCUMENTS 375 520 Al 470 569Al 566 24.5Al 567 816Al 627 226Al 679 373A2 wo90113332 wo 91/10424 wo 91/11193 wo 91112779 WO 92f12717 WOW15286 wo93106792 w094/21308 wo 94Q4%1 wo95io3036 w095/03795 6/1990 211992 lo/1993 lUl993 211994 1111995 1111990 70991 8/1991 9/1991 ff.‘ 1992 9/1992 911994 1111994 21199s 2/1995 Europan Pat. Off. . bu‘ q=a~ Pu. off. . Enmpum PP off. . Euroum Pat. off. . Eum&n Pat. off. . E~lmpan Pat. off. . QIIPb. WIPO WIPO. WIPO. WE’ . 0 WPO WIPO. WJPO I WwO . WIPO

1301 [Sl]

id. 19, 1994 [wol [52]

. . ..... ... . . ..- PcwC494Q0373

Int CL6 .. ... ... ..._I......... A61K 3lf335; A61M 29/00 U.S. CL ... .. ... ... ... ....._..... 514/449; 12&‘ 898; 526/304; 5281421; 604153; 604I2& 60W21; 604/%; 6041269; 604’ 198; 623112 [58] Weld of Search .. ... ... ... ..._..._.........w.. 604/53. 20. 21. 604f96.269; 128f898; 514/449; 606/198;
623/12;528/421; 52#304

411993 WIPO

1561

References Cited U.S. PATENT DWUMENTS

4300,244 1111981 Bokms .....--..._...._e..........._..._ 1 3/l .4 4531,936 711985 ciodal “. ...-..................-- ....” ... 604149 .... 4542,025 9f 1985 Tice et al. ....... .._....m......w.... 424fi% 4580,568 4ll986 Gianlulco .... ........._“.............. 1281345 4,733,665 311988 edmm ..“^ .. ............... . .. ... .-. 1281343 4,739,762 40988 Pahu ..... ._........ . .I ... .._w..“. 1281413 4.776137 lo/1988 pahaz ..u........._...e................. 128/343 . 4.800.082. . 111989 G~~UUIOO ._........_.....w.. .... .v. 12StM3 “. ...... . . ... . ...... .” ... 604153 4,824.436 441989 WW 4.834.755 -5n9a9 sjbcwini et al. .-.- ._........... 623113 4.955.878 9n990 see et al. ... ..... .. . . ““. . ... . ... 604/181 4;960,790 lam990 skna et al. .. . .. I.. .... ..*.--.. ..... 514M49 4.989,601 2/1991 Mach&y et al. .-..... .. -.-. .... 12SI399 “.^ .- ...“. .. ...“. ...” ... . w191 4990,155 2p91 Whff s,o19,OsQ Y1991 Piadluk ... ..“. ... . ...“.“” .*. ... I .. 606w4 s,O49,132 911991 ShEa et al. ll.._..._ .. -- .. &XUOl .... .. 5.053.04s lo/1991 Pin&l& ........ . .” .... ........ . . . 623n 5,059,166 10/1991 Fiscbdl u al. . . ..... .w_........... ..... 6oon 5059.178 1011991 Ya .“.“. .“. .. ..“. ... ..“.....” ..“. ... . . w101 . @J&198 5.059211 lo/1991 Slacketd. “...““I.“..“.““..... S.092.841 3/1992 spaas .. . ... ..“. ... “” .. .. ... “..I ... 604i96 S.lrn.417 4i1992 Palmtz ... .... ... .“.............“..“I 60&l% s,171+217 12M92 Maldl et al. I.” ..“. .... “.“....... ... 6O‘ w53 . 5,171262 131992 Mecolegclr . .. ........... ...“f...” ... 623/l u d. .. . . ..... *........ .. 4wl.l 5.176.617 111993 Fii 5jl33, 50993 slepipl et al. ... .. I._ .................. 62311 5234,456 s/l993 siivesaini ... . ..”. . .... .”...I.. ... 606/M spa.54 2f1994 sbapdand et al. .......... . .“. ...... 604m 5292~12 3f1994 sdr.xfa et al. .. ..“. .... ....“. .... 424b401 5304,121 4Il994 Salvujinn .“. ....... ................ I .. . 604/53 s314.688 511994 Icau&Mn et al. ...“” .-.......... . 424423 891.1 $342348 8/1994 Kpplm I.._......._._..-__ ..... . @4.‘ 5342,621 al1994 B=Y ........ . ... . ........ “.“.” ..I .... w423

CYl’ HER PUBLICKl-iONS Walter et al., “Iutrrstitial Tax01 DcIivcrcd from a Biodegradabie Polyma Implaut against Experimental Maliguaut

Glioma.” Cancer Research 54~2207-2212. 1994.
Bartoli et al.. “ In vitro and in vivo antitumcral activity of ‘ free. and encapsulated taxoL” Journal of hiic~naqpsukation 7(2): 191-197. 1990. Bisset! aod Kaye. ‘ Tax01 and Taxotere-Current Status and Future Prospects.” European Journal of Cancer 29A(9): 1228-1231, 1993.

De Scheader et al.. %icwmpatiiility

of polymer-coated

ovasizedmetallicstentsirnplanMinwrmalpcrcinccoronary attexies.“Arhemckwsis 114: 10.5-114. 1995.

(List continucdon

next page.)

Primary EkaminerGfihailen&a Kumar Attomq Agent, or Fima-Sccd aud Berry W
[57l

ABSTRACT

The present invention provides compositions con@stig an anti-angiogcnic factor. and a polyrnaic carrier. Representative examples of anti-angiogcnic factors include AntiInvasive Facfca. Rdinoic acids and daivatives thereof. and paclitaxel. Also provided are merttods for cmboiizing blood vessels. and elimiuating biliary. urethral, esophageal. and trachcaubfonchial obstruuions. 18 Claims, 75 Drawbg Sheets

5,716$81
Page 2 OTHER PUBLXXlONS Flay et al.. ‘ Design of Biodegradable Polymer Systems for Controlled Releaseof Bioactive Agents,” in El-Nokaly et al (eds.). Polymeric Lklivery Systems: Pmperdes and AppLications, American Cbemieal Society. Washington, DC.. 1993. pp. 154-167. Holland et aL. “Polymers fa Biodegradable Medical Devices. 1. The Potential of Polyesters as Controhd Macromolecular Release Systems.” 3ownal of Contmlkd Rekmse 4: 155-180, 1986. J&L R. “Biodegradable Poly(LaUic Acid) and Poly(Lactide<o-Glycoiide) Polymers in Sustained Drug Deliveq.” Drug Develqment and Industrial Pharmacy 16(16): 2353-2367.1990. Kohkr and Goldspiel. “Evahation of New Drugs. Paclitaxel (Taxol).” Pburmacotkqy 14(l): 3-34.1994. Kuhn. J.. “pharmacology and Pharmocokinetics of Paclitaxel.” Annals of Phamacothe~ 28: S15S17, 1994. hger. R. “1994 Wbitaker Lecture: Polymers for Dmg Delivtsy and ‘ IIssue Engineering.” Anna& of Biomedical Engineering 23: 101-111. 1995. Pas&W.. “Synthetic Polymas in Modem Pharmacy.”Pmg. Polym Sci. 14(S): 629-677. 1989. Rowinsky et al.. ‘ Taxol: A Novel Investigational Antimiemtubule Agent,” Jourmi of the National Gmcer Institute 82: 1247-1259, 1990. Saoro et al.. ‘ The Anti-‘ hmor Agent, Taxol. Attenuates Conttadile Activity in Rat Aatic Smooth Musck.” Life Sciences 56(7): PL, 157-161.1995. Schindia et al., “Biodegradable Polymers for Sustained Drug Delivery,” Contemp. Tbp. Polym Sci. 2: 25 l-89.1977. Sinko and Kolm. “Polymeric Drug Delivery Systems. An ovavietv.” in El-Nokaly et al. (eds). Polymeric Delivery Systems: Pmperrits md Appiicatio~ American Chemical Society, Washington D.C.. 1993, pp. 18-41. Sollott et al.. ‘ Tax01 Inhibits Neointimal Smooth Musck Cell Accumulation after Angioplasty in the Rat,” Joumf of CZinicuZInvestigatZm 95: 1869-1876. 1995. Sonnlcbsen and Relling, “Clinical Phamxieokinetics of PaclitaxeL” C&z. Phmmacokhet. 27(4): 256-269. 1994. Spencer and Faulds. “PaditaxeL A Review of its Pharmacodynamic and Pbarmacokinetic Properties and Therapeutic E pne Treatment of Cawa.” Drugs 48(5): Vainion& et aL. ‘ Surgical Applications of Biodegradable Polymers in Human Tissues.” Pmg. PoZym Sci. 14: 679-716.1989. Yasumai et al.. ‘ placement of Covered Self-Expanding Metallic Stats in the Common Bile Duct A Feasibility Study.” Jowml of biascukzr andlnterventional Radiology 4: 773-m. 1993.

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POLYLACTIC ACID MICROSPHERES COATED IgG F127/lgG UNCOATED

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5,716:98 1 1
ANTI-ANGIOGENIC COMPOSITIONS METFIODS OF USE AND

2

Othershavesuggested Useof antibodiesin the treatthe meat of cancer. Briefly. antibodies he developed may which recognizecertaincell surfaceantigens are eitherunique. that CROSS-REFERENCE RELCl??D ‘ l-0 or mae pcvalent on cancerceils compared to rmmal c&s. s These ~tiies, a “magic builets.” may be utilized either AF’ FUWONS alone a conjugatedwit& a toxin in o&r to specifically This applicationis a division of U.S. patent applicati0n target and kill turn- cells GMlman. ‘ Antibody Therapy.” Ser. No. W417.160, filed Apr. 3, 1995. now abandoned; Frincip&s qf Cancer Bidwwpy. Oldham (ti). Raven which is a contirmation-in-part U.S. patent application Press. of hi.. New Yort 1987).However.onedifkiarltv is that I Ser.No. CRVW536, filed Jul. 19. 1993,now abandoned 1o most mormclonal antiies are of murinc origin. and thus hypersensitivityagainstthe murine antibody may limit its TECHMCALFIELD efficacy.particularlyafier repeated therapies. Commonside The psent invention relates gencdly to coqositiotks effcetsin&de fever.sweats chills. skin rashes.arthritis. aad and methods for treating cancer and other angiogenic- and nervepalsies. dcpeudent diseases. more sptcificahy. to compositions and One additionaldifficulty of presentmethodsis that local comprisingaoti-angiogenic factors and polymeric cauias, IS recutrcnceandlocal disease control remainsa major chalstern.5 which havebeen coatedwith such compositions.as leugein the treatment malignancy. particular.a total of of In well as methodfor utilixing thesestentsaud compositions. 630,ooO patientsannually (ia the U.S.) have localixed disease(PO evidence distantmetastatic of spread) the time of at BACKGROUND OF THR INVRNTION 1. presentation; represents of al those patientsdiagthis 64% Angiogenesis-dependent diseases(Le., those diseases nosed with malignancy doesnot include nonmelanoma (this which require or induce vasculargrowth) repaesent siga skin cancaror carcinomain situ). For the vast majority of ni.6cant portion of all diseases which medicaltreatment thesepatients, for surgicalresection the disease of represents the is sought.For example.canceris the secondltadiag cause greatest elm@ for a cureand indeed428.000will be cured of deathin the united states.andaccounts over one&M 25 after the initial ttWment428.000. Unfcxtuaatcly,202.ooO for of the total matality. Briefly, canceris characterkd by the (or 32% of all patientswith lokalixcd disease)will relapse uncontrolleddivision of a populationof cells which, most after the in&l treatment. thosewho relapse.the number of typkdly. leadsto the formationof oneor moretumors.Such whowillrelapseduetolocalrecmrenceofthedisease tumorsarealsocharacte&dbytheingrowthofvasculature amounts to 133.000 patients aapually (or 21% of ail those which provide various factcrs that permit continuedtumce 3. with localizeddisease). aumbtxwho will relapsedue to The growth. Although cancer is genaally mace readily diagdistantmetastases thedisease 68.000patientsannually of is nosedthan in the past. manyfcems, evenif detectedearly, (11% of all thosewith kxAized disease). Another 102.139 are still incurable. patients annually wiIl die as a direct result of an inability to A varieryof methodsarepresentlyutilized to treat cancer, cord& the local growth of the disease. including for example. various surgical procedures.If s Nowhere is this problem more evident than in breast treated with surgery alone however, many patients cancer,which affeus 186.000womenannually in the U.S. @arIiculatiy those with eertaia types of cancer. such as and whose mort&ty rate has remainedunchanged 50 fa bre.asLbrain. colon and hepatic cancer) will exprzience years. Surgical resection of the diseasethrough radical recurrence the cancer.Tbaefore. in additionto surgery. tn@eUomy.modified radical mastectomy. lumpectomy of or many cancers also treatedwith a combinationof t&a- 40 remains the mainstay of treatment for this condition. are pies involving cytotoxic chemotherapeuticdrugs (e.g.. UnfatunatelY. 39% oftbose treatedwith lunmectomvalone vinuistine. vinblastine,cisplatin.methotrexate. 5-FU, etc.) will develcpbrecuuence the disease, &prisir&y, so of and and/a radiationtherapy.One di5culty with this appwacth will 25% of thosein which the resectionmargin is found to however, is that radiothcrapeuticand chemotherapeutic be ckr of tumor histologically.As many as 90% of these agents arc toxic to ncamaI tissues, and often aoatc life 45 local recanren ceswillocauwithin2cmoftheprcvious thmtening sideeffects.In additiion,theseapproahesoften excisionsite. have extremelyhigh failurckuission rates. Shihrly. in 1991.over 1l3,ooOdeathsand 238.600new In addition to mgical, chum+ and radiation therapies, casesof liver meW&sis were npgtul in North America othersbaveattempWtoutilkeanindivi~sownimmune alone. The mean survival time for patients with liver systeminordertoeliminatecancerouscells.Fcrexample, x, memtascs is only 6.6 months once liver lesions have some have suggested the use of bacterM ce viral compo developed. Non-surgicaltnatmUlt for hepatic m&stases radiation. nentsas adjuvants adex to stimulatethe immune system include systemic chemotherapy. in to destroytuma cells. (SeegenerallyTrinciplea of Cancer chemoemhohxation. bepatic arterial chemotherapy,and Biotherapy,” Oldham(ed), RavenPress,New Ya 1987.) iam radiation.However,despiteevidencethat such Suchagentshavegermrallybeenuseful as adjuvantsand as 55 treatments transientlydecrease size of the hcpatk can the nonspecific .uimuhts in animaltuna models,but havenot lesions (e.g.. systemicchemotheqy and hepatic arterial as of yet provedto be genaally effective in bumarm chemothempy initiaUyreduces lesionsin H-2046. and 80% of patients.respectively),the lesions invariably reoccur Lymphoki.r4es also beenutilized in the treatmentof have Surgicalrcscetionof liver mctastases representsthe only cancer.Briegy. lymphokines are seueted by a variety of is cells, and generallyhave an efFecton specilic cells in the 60 possibilityfor a cure.but sucha procedure possiblein only 5% of patientswith metastases. in only H-208 of and gcllcratiotlof an immune response. Examplesof lympbolriJJc!s include Intffleukins (IL)-1. -2. -3, and -4. as well as patientswith primaty bcpatic cancer. One methodthat haskfsi attempted the treatmentof for colony stimuh% factors such as GCSF, GM-C!% and tumas with limited successis ttlaapcutic emboIimiocb M-W? Recently,one grouphas utilixed IL2 to sbmulak peripheralblood cells in orderto expandand producelarge 65 BrielIy, bloodvessels whi& nourisha tumor aredcliiately blockedbyhjeuion of an embolicmat&al into the vessels. quantities of cells witida arc cytotoxic to tumor cells (Rosenberg al., N. EJsgL Med 313:1485-1492,198s). A varie4yof mate&Is have been attemptedin this regard et J.

5,716,981 3
inciuding autologous substances asfat. blood clot and such choppedmuscle fhcgrBellts. well as altiflcial maaials as such as WOOL cotton. steel balls. plastic ar glass b&s. tankdmnpowder.siliconecompounds, radioactiveparticles. StKile absorbaMe gehlia sponge(st&pott. GtWam), oxi- 5 dized oeUulost (Ox@). steel coils, alcohoL lyophilixcd human dura mater (Lyodura), microlBriUar collagen (Aviteac). collagen fibrils (Tachotop),polyvinyl alcohol sponge(PVN, Ivaloa). Barium-impregnated silicon spheres 10 (Biss) anddetachable balloons.The sizz of l&r metastases may be temporarily&~easal utilizbq such methods.but tumors typically respondby causing the growth of new blood vesselsinto the tumor. A relatedpoblcm to tumor formationis the devel~meat of caacerous blockageswhich i&ii the fiow of material 15 through body passageways, as the bile ducts.uachca. such esophagus. vasadatureand urethra One device, the stcnf has been developedin order to hold open passageways which have been bloclad by tumors or other sukttanccs. Rqrcsentstive examplesof common stems iacludc the 20 Walkcat. Strecka steat. Gianmrcosteak and the Pahnaz stent The major ~obkrn with steats.however,is that they do not prevent the iBgrowI of tumcr or iat3ammatory mataial throughthe intastices of the steat.If this material reachesthe iasidc of a stcat and compromises stent 25 the lumen. it may result ia blockageof the body passageway into which it has bcca inserted.In additioa psencc of a stcnt the body may iaduccrcactivcn inflammauuytissue in (e.g., blood vessels.fibroblasts.white blood cells) to eata the steat tureen.resultingia partial cc compktc dosure of 30 the stent. The presentinventionprovidc~compositionspad mcthcds suitable for heating cancers.as well as other nontumcrigenic angiogencsisdcpca&atdiseases, fmtha and 35 providesother relatedadvantages. SUMMARY OF THE INVENTION Briefly stated, the present iaventioa provides aatiangiogeniccompositions, well as m&hods and dcviccs 40 as whkil utilize suchcompositioBs the treatment CancQ for of and other angiogeaesis-dcpcrideat diseases.Within one aspectof the presentinventioa impositions arc pvidcd (anti-angiogeaic compositions)comprising (a) an antiangiogenic factor and(b) a polymuic c&a. A wide vroiety 45 of molc4ulesmay be utilized within the scopeof theFcseat iavcntioa BSanti-aagiogcaic factors,induding for example Anti-Invasive Fador. retiaoic acids and $hcir derivatives. paclitaxd including analoguesand dcrivativts thereof, suramia. msuc Iahibitot of Metalloproteinnsc-1, Tiisuc so Inhibitor of MeUoproteinase-2. PiasminogeaActivakr lnhiiitor-1 and Pla.uninogtoActivator Inhibitor-2, and lighter Y group” transitionmet&. Similarly~ wide variety P of polymeric carriers may be utilized. rcprcscntative examplesof which include poly (cthyleac-vinyl acetate)SS (40% cross-linked).poly (D.L.lactic acid) oligomcrs and polymas. poiy (ihctic acid) oligotnus andJJO@IlKS, poly (glycolic acid). copolyn~~~ lactic acid and glycolic acid of poly (capolac&ne),poly (valr!rolactone). (anhydrides), poly t-apolymKsof poly (cqxolactone)or poly (lactic acid)with 60 jrolyethyleneglycol, sad blendsthereof. Within ccatainfrefand ca&o&mctits, the compositioas comprlsca compouad which disruptsmicrotubulefuadion, sucll as. for example.pacliuuctl. c.stramustiae. colchicjne, mcthokcxatc,curacin-A.cpothilone.vinblastineor BC!EV. 65 within otha preferred cmbo&unts. the compositious com@se a polymeric aria and a lighter d gropu transition

4
metal (e.g.. a vanadium species. molyb&rium species. tungsten species.titanium spcclcs.niobium species[x bintalum spccles)which inhibits the form&on of new blood vessds. witbin oneembodiment the invention.the composition of has an average size of 15 to 200 pm. within other emlmlheats, the polymeric cania of the compositionhas a molecularweightranging from less than LOGI daltoosto lzrcata than 2amoo to 3oo.coo daltolls. withill vet other mbodimeats.the comqitions provided herein-may be
klXtlCd blt0 fib With 8 lbi&eSS Of b&WCCB 100 p and

mm, or thamologically active compositionswhich are liquid at OBC tunpahue (e.g.. above45” C.) sad solid or semi-solidat snother(e.g.,37OC.). wil aaodla aspat of the presentinvcutioa methods for embolizinga blood vesselsre provided comprisingthe step of deliveringinto the vasd a therapeutically&ctive amount of M anti-angiogenic composition (as dcsaibcd above).such that the biti vessel is effectively occluded. Within one embodimentthe anti-aagiogenic compositionis delivacd to a blood vesselwhich nourishesa tumor.
2
within yet Moth+% aSpWr Of the PSCBt iDVClltiO0. StCDtS are provided comprising a geacmlly tubular struc~urc. the surface being coated with one cx mom anti-angiogcnic

compositions. within OthK asof the prtscnt inveatioa. methodsare providedfor expandingthe tureen of a body passageway. comprising inserting a stent into the passageway. stmt having a gcncraily tubular skucture, the the smfsoz of the skuclure being coated with an antiaagiogcnicamposition as dcsa-iid above.such that the passageway expanded. is within various embod&ats of the iave~tion.methodsare providtd for eliminating bilisry ohstructioas.comprisiag inserting a biliary stcnt into a biliary passageway; chmin&ng urethral obskuctions. for comprising insating a urethral stcnt into a urtthra; for eliminating esophageal obskuctions. comprising inserting an esophageal into an csopbagus; for elimiaatiag stem and kachcaVbroachial obskuctions. comprising inserting a tmchcalibroadtialstentiuto the tracheaor bfonchi. In each of these embodiments. dent has a generally tubular the StruUure.the surface of which is coated with an aatias dcsaii above. aagiogenicCOlBpO&OB W&in antier aspectof the presentiavcntion. mcthcds are provided fCr treating tumor excision sites. coqaising adminiskaingan anti-anglogcnlccompositionas dcscribcd above to the resaxioa margins of a tumor subsequeut to excision. such that the local recurmaceof cancer aad the formation of new blood vesscis at the site is i&Wed. wlthia yet Motba aspwt of the iaveatioa. mctbods for keating coracal ocovascularizatJoll provided, amlplisare iag the step of administering a patient a tbcrapcutically to cffccrive amouat of sa anti-angiogenic compo6itioa as dcsetihcd aboveto the coma. such that the formation of blood vessels is inhibited. Within one embodiment.the anti-aaglogtmlccomposhion fuxtlux compiscs a topical corticostaoid. Withia anotha aspectof the present invention, methods are provided for inhibiting angiogenesis patients with in non-tumorigcaic,angiogenuisdcpcadcnt diseases,comprising adminiskriagto a patient a tbaaptuticaUy effective amount of paclitaxel to a patient with a non-tumorigenic angiogeuesisdcpcndant disurst. such that the formationof new blood vessels inhibited. Within other aspects. is mcthads an pcovidcd for cmbolixiug blood vessels in aontumorigeBic,mgiogeaesis-depen&nt diseases, comlxising &lhmiBg to tilt vessela thcrapWticallyeffcctivc amountof a compositionatmprising paclitaxcl. such that the blood vessc.l cffcctivcly occluded. is

5,716,981
5 6
Within yet otheraspects thepresent of invention,methods image takenwith a stereomicroscope. JivJng,unstained of areprovided expanding lumenof a body passageway, cqiJlaries (1040x). FIG. 1C is a photograph a corrosion for the of comprisinginsetting a stent into the passageway, stent castingwhich showsCAM miuovasculaturethat arc fed by the having a genemdlyMxJar structure.the surface of the Jarga. urtderJying vessels.(arrows; 1300x). FIG. ID is a structure being coated with a composition comprisJng s photograph which depictsa 0.5 nunthickpJastic sectioncut paclkaxel. such that the passageway expanded. is Within transvasely through the CAM, and recorded at the light variousembodiments of&beinvention.methods provided micruscopeIed. This photogr;aph are showsthe composition for eliminatingbJJiary obstructions, compiisingJnsating a of the CA&I, including an outer double-layered ectoderm bilhry stent into a bihary passageway; ehminating (EC). a mesoderm contai&g cqiharies (arrows) and for @I) urethraJ 0bstmctJons. com&Jng insating a urethraI stent 1. scat&redadventit& cells. and a single layered endoderm into a urethra; for eEminating esophageal obstructions. (J3) (400x).FIG. I.3 is a photographat the eJectron microampxising insuting an esophageal into an esophagus; scopelevel (3500x) wherein typic& capibary structureis stent presented showing thin-walled endothelial cells and for eJimJnatin8 tracbeaUhronchiaJ obstructions,com@sing insettinga tracheal/bronchial into the &a&a or dent (arrowheads) an associated cyte. and pen’ bronchi.Within eachof theseembodiments stenthas a the FIGS. 2A2B. 2C and 2D am a saics of digitized Jmages genuaJJy tubularstructure, surface the structurebeing t5 of four diffaent. unstainedCAMS taken after a 48 hour the of coatedwith a compositioncoquJsJngpachtaxet exJxmre to digitized Images of four different living. Within anotheraspectof the prcscntinvention.methods unstained CAMS wac takenafter a 48 h exposureto 10~ are providedfor treatinga tumor excisionsite. comprisiig pacJitaxe1 10 ml of methylceJJulose. transparent per The administuiog a compositioncomprisktgpacJJtaxe1 the to methyJcclh&se disk (+) conraining paeJitaxeJ presenton is resectionmar@ of a tumor subse8utatto excision. such m eachC~bf and is positionedover a sing&w avas&ar zone that the localrecurrence cancerandthe formationof new of (A) wJth surroundingblood islands (Is). These avascular blood vessels the site is itdriited. within orheraspects, at areasextendbeyondthe disk and typically have a diameter methodsare provided for treating neovasctdar diseases of of 6 min. FJG. 2D illustratesthe typical “elbowing” effed the eye,cotqxising adminJsterJng a patient a thaapeutito (arrowheads) both .smaJJ large vesselsbeing rediof and caJJy effectiveamountof an anti-angiogenic factor (suchas a rectal away from the peripheryof the avascularzone. a compoundwhidt disrupts mJuotubuJe function) to the FIG. 3A is a photograph (Mag==x) which showsjust eye.su& that the fcrmation of new vesselsis inhibited. avascular WitJtJo otheraspects the present of invention.methods are peripheral to the numerous zone. that capillaries exhJbJt endothelialceJJs arrested in povided for treating in&mmatory a&ritJs. comprising (amowheada) (Ec); Mesoderm (M); Endoderm(Rn). administeriog a patienta therapeutically to &ective amount mitosis. of an anti-angiogenic factor (such as a compoundwhich x, FIG. 3B (Mag=tOOx)showsthat within the avascular zone structurehas been cJiminated dhupts microtubule function).ora composition comprising proper the typical capiJJary and there are numerous extravasatcd blood cells an anti-angiogenic fador and a polymericcauier to a joJnr (arrowheads). 3C (Mag==x) showsthatJnthe centrat FIG, Within preferredembodhnents, anti-angiogedcfactor the area of the avascularzone. red blood ceJJs disposed are may be a compoundwhich disruptsmicrotubulefunction suchas padJtaxe1. an demerit from the lighter ‘ grollp’ ” throughoutthe mesoderm. or d transition metals. such as a vanadiumspecies. FIGS. 3A. 333and X are a seriesof photogra@ of 0.5 Witbin yet aootha aspectof the invention.pharmaceuti- mm thick plastic sections transversely cut through a al products provided,contpisiog (a) a compound are which pachtaxei-treated CAM at three differcot locations within disrupts microtubuJe function. in a container.and (b) a the avascular zone. notice associated wJtJr container form prescrii by a 4o FXGS. 4B and 4C arc series of electronmicrographs the in 4A. goveromental agency regulating the manufacture. use. or which wae takenfrom locationssimilar to thatof FIGS. 3A. sale of pharmaceuticals. whJch notice is reflective of . 3B and 3C (respe&veJy)above. approval by the agency of a compoundwhich disrupts FIG. 4A (Mag=22OOx)shows a srnah capJJJary lying mJcrotubuJe tirnctioe for humanor vetetimuy administra- subjacentto the ectodermaJ Jaya (Ec) posse&JagtJuee tion to treat eon-tumcrigenicangiogenesisdependent 45 cndotheliatc&s arrestedJn mitosis (+). Severalother ceJI dJaeases as.for example. such intlammatoq arthritisor neovas- typesinbotJtthtectodermandmesodermareaJsoarrested cular diseases the eye.BriefJy,FederaJ requiresbrat io mitosis. FIG. of Law the useof a pharmaceutJcaJ Jn the therapyof humans phase contains43 (Mag=Z.8OOx) shows the early yvasasiar agent extravasated blood cells subjacent to the kc approvedby an ageney of the Federal govunmcm ectd, theseblood celtsarei&mixed with presumptive Responsibility enforcement the Ussited for (in States) with m cndothellalcells (*) and their processes. is DegratJve cellular the Fooda6dIhug Administration,which issuesappqrJate vacuoles(arrowhead). 4C (Mag==2.8OOx) thatin FIG. shows ngulationsfor securingsuchapJn0va.L detailedin 2I U.S.C. responseto pacJitaxe1. ecto-methe iutuface has ~$301-392. Regulationfor bJoJogicaJ mataiaLscomprising beumepopdated with cellsin variousstages degradatJon of pducts madefrom the tissuesof animals.is also provided containittgdensevacuolesand granules(auowheads). under42 U.S.C.$262.SJmiJar approvalis requiredby most 35 FIG. 5 is a bar graph which depictsthe size distrJbutJon countries,although.regulationsmay vary from country to of microspheresby number (5% poiy (ethylene-vinyl country. acetate) with 10 mg sodiumsurammJnto 5% WA). TJmc and other aspects of t&e presentinvention will depictsthe size distribution bwme evident upon referenceto the foJJowJng detaJJed of FIG. 6 is a bar graphwhichpoJy(ethylene-vinylantate) 60 microspheres weight (5% by dewiption and attacheddrawings. In addition, various references set forth beJowwhich de&be in moredetaiJ with 10 mg sodiumsuramininto 5% WA). are FIG. 7 is a graph which depicts the weight of ennrpsucatain proccdurrs. devices or compositions, and arc thcrclation of Sodium Suramin in 50 mg poly (ethylene-vinyl fare incclrporated by reference their entirq. in -1. BRIEF DRSCRR’ TIONOF THE DRAWINGS 6s FIG. 8 is a graphwhich depictsthe percentof encapsulation of Sodium Suraminin 50 mg poly (ctJrylen*vinyJ FIG. lA is a photographwhich shows a shell-lessegg cultureon day 6. FIG. 13 is a digitized computerdispJayed acetate).

5,716,981 7
RG. 9 is a bar graph which depictsthe size distribution

8
is unaffected. FIG. 19B is a photograph 20% paclitaxelof loaded thamopaste on a CAM. Note the disnrptionof the

by weight of 5% ELVAX microspheres containing 10 mg Y8sculattlre when compartd to the smndiog tissalcs. The sodium suraminmadein 5% PVA conmining 10% NaCL drug loadedpaste has blockedthe growth of the capihxies. FIG. 10 is a bar graphwhich depictsthe size distribution regression the largervessels.andcreated a region of by wdght of 5% microspheres containing 10 mg sodium 5 paused of avascular@ the CAM assay. on PlG. 19Cis a photograph suramii madein 5% PVA coataioing10% NaCL of 0.5% paditaxel-loaded tbemxmstc on a CAM (Man.FIG. 11 is a bar graph which depictsthe sire distribution 40x). B&ly. the paclitaxel&adcd thermopa& zisk by numberof 5% microspheres containing 10 mg sodium induwd an avascular wtle memrhg6mrniodiametaon suraminmadein 5% PVA containing10% NaCL 1o the CAM. This avascuhrr region was inducedby blocking new capWirygrowth andoccluding.disrupting.andregressRG. 12 is a line graphwhich depictsthe time courseof ing the existing blood vessels found within the treated sodium suraminrelease. PIG. 13 is an illustration of a representative embod&mnt region. FIG. 19D is a photographof control (unloaded) Thermopaste a CAM. Briefiy. after a 2 day exposure. on the of hepatic tumor aribolizatiw. blood vesse1 cu8anirationof the CAM (Mag=50x) treated RG. 14 is an illustration of the insertion of a repcesen-1s with the contrcl pasteshowsncrmal Moodvesselorganiratative stent coatedwith an anti-angiogenic composition. tion. Functional~easds arc locatedimmediatelyadjacentto FIG. IsA is a graph which shows the c&t of the tileunlwdedpaste. EV&PM polymer blend ratio upon aggregatioo microof FIGS. 2OA and 2oB are two photographsof a CAM spheres. FIG. 158 is a scanning electronmicrographwhich with control(unloaded) thermq~ste. 20 haviog a arrn~ treated showsthe size of “smaU”miuosphaes. FIG. 1sC (which Briefly. in FlG. 20 A the centralwhite massis the tumor indudes a magnitiedinset-labeUed WC-inset”) is a scan- tissue. Note the abundance blood vesselsentering dre of ning elexthonmiaograph which showsthe size of “large” twtm from theCAM in ali directions. tumorinducesthe Tire microsphaes. PIG. MD is a graphwhich depictsthe time ingrowth of the host vasculature through the productionof course of in vitro pa&axe1 release from 0.6% wlv ‘ kngiogeoic factors.” The tumor tissue expandsdistaIiy paditaxel-loaded50~50E!VAzEXA polymer blend micr+ 25 along the blood vesselswhich supply it FIG. 2OB is an spheresinto phosphate bufferedsaline (pH7.4) at 37’ C. undaside view of tireCAM show0 20A. Briefly. this view in Opeo circles are “small” sized microspheres. dosed and demonstrates radial appearance the blood vessels the of circles are “large” sized miuospheres.FIG. l5P is a phowhidt eata the tumor like the spokesof a whcd. Note that tograph of a CAM which shows the results of paditaxel the blood vessel densityis greatain the vicinity of the tumor releaseby microspheres (“MS”). FIG. l5P is a photograph30 thanit is in the surrounding normalCAM tissue.FIGS. 2OC simihu to that of SE al increase magnificati0~ andaODaretwophotograpbsofaCAMhavingatumar PIG. 16A is a graph which showsrdease rate prorilts treated with 20% paditaxd-loadedtbermopastc. Briefly. in tiom polycaproIactone microspberes contahliog 1%. 2%. FIG. ZOCthecentralwhite massis the tnmu tissue.Note the 5% or 10% paditaxd into phosphate buffaed salineat 37O paucity of bloodvesselsin the vicinity of the tumor tissue. C. RG. MB is a photograph which shows a CAM treated 35 The sustainedrelease of the angiogenesisinhibitor is with control microsphaes.PIG. 16C is a photograph which capable ovacomingthe angiogenic of stimulusproduced by shows a CAM treated with 5% paclitaxeI loaded miuc+ the tumu. The tumor itself is poorly vascularhedand is spheres. progressivdydtmeahg in size.RG. #ID is takenfrom the the PIGS. 17A and 17B. respectively. two graphswhich 40 undaside of theCAM shownin MC. anddemonstrates are Dixon of blood flow into the tumor whencomparui to show the rdase of paditaxel from EVA films. and the c~ntml tumor tissue. Note that the Mood vcssct dcosity is pacent paclitaxl rcmaimngin thosesamefilms ova time. FIG. 17C is a graphwhich shows swell@ of EVA/P127 rcduccdinthevicinityofthtturnor~disspiaserthanthat the of the normal surrounding CAM tissue. iilms with DO paditaxel ow time. FIG. 17D is a graph whidl shows the swdlitlg of Evmpall80 films with no 4s RG. 2L4 is a graphwhich showsthe effectof pacliuuel/ paclitaxel over time. FIG. 17E is a Bnph which depictsa PCL on tumorgrowth, FIGS. 21B and 21C arc two photostressvs. strain curve for variousEVA/FU7 blends. graphs which show the effect of control. 10%. and 20% paditaxel-loaded thm-mopaste tumor growth. on RGS. 18A and 18B arc two gra$s which show the mdting point of PCUhlepEo polymerblendsas a function FIG. 2% is a photographof synotium from a PBS of of % MePEG in the formulation (MA), and the percent so injectedjoint. PIG. ZZB is a photograph synoviumfrom a microsphere injected joint. FlG. 2X is a photographof inacastintimentededforpcL.pasteat6ooC.tobeingto cattihsgetirn joints injected with PBS. andFTG.22D is a solidify as a function of the amount of MePEG in the photographof cartilage from joints injected with micro formulation (18B). FIG. 18C is a graph whic!b depicts the spheres. softoessof varying PUfMePEG polymer blends.FIG. 18D is a graphwhich showsthe percentweight change ova time 55 PIG. 23A is a photograph of a 03% Paclitaxel Ophfor polymer blendsof variousMcPE!G concentrations. FIG. thalmic Drcq Suspcnsioa00 a CAM (Mag.=32x). The 18Eisagraphwhichdepictsrheratcofpaditaxdrrlew plastic ring was usedto localizethe drug treatmentto the over time from various polymer blends loaded with 1% CAM. Note the lack of blood vessels kated within and paclitaxel. FIGS. 18F and 18G are graphswhich depict the imu&iaWy adjacentto the ring. The functional blood elfea of varying quantitiesof paditaxel on the total amount a vessels bordaing the avasw zoac are definedby their of pa&axe1 released from a 20% McPEG/XL Mend.F’ lG. “elbowing” morphologyaway fom the dmg source. FIG. ISHisa~whichdcpiasttrc~~ofMePEGonthc 23B is a pbotogmpbof a control (unloaded)ophthalmic tensile streogthof a MePEGKL po!yrner. Drou Susnensi~ on a CAM Na~32x). Note the normal PIG. 19A is a photograph which showscootroi (unloaded) org&a&o of the CAM blood v&s& hodthe abundance thamopaste on a CAM. Note that both large vesselsand 65 of functional vessels locatedwitbln the ring. FIG. 24A is a photogmphof a 2.5% Paclitaxel-Loaded .smaU vessels (cxpibriea) arefoundimmediatelyadjacent to the paste.Blood Bow in the arcaaroundand underthe paste Stcnt Coating (Mw26x). Briefiy, the blood ~cssels sur-

$716,981 9
rouodingthe avascular zonem moiphologicailyredirected awayfrom the paclitaxclscurce;this producesan avascular zone which can measureup to 6 mm in diamucz The disrupted vascular remnants which represent vascular regression be seenwithin the avascularzone.FIG. 24B 5 caa is a control (unloaded)Stent Coatiag (Mag=26x).B&fly. the blood vessds of the CAM are found immediately adjacent the stult anddo not illustrate ahoy to 1~aph010gicd
(ce&andpaclitiuclat28pM);n3.

10
CFPD crystals (50 rnghl,. Bffcct of pa&axe1 at (0) no pad&cl, (‘ 28 p&f, (A) Control (cells atone), (A) Control )

FIG. 34 is a graphwhich depictsproliferationof synoviocytes at vaious concentrations paciitaxel. of FIG. 35 is a bar graphwhich depicts the cytatoxicity of paditaxcl at various coaccntrations to pro&rating synoviocytec alta;ltionS. FIGS. 36A. 36B. and 36C arephotographs a seriesof of 10 FIG. 25 is a photographof a control stint. Briefly, this gels which show the effcu of various conczntrations of image shows the longib~dinaiorientationof a nylon stent paclitaxelon c-FOSexpression. incorporatedwithin gliosarcoma tissue of the rat liver. FlGS. 37A and 37B are photographs a seriesof gels of Ingrowthwithin the nylon stmt is evident. which showtheeffectof variousconcentrations paclitaxel of FIG. 26 is a photographof a control stcnt. Briefly, this 15 on collagemscexpression. imagealso iuustratcstumor ingrowth WithiJlthe tureenof FIG. 38 is a bar graph which depicts the effects of the nylon stent. paditaxel on viability of namal chondrocytcs vitro. in FIG. 27 is a photograph of a lung. Briefly. in additionto FIG. 39 is a gap41 which showsthe parentage of padilargeliver tumors.metastasis the lung is amunon. Such taxel rdease basedupon gdatinixcd-pditaxcl of either a to mctastas.zs evident by the presenceof small white 20 large (7200 pm) or SmalJ (2100 pm) size. arc lobole seenthroughoutthe lung, FIG. 40 ls a graph which showsthe effectof gelatinand/or FTG. #IA is a photographof Suramin and Cortisone scdium chlorideon the rfkdsc of paclitaxtl from FTL. Acetate on a CAM (Map*). Briefly. this imageshowsan FIG. 41 is a graph which shows the release of paclitaxd avascular mne treatedwith 20 pg of suraminand70 pg of from PDLLA-PEG-PDUA cylinderscontaining20% padicortisoneacetatein 0.5% ~nethylcelklosc.Note the blood 25 UXCL vessels iocakd at tbc puipbuy of the avascular zonewhich FIG. 42A is a graph which depicts the time course of arebeingrcdireded awayfrom the drug source. FIG. 28B is paditaxei release from 2.5 mg pellets of PU. F’ 423 is IG. a photograph which showsthe vasculardetail of the dkcted a graphwhich showsthe percent paclita~elremainingin the rcgirm at a highu magnilication(Mag==2Ox). Ncte the avaaatlar regionsand the typical uclbowing”effectof the blood 30 pdlct, over time. FXG. 43Ais a graph w&b showsthe cl&t of MePEGon vessels bordcriagthe avascularzone. paditaxcl rdcasc from PCL paste lea&d with 20% pacliFIG. 29A is a graphwhich shoWs chemiluminescencc the taxel. FXG. 43B is a graphwhich showsthe percentpacliresponse of neutrophils (Sx106 cells/ml) to plasma taxel -g io the pdl#. OVCI time. qJsonized CFPD crystals(50 q/ml). Effect of paclitaxd at FIGS. 44A and 4dB are graphs which show the effect of 35 (@&no p~litaxel. (.) 4.5 JIM.(A.) 14 m. (A) 28 @t (0) 46 various amccntions of MePE!Gin PCL in terms of :n=. melting point (44A) and time to solidify (44B). FIG. 29B is a gqh which showsthe time courseconFIG. 45 is a graph which shows the effect of MePEG centrationdependence paclitaxel inhibition of plasma of cpsonizcdCPPD crystal inducedswtrq~hil chcmilurnine~ immpmdion into FCL on the tensile strengthand time to 40 fail of the polymer. ceoa. FIG. 44 is a graphwhich showsthe effectof irradiationoo FIG. 30A is a graph which shows superoxidt anion paditaxel release. productionby ncutrophils(5x10* c&s/ml) in responseto FIGS.47A, B. C. D sod E show tbc c&‘ed of MTX release plasma opsonizcd CPPD uystals (50 mglml). Effect of from PCL ovcztime. paclhaxelat (0) no pa&tax& (‘ 28 pM. (A) Control (cells ) alone);n=3. FIG. 30B is a graphicwhich showsthe time 45 FlG. 48 is a graphof partide diameter(pm) &term&d by a Co&a@ LS130 Partide Size Analysis. courseconcentration dependence paditaxd inhibition of of plam opsonized CPF’ uystal induced neuhqhil supaD PIG.49isagraphofpartidediamder()un)dckrmined responseof ncutrophils (5x10” ccWml) iu responseto plasma opsonizcd zymozan m&l). EiRct of paditaxe1 (1 at (0) no drug, p) 28 pM; x1=3. FIG. 31B is a graph which shows plasmaops5nized zymosan inducedncutrophilsuperoxide anion production. Effect of paciitaxel at (0) no 55 pacli~cl. p) 28 pM. (A) Control (cells alone);II 3. FIG. 32A is a graph which shows mydopcroxidase rekase from neutrophils(Sxld c&/ml) in responseto plasma opsonizedCPPD crystals (SO r@ml). Effc& of paclitaxelat (0) no pad&ax& (‘ 28 pM. (A) Con&d (cells 60 ) alone).(A) Control (cells witb paclitaxel at 28 JIM); n=3. FIG. 32B is a graph whid~ shows the co~~centration &pendence of paclitaxc.l inhibition of myclopcroxidase releasefrom ncutrophilsin responseto plasmaopsonized CPPD aystals; n 3. 65 FlG.33isagrap which showslysozymc rckase from mteophib (5x10 /ml) in responseto plasma opsoniizd
by a CoultacQ Is130 Farticlc Size Analysis. oxide anion production: n 3. FIG. 50 is a graphwhich showspaclita~elreleasefrom FIG. 3lAis a graphwhich showsthe d~cm&tminesocr~~e 50

variouspolymuic fcXmulations. FIG. 51 is a gtl?pbwhich shows the effect of plasma opsonlzation polymuic microspheres tit chemilumiof OP neLesceMx response neutr of (20 m&d microsphcrcs ip 0.5 ml of cells (cont. 5x1F cells/ml) to PCL microsphcres. FIG. 52 is a graph which showsthe dfcu of precoating plasma #-2% @Ironic Fl27 on the dlemilumirlesceBcc mpoose of neutrophils(5x106 ccll&lll) to PCL microsphacs. FIG. 53 is a graphwhich showstbt dku of prccoating plasma if-2% plurotic Fl27 on the chemiluminesccnce rcsponscof ncutropbils{5x10* cc.lWml) to P&WA microspheres. FIG. 54 is a graph which shows the effect of pccoating plasma i&2% plumoic Fl2’ 03 the chcmihukxence 7 responseof tsmtr@& (5x10* CWml) to PLA microsphacs.

5.716,981 11
FIG. 55 is a graph which showsthe effed of precoatiug plasma %2% pluronic Fl27 on the chemiluminescen~ respouse neuhcphils(Sx106 of cellsIn@toEvAL~miaospheres. FIG. 56 is a graph which shows the effect of precoating 5 IgG (2 mghl). or 2% pluronicF127 thenIgG (2 m@nl) on the cbemiiuminncence responseof neutrophilsto PCL microspbaes. FIG. 57 is a graph which showsthe effect of prccoating ZgG (2 rngfml), or 2% pluronicFl27 thenI@ (2 @ml) on 10 the &emiluminescence response neutropbilsto PMMA of miaosphaes. FIG. 58 is a graph which showsthe effect of ~cecoating JgG (2 mghnl), or 2% pluronicF127 thenIgG (2 mglml) on 15 the chemiluminescence responseof aeutrophilsto WA
miaosphacs.

12
sampleto he testedmy be readily datamid by visualimtion of the chick chorioallantoic membrane theregion in surrounding methylcellulosedisk Inhibition of vascular tbt growth may also be detedned quantitatively.for example. by detaminiog the numberand size of blood vesselssurroundingthe methyl cellulosedisk ascompared a conmi to methyl celbtlast disk. Although anti-augiogeuic factors as desaii herein are considered inhibit the formation of to new blood vesselsif they do so in merely a statistica~y significantmanna. as comparedto a control, within prcfenad aspectssuch anti-angiogenic factorswill completely inhibit theformationof new blood vessels. weU asreduce as the sizeand numberof previously existing vessels. ln additionto the CAM Way described above.a variety of o&a assays alsobe utilized to &amine the efticacy may of anti-angiogenic factorsin viva. including for example. mousemodelswhich havebeendeveloped this purpose for Res. In (seeRoberston aL. Cizncer. JL:1339-1344.1991). et addition.a variety of qsentative in vivo assaysrelating to variousaspec&of the inventionsdescribedherein have also beendcwibcd in more detail below in Examples5 to 7. and 17 to 19. As noted above.the mnt inventionprovides compositions comprising an anti-angiogenic factor. and a potymaic carrier. Briefly, a wide variety of anti-angiogeuic factas may be readily utilizd within the context of the presentinvention. Representative examplesinclude AntiInvasive Factor, retinoic acid and derivatives thaeof. paditaxd. Suamin,Xssuelnhibitw of Metalloproteinase-1. TissueInhibitor of Metalloproteinase-2. Plasmiuogen Activator Inhibitr~-1, Plasminogen Activator Inhibitor-2. and various fm of the lighta “d group”transition metals. Theseandotba anti-augiogenic faaorSwill be discusstdin mae detail below. BrielIy.Anti-InvasiveFactor,or “ AIF’ which is prepared ‘ from extractsof cartilage,containsconstituentswhich are responstble i&Wing the growth of new blood vessels. for l&se constituentscon4aisc a family of 7 low molecular weight wins (dO.ooO daltoos) (Kuettner and Pauli, “ Inhiiition of oe.ovasculariration a cartilage factor” in ‘ by Foundation Symposium IOO), 163-173.1983). pp. including a varietyof proteinswbicb have inhibitory effects againsta variety of proteases (Eisentein et al. Am. J. Pothol. 81337-345.1975; Langa et al.. Science193:70-n. 1976: and Horton et al., S&ICC lQQ:l342-1345, 1418). AIF suitablefor use within thepresentinventionmay be readily prepared utiiizing techniques known in the art (e.g.. E!isentein ai, sup: Kuettna apdPauli,supra;andLanger et et aL.SW). purified constituents AIF suchas Cacti@* of Derived Inhibitor (“CDP’ (SW Moses et at.. Science ) 248:1408-1410,1990) may aisb be readily preparedand utilized within the context of the presentinvention. Retinoicacidsaltertbemetabolism extracellularmatrix of eomponeuts, reMting in the inhibition of angiogeneds. Addition of poiine analogs. ang$ostatic steroids,or hcpariu may be utilized in OrdM to synagisticaliy increase the anti-angfogenic effectof mic acid.Retinoic acid,as well as daivativa thereofwbicb may alsobe utilized in the contut of the presentiovention, may be readily &&ted from commerc+l sources,in&ding for example. Sigma Chemid Co. (+RX25). Pa&axe1 is a highly dcrivarizeddimoid (Wani et aL, f. Am Ckcm.Sot. 93:2325,1971)which has been obtained from tite harvested and dried bark of Taucs bm@dia (Pacific Yew.) and Tamnyces Andrrmrac and Endophytk
Devclqineat ofthe Varcdar Sy+rn. Pitman tiooks
(CIBA

FIG. 59 is a graph which showsthe effed of ~ecoating IgG (2 tug/ml). or 2% pluronic F127thenIgG (2 mglmi) on
the cbemiluminescenctresponse
miCKOsphaeS.

of neutfophib to JXlkPLA

20

FIG. 68 is a photographof 10% methotrexate (WILY’ ) loadedmiuospberesmadefrom a 5050 ratio of PLA:GA loadedvanadyi sulfate from FCL. FfG. 62 is a photographof hyalurouicacid miaospheres containingvanadiumsulfate. FTG.63A is a graphwhich depictsthe &ease of organic vanadatefrom PCL. FIG. 63B depicts the percentage 30 of aganic vanadate remainingova a time course. FIG. 64 is a photographshowingpoly D.L. lactic acid microspheres containingwganic vanadate. FIGS. 65A and 6SBarephotographs control(uncoated)3s of stentswhich show typical epithelialingrowtbseenat beth 8 weeks (A) and at 16 weeks (B). Indentationsof the stent tines (t) and narrowing of the lumen (lu) are &own. There is progressive epithelial overgrowth the stentsurface of ova
this time hy fibrous and inflammatory tissue.
40

(Iv 0.78). FIG. 61 is a graph which depicts the releaseof 10%

2s

FIGS. 66A. 66B. 66C. and 66D are a seriesof photo. graphswhich show control and paciitaxe&oatedWary stents. FIG. 66A illustrates the cbliteratioo of tbe ‘ stent lumen by the processof benigncpithelial o~agrowtb.~At higha magnidcation(66B). the fibrous and intlsmu#ory 15 tissue is evident with little luminal spaceremain&. like paclitaxel-treatedhi&By dna remains patent (66c). At higher nqnification, ncmal biliary tract epitbelium # presentwith only minimal alterationof the mucosallining by the mated stent tines (t). so DEAEEDDEXRXFIT ON OFTHE RWENTlON As notedahove. the presentinventionpvides metheds and compasitions which utilize anti-angiogenicfactors. 5s Briefly, witbin tbe context of tbe prcseatinventioa antiangiogcnicfactors should be UrldcIstoWJ include any to prcrdn. peptide.CbemicaL othermolecule,wbicb ads to or inhibit vascular&row&.A vanity of methods benadily may utiliml to determinethe anti-angiogenic activity of a given 60 facts. including for example,chick cbtioallantoic membane (“CAM” j assays. Briefly. as described man?&tail in below ia Examples2A and2C. a portion of the shell from a fleshly fatilized chicka~ egg is removed,and a methyl cellulose disk containing a sampleof the anti-angiogenic65 factor to be testedis placedon the membrane. After several days (e.g., 48 hours), iubiiition of vasculargrowth by the

5,?16.981 13
Fungus of the Pacific Yew (Stierle et al.. Science 60~214-216.1993). Generally.paclitaxel acts to stabilize microtubularstructures bindingtubuknto form abnormal by mitotic spindles.“Paclitaxel”(which shouldhe understood herein to include anaiogues dcrivativcs such as. far 5 and example. TAXOL@.TAXCYIHRII@, l@desacetyI analogues of paclitaxel and 3Ndesbettzoyl-3%t-butoxy carbonyl analogues paclitaxel)may be readily preparedutilizing of techniques known to thoseskilled in the art (seealso WO 94I07882. WO 94107881. WO 94lO7880. WO 94/1)7876,10 WO 93/23555.WO 93/10076,U.S. Pat. Nos. 5294.637,
5983.253. 5279.949. X274.137. 5.202.448. 5.200534.

14
as vanadyl &fate mono- and trihydratcs. Representative examplesof tungstenaud molybdenum c0mp~exe.s itdude 0x0 complexes.Suitable0x0 Nngah sten complexes include tungstateand tungstenoxide complexes. Suitabletungstate(i.e.. WO,*3 complexesinclude ammoniumutngstate(i.e.. (NH&WOJ. calcium tongstate (i.e., CaWO,). sodium tungstate dihydrate (i.e.. Na,WO,.2H,O). and tuugstic add (i.c. H,WO,). Suitable tungsten oxidesincludetunp;steo oxide (i.e.. WO,) and (IV) tun&en (VT) oxide (i.e.. WOa). Suitable0x0 molybdkwn complexes include molybdate. molybdenum oxide. and molybdenylcomplexes. Suitablemdybdate (i.e.. MOO,“) complexes include ammonium molybdate (i.e.. (,NH,) &io04) andits hydrates. sodiummolybdatc(it.. Na,MoO,) and its hydrates.and potassiummolybdate (i.e., I&MOO> and its hydrates. Suitable molybdenum oxides -include mofybdenum(VK) oxide (i.e.. MoO,). molybdenum(VI) oxide (i.e., MOO,). andmolybdlc acid. Suitablemolybdenyl (i.e..MOO,*+) cornmexes include.for examnle, molvbdenvl tktyiace6natc. Other suitable tungstenand molybdenum complexesinclude hydroxo derivativesderived from. for example.glycerol. tartaric acid. and sugars. A wide variety of other anti-angiogenic factors may also be utilized within the context of the present iuveation. Representative exampiesinclude Platelet Factor 4 (Sigma Chemical Co.. #Fl385); Rotamine Sulphate (Clupeine) (Sigma Chemical Co.. rCP4505);SulphatedChitin Duivatives @repared from queencrab shells). (Sigma Chemical Co.. K3641; Murata et al.. Cancer Res. SEX?-26, 1991); Suiphated Polysaccharide Peptidoglycaa Compkx (SP-PG) (the function of this compoundmay be euhancedby the presenceof steroids suchas estrogen, tamoxifea citrate): and Staurospotine (SigmaChemical Co.. +&i400); Modulators of Matrix Metabolism. including for example.proline analogs { [(L-azetidine-2-carboxylic acid (LACA) (Sigma Chemical Co.. #A0760)). CishydroxyproIine, hL-3.4dehydropsoline (SigmaChemicalCo.. #DO265).Thiaproline (Sigma Chemical Co.. aO631)J. a.a-dipyridyl (Sigma Chemical Co.. #D750S), ~aminopropionitrile fumarate (Sigma ChemicalCo.. #A3134)1]: MDL 27032 (4propyl5-(4-pyridinyI)-2(3H)-oxazoloae; Merioo Merrel Dow Methotrexate(Sigma Chemical Co.. ResearchhtiNte); #A6770; Hirata et aI.. Arthritis and Rheumatism 32X%5-1073.1989); Mitoxantrone(Polverini and Novak. Biochem. Biophys. Res. Comm 140:901-907): Heparin (Folkttm, B&Y. Phar: 34905-909. 1985; Sigma Chemical Co., #P8754); Intetferoos (e.g., Sigma Chemical Co.. #13265); 2 MauoglobuIin-serum(Sigma Chemical Co.. WM7151); ChIMP-3 (Pavloff et al.. J, Bia. Chrm. 267:17321-17326.1992); Chymostatin (Sigma Cbcmical
and vaaadyi Sdfate inChIding vanadyl sulfatehydratessuch

~kU29, and HP 590267).or obtainedfroma variety of ammercial sources. includfngfor example. Sigma Chemical Co.. St. Louis. Miss. (T7402---fromTearsbrcvtifoli). I5
Suramir~is a polysulfo~atcd naphthylurca compound that is typicaUy used as a trypanocidalagent Briefly, Suramia

blocks the speciiic cell surfacebinding of various growth factors such as platelet derived growth factor C’ PDGF”), epidermalgrowth factor (‘ EGF”). transforming growth fac- 20 tor (‘ TGF-r). insulin-like growth factor (“IGF-I”). and fibrobiast growth factor C’ fIPGF”).Suraminmay be pepared in accordancewith known techniques.or readily obtained from a variety of commercial sources. includingfor exampleMobay Chdcd Co.. New York. (seeGagliardiet 25 al.. CancerRcs. 525073-5075.1992;andCoffey.Jr.,et al.. J. of Cc& Phys. 132:143-148. 1987). Tissue Inhibitor of Metalloproteiuases-1 (LTIMP”) is secreted by endothelialcells which also secrete MTPases. TlMP is glycosylatedand has a molecularweight of 28.5 30 kDa. TIMP-1 regulates angiogenesis bindingto activated by metalloproteinases. thereby suppressing invasion of the blood vessels the extraccllularmatrix. ‘ into Issue Inhibitor of Metallcproteinases-2TIMP-2”) may also be utilized to (‘ inhibit angiogenesis. Briefly. TIMP-2 is a 21 kDa nongly- 35 axylated protein which b&is to mewoproteiuases both in the active and latent.procnzymefcrms. Both TIM&l and TIMP-2 may be obtained from commacial sourcessuchas Syoqen. Boulder.Colo. 40 PlasminogenActivator Inhibitor-l (PA) is a 50 kDa glycoproteiuwhich is present bloodplatelets, canalso in and be synthesized endothelial by cells audmusclecells. PAIinhibits t-PA and urokinaseplasminogenactivetar at the basolatcralsite of the endothelium. additionallyregu- 45 aud lates the fibrinoiysis process. Plasminogen Activator Inhibitor-2 (PAI-2) is generallyfound only in the blood unducertain circumstances such as in pregnancy, and in the presence tumors.Briefly. PAI- is a 56 kDa proteinwhich of is secreted mouocytes macrophages. is believedto so Co., uc7268: Tomkinson et al., Biochem J. 286:475-480. by and It regulate fibrinolytic acfivity,andin particularinhibits uroki1992); ~clodextrin TetradecasuIfate (Sigma Chemical nase plasminogeo activator and tissue plasminogen Co.. UC4767): Hponemycin; C.amptothccin;Fumaglllln activator, thereby preventing filxinolysis. (SfgmaChemicalCo., #F6771;Canadian PatentNo. 2.024. Lighter “d group” traasition metals inclu&. for example, 306;Iogberet aL. Nararc348:555-557,199O); Sodium Gold vanadium,molybdenum mngstcn.titanium, niobium. and 5s Thiomalate(WV”; Sigma:G4022: Matsubaraand-ZfI. J. tantalum species.Such transitionmetal speciesmay form Ciin. invest. 79:1440-1446. 1987); (D-Penicillamine transition metal complexes.Suitable complexes of the (“CDPR Sigma Chemfcal Co.. #P487S or P5OOqHCl)); above-mentioned transitionmetal species include 0x0 tranpl-anticolla~enase-serum: a2-antiplasmin (Sigma Chat sition metal complexes. C0.:A0914: Holmes et al.. J. Biol. Chem. 262(4) Relxeseutative examples vanadium complexes of include 60 :1659-1664;1987);Bisantrenc(National CancerInstitut& oxo vanadium complexes such OS vanadate and vanadyl Lobenzarit disodium (N-(2)-carboxyphenyl-4chloroaothronilfc disodium or ‘ acid YICA”; Takeudri et al. complexes. Suitablevanadate complexes include mctawnadate(it.. VOs-) andorthovanadate VO,‘ complexes (i.c, -) Agenrs Actions36:3%316.1992);Tbalidomidc;Augostat.k such as, for example. ammonium metavanadate(i.e., steroi& AOM-1470; carboxymiminolmidazo1e; metallopro NH,VOs). sodiummetavauadate NaVO,), and sodium 6s teinase inhibitors such as BB94 and the pcptide (i.e.. catt~ovamdate Na.,VO,,). (k. Suitablevauadyl(i.e.. VO*+) CDPGYIGSR-NH,(SEQUHNCHID NO. 1) (Iwaki Glass. complexesinclude. fcf example,vanadyl acetylacetoaate Tokyo. Japan).

5.716.981 15
Althcugh the above aoti-angi~genic factors have been jxovided for the purposesof ilhMMion. it shouldbe understood that the present invention is not so limited. In pmticular. althoughastain imti-angiogenic factors xc specifically referredto above.the presentinvention shouldbe 5 understood include smalogues. to derivativesandcoujugates of such anti-angiogenlcfactors. Fn example. padibrxel should be understoodto refes to not only the commoo chemicallyavailableform of paclitaxel,but analogues (e.g.. taxotue. as noted above) and paclitnxel conjugates(e.g.. 10 paditaxd-FEG. pactitaxel-dextran, paclitruel-xylos). or Aoti-angiogeniccompositionsof the presentinvention may additionallycomprisea wide varim of compounds in additionto the anti-angiogenic factor andpolymeric carrier. For example.anti-angiogeniccompositionsof tie present 15 invention may also. witbio cat&o embodhents of the inveotioll, also comprise one or more aotiiotics antiidumnaties. anti-vital agents.anti-fungalagentsand/or anti-protozoal agents.Representative examples antibiotof ics included within the compositions described herein 20 include: penicillins; cepbalosporinssuch as cefadroxil, cefazolin. cefaclor; aminoglycosidessuch as gcntamynh and tobramycin; sulfonamidessuch as sulfamethoxa~ol~ and metronidaxole. Representative examples of antiinflammatories include: steroids such as prednisonc. 25 prednisolone. hydrocortisone. adrenccorticotropic hormone. and sulfasalazioe; and oon-staoidal a&-lngammatory drugs (“NSAJDS”) such as aspirin. ibuprofen. nqxoxen. fenoprofen,indomethacln.and phenylbutazone. Relxesentative examples of antiviral agents include acyclovir. 30 gaocidovir. xidovudiue. Representative examplesof antifungal agentsiocludeznystatin.ketoconazole. griseofulvia flucytosint. micooaxole. clotrimazole. Representatfve examples of antiprotozoal agents include: pentatuldine isethionate. quinine. chloroquine.and mc5oqttine. 3.5 Aoti-angiogcnic compositionsof the presentinvention may also contain one or mcxe hormooessuch as thyroid hormone.estrogen. progesterone. ccatisoneand/or growth hormone. other biologically active molecules such as insulio. as well as TH1 (e.g., Merleukins-2, -l2, and -15. 40 gamma ioterfaoo) or Tr,2 (e.g.. Interleuhin~ and -10) cytokioe5. Within certainprefared embodiments the invention. of aoti-angiogeoic compositions are pr0vided Which Contain oneor Inorecompounds which disruptmiuotubuk function. 45 Representative examplesof suchcompounds includepaclitaxel (discussed above), estramustine (available from Sigma: Wang and Steams CMcrr Res. 48:6262-6271. cumcin-A. col&icine. meth0trexiu.e. vln1988).epothilone. blastine and 4-tat-butyl-[~2chIoroctbyl)unido)~~~ 50 (‘ %BCEW). hti-aogiogenic compositionsof the presentinvention may alsocontaina wide variety of othercompcnmds, lndudiog for example:a-adreoagic blocking agents.angiotensin II receptor ~tagouists aod receptor antagonists for 55 histamioe.serotonio,endothc~, i&bitom of the sodium/ hydrogen anfiPerta (e.g.. amllcxide and lts derivatives); agentsthat modulate intracellular cd+ trampa? such as I+e (e.g., diltfazem. nifedipine. vempamil) or T-type Ca + channel bfockas (e.g., nmiloride),calmodulinatttagct 60 nists (e.g.. H,) and inhibits of the sodium/caWrmantiporta (e.g.,amilaidc); apl inhibitots (for tyrosinekinases, urotein kinase C. mvosin light chain kittase, Ca2+/
L calmQdulinkinaseD;&inldna;eH);Mti~&(~g.

16
G-CSE epidamal gmvtb factor. transfamiug growth fsctars.@&a and beta,TNF. aud antagonists of vascularepithelial growth factor. endothehaigrowth factor. acidic or basicfibroblastgrowth factors.andplateletdervivedgrowth factor); inhibitorsof the Ps receptor(e.g..heparin); protease and collagenase inhibitors (e.g..TIMPs. discussed above); nihovasc&lators (e.g.. isosorbide dir&ate); anti-mitotic agents(e.g..colchicine.anthracydinea otherantibiotics, and folate antagonists and other anti-metabolites, vinca alkaloids. nltrosoureas. DNA alkylating agents. topoisomerase inhlbitcxs. purine antagouists analogs. and pyrimidioc aotagonists analogs.alkyl sulfonates): and immunesuppressive agents (e.g.. adrenocorticosteroids, cydospaine); sense or antisenseoligonucieotides(e.g.. DNA. RNA. nudeic acid anatogues (e.g., peptidenucleic adds) or any combinationsof these): and i&&hors of transuiption factor activity (e.g.. lighter d grouptransition metals). Anti-angiogeniccompositionsof the presentinvention may alsoamprise additionalingredients assurfactants such (tither hydrophilic or hydrophobic;seaExample 13). antineoplasticcc chemo~c agents(eg.. 5-fluorouxacil. virwaihe, vinblastine,cisl%atin. doxyrubicin.adriamycin.
or tamodfen), radioactive agents (e.g., Cu-64. Q-67.

Ga-68. 2.89. Ku-97. Tc-99m Rh-105. Pd-109, In-111. 1-123. I-125, I-131, Re-186. Re-188. Au-198. Au-199. Pb-203,At-211. Pt+212 and Bi-212) or toxins (e.g.. &in. abrin, diphtheriatoxin. cholera toxin. geloain. pokeweed antiviral protein, ttitin, Shigella toxin. and Pseudomonas exotoxinA). . As noted above. anti-angiogeniccompositionsof the preseotinventionco@ an anti-augiogeuic factor aad a polymezic cania. I0 addition to the.wide array of antiaogiogenicfactors and other compounds discussed above, anti-aogiogenic compoaitioosof the presentinvention are provided io a wide variety of polymeric car&s. including for example both biodegradableand non-biodegradable compositions. Representative cxamplcs of biode~dable compositionsinclude albumin. gelatin. starch. cellulose, dextmns,polysaabarides, 6ogeo. poly (DL lactide). poly (DL-&tide-coglycoli&). poly (glycoli&). poly (hydroxybutyrate), poly (alkykarbooate) and poly (os?hoaters) generally.Ill&n, L. David& S. S. (eds.) (see ‘ Tolyrmxs in controlled Drug Delivery” Wright. Bristol. 1987; Arshady. 1. Com&d Release 17:i-22. 1991;Pitt ht. J. Phm 59:173-l%. 1990;Hollandet al., 3. C’ cmtmlkd Reimse 4:155-0180, 1986). Representative examplesof nondegradable polymersinclude EVA copolymers. silicone ~bber and poly (methylmethactylate).Particularly preferred polymeric carriers include poly (ethylene-vinyl acetate&W% cross-linked),poly (DMactic acid)otigomers andpolymers.poly (L-lactic acid) oligomersandpolymas, poly (glycelic acid). qx+urs of lactic add andglycolic acid, poly (caprolactooe). poly (valerolactone), poIyaohydrides, copolymas of poly (cqXolactone)or poly (lactic acid) with polyethylene(#ycol and blendsthereof. Polymericeauiersmay be fashioned a varietyof forms. in induding for example.rod-shaped devices, pellets.slabs.or capsules(see. e.g., Goodell et al.. Am 3. Hosp. Phann 43:1454-1461,1986,Langa et al.. “controlled releaseof macromoleatles from polymas”. in BiomediccIpolymers
Pdymen~c materials and phanmceuticals for biomedical WC, ~dbesg,EP.,N~~k(eds.)AcademicRess,W 1%137, 1980; Rhine et al., J. Pliam 5%. 695?65-270. 1980;Brown et al., 1. Pham sci. 72:1181-1185.1983; and Bawa et al.. J. ConrmucdRelease 1:259-267.1985). Antl-

amytriptyline. fluoxetioe, LWOX@ and PAXTL@); cytok- 63 ine and/or growth factors. as well as their respective aogiogenic kctors may be linked hy ocdusion ia the mafrireceptors.(e.g.. tbc interleukins, a. p cx ~IFN. GM-CSl?

5,716,981 17 18
ces of the polymer.bound by covalentlinkages.cr cncap. to 100 pm thiclc Such fihns are preferablyflexible with a sulated in microcapsules. Within certain preferred good tensile strength (e.g.. greater than 50. preferably embodimentsof the invention. anti-angiogeniccomposi- greaterthan 100. and mate preferably greater than 150 or tions are provided in aon-capstdat formulations such as 200N/cm2), goodadhesive prop&es (i.e..readilyadheres to microspl~aes(rangingfrom nanometers micrometers s moist d wet surfaces).and has controlkd pameability. to in size),pastes.threadsof various size. films and sprays. Representative examples suchfilms areset forth belowin of Preferably.anti-angiogenic compositionsof the present the Examples(set e.g.. Example l3). invention (whicfi comprise one a more antI-angiogenk Representative examplesof the incomomtion of antifadm. and a polymeric carrk) are fashionedin a manner angiogenicfactors such as those describedabove into a appqxiate to the intendeduse.WQhincertainaspects the 10 polymctic cauias is desuii in more detail below in of presentioveatioa the anti-sogiogenic compositionshould Examples 3.4 Md 8-15. be biocompstiblc.and release oae or more anti-angiogcnic POL~CURRIERS FORTHERELEASE daysto months.For exampk, factors over a periodof several OF HYDROPHOBICCOMPOUNDS “quiclt release” “burst- anti-angiogenic or compositions are of providedthat releasegreeterthan IO%, 20%. oc 25% (w/v) IS W&i0 furrhu aspects thepfueot inveotioo.polymeric cactias are provided which are adaptedto contain and of an anti-angiogenic factor (e.g..paclitaxel)over a p&d compound. carriercontainingthe the of 7 to 10 days.Such“quick &case” compositionsshould, releasea hydrophobic hydrophobiccompound in combination with a carbohydrate, within cerrainembodiments. capableof rekasing chebe protcio a poIypeptidc Within cectaio embodiments. the mothaqmtic levels (where applicable)of a desiredantia angiogenic factor Wlthin other embodimtnts. “low rekase” 20 polymeric anicr conrains comphes regions.pockets.or granules of one or more hydrophobic compounds.For anti-angiogcnic compositions provided that rekase kss are example,whhio one embcdmeotof the invention, hydrothan 1% (w/v) of an anti-angiogenic factor over a period of phobic compoundsmay be inccapceared within a matrix 7 to 10 days. Further.anti-angiogenic compositionsof the the hydrophobiccompouad followed by presentiovcntioa should pzfcrably be stable fa several which conmonthsandcapable bcingproduced maintained of and unda 2s incorporation of the mati within the polymeric cmia. A variety of matricescan be utilized in this regard,including stuik conditions. for example, carbohydrates polysaccharides and such as Within certain aspectsof the presentinvention, antistarch, cellulose.dextran.methylcelhrlose. hyaluronic aud angiogenic compositionsmay be fashioned in any sixe acid.proteinsor polypeptidcs as albumin.collagenaad such ranging from 50 mo to 500 pm. dcpendiog up00 the particular use.Fcr example. whenusedfor thepurpose tumcn 30 gelatin (see e.g.. Example 31). Within alternative of embodbwnts,hydrophobiccompouOds be contained may embolization discussed (as below),it is generally pmfembk within a to fashionthe anti-angiogenic compositionin microsphcres witbin a hydrophobiccore.andthis core contained hydrophilic s&R. For example. described &ample 38. as in of between15 and500 pm preferablybctweeo15 and 200 paditaxel may be in-ted into a hydrophobiccore pm, and most preferably. between 25 and 150 pm. Alternatively. such compositions may also be readily 3s (e.g.. of thepoly D.Llactic add-FE0 or MeFEG aggregate) appliedss a “spray*. which solidifiesinto a film or coating. which has a hydrophihcshell. A wide variety of hydrophobic compoundsmay be Such spraysmay be preparedfrom microspheres a wide of released from the poiyme@ccan&s de&bed above. amy of sixes.includingfor example, from 0.1 pm to 3 pm. induding fcr exampIe: certain hydrophobic compounds fiomlO~to3O~andfrom3O~to1oO~(see 40 whiti disrupt microtubulefunction such as paclitaxcl and Example8). hydrophobicproteins such as my&in basic Anti-angiogeolc compositionsmay also be prepared estramustine; given,thc disclosureprovidedherein,for a variety of other protein. peoteolipidproctios of CNS myelin. hydrophobic cell wall protein, podns, membraneproreios (EMBO 1. applications. example.for administration the come& For to myelin oligodeodrocyte glycoprc+ the aoti-angiogcnic factors of the presentinventionmay be 4s 12(9):3409-3415.1993). tein (“MOO”) (Biockm andhfol. BioL in!. 30(5):945-958, ioaxporatedinto moco-adhesive polymus (e.g.,polyaaylic 1993, P27 Cancer Rcs. 53(1?):4096-4101, 1913. acidssudt as(CARBOpoLO. dextton,polyme&aaylate,oc bsUuioopsio. human surfactantprotein (“HSB”: 1. Bioi. starch (see LcMmg and Robinson,3. of CoMoilcd RCL 1993). and SP-B or SP-C Ckm. 268(15):1116&-11166. 5:223. lQ88)), a nanometer-sizedmicrospheres (see 1992). Kreuu J. Control&d Release l&169-176,1991; so (Biuchhica ct Biophpica Acta 1105(1):16X-169. generally. Couvreurand Vauthia. J. Comviled Relcarc 17:187-198, ARTERLGEMBOLZWl-ION 1991). In addition to the compositionsdesaibcd above. the Anti-aogiogeniccompositionsof the presentinvention preseatiovenrho alsoprovidesa vat&y of methodswhich mayalsobeprrpgledinavarietyof’ paste” orgelforms.Prr utilize the above-descrii anti-angiogenic compositions. In example, within one embod&znt of the inventiw, anti- 5s particulpt, within ooe aspectof tbe present invention metbangiogcniccompositionsare provided which are liquid at ads am providedfor embolizinga blood vessel.comprising one tunpnaarrr (e.g.,temperature greater than 37OC., such tbe step of delivering into the vessei a &rapeuticslly as 40’ C.. 45’ C.. 50’ C., 55’ C. a 60” C.), and solid oc effective aolouor of an anti4ulgiogeniccompositiotl (as semi-solid at another temperature(e.g., ambient body dcscrihcdabove),such that the blood vesselis cffcctively tempemtute. any temperature or lower than 37OC.}. such 60 occluded. Thaapeutically effective amounts s&able for ‘ yhernropastes” be readily made given the disdwure may ocdudiog blood vesselsmay be readily detmminedgiven pvided herein (see.e.g.. Examples10 and 14). the disdosureprovidedbelow,aedas described Example ia 6. Wirhin a proticolatly prefm rmbdment, the aotiwithin yet other aspects of the invention, the antiangiogcniccomposition deliveredto a blood vesselwhkh is aogiogeolccompositionsof the presentinvention may be formed as a filoL Preferably,such8lms are genaally less 6s nourishes a tumor (seeFIG. 13). than$4.3.2, or 1. mm thi&. morepreferablylessthan0.75 Briefly. the& are a oumlsr of c&d& shwions (e.g.. mm or 0.5 mm thick, and mostprefemblyless than 500 pm bleeding,tumordevelopment) whereit is desirable reduce to

5,716,981 19 20
through the catbcter, until flow is obsuvcd to cease. Occlucc abolish the Mood supply to an orgao or region. As &saii in greater &tail below. this may be accomplished sion may be confirm4 by npeating the angiogram. by iojutiog aoti-angiogenic compositions of the present FiubboWoo the.rapygeoaally rcsuhs in the distriiutioo invention into a desired blood vessel through a salcctivcly Of composi~oos cootaidg anti-angiogenic faders throughJxJsitioncd catheter (see Jm. l3). The compositioo travels 5 out the intmZic42s of the tumcx or vascular mass to be via Uxe blood stream until it becomes wedged in the treated The physical bulk of the cmb0lic particles &gging vasctdauuc. thereby physically (CT cbemicaJJy) occluding the arterial Jumeo results in the occlusion Of the blood the blood vessd. The reduced or abolished blood flow to the Supply. In add&ion to this affect. the presence of an antisekcted area ma&s in infarction (dJ death doe to an aogiogeoic facto@) prevents the formation of new blood inadequate supply of oxygen and outrknts) or reduced blood 10 Vessds to supply the tumor Q vascular mass. enhancing the loss from a damaged vessel. devJmlizing effect of cutting off the blood suppiy. For use in anbo~oo thcqy. anti-aogiogenic camp Therefcrc. it sbould be evident that a wide variety of sitions of the present invention are preferably non-toxic. tumors may he embolized utilizing the compositiolts of the thrombogeoic. easy to inject down vascuk catheters.radiopresent ioventioo. Briefly. tumors arc tvpically divided into op;lquc, rapid and permanent io efkct. sterile. attd readily 1s two dasscs: benign and maJJguant Jn a benign tumor the available in different shapes or sizes at the time of the ceJls retain their dilfercntlatcd features and do not divide in prmdore. Jo addition. the compc&tJons pr&rabJy result in a compJctcly uncootrokd manner. Jn addition. the tumor is the slow (id&y. over a paiod of scvadl we& to months) Jocalkud and oonmctastatic. Jn a malignant tomor. the cells rchse of an anti-angiogenk factor. PartJcuJarJy preferred become oodBumtiatcd do not respond to the body’ s anti-at~giogeoiccompositioos shouJdhave a predictable size 20 growth and hormonal signals. and multiply in an unconof U-200 pm after being injected into the vascular system trokd manob; the tmncr is invasive and capable of spreadReferably. they should not clump Jnto Jargcr particles eJtJuz ing to distant sites (mctastasiziog). in soJution cr once injecttd. In additiw. pcfaabJe compoWithiit we aspect of the present invention. mctastascs sitions should not change shape or physical prop&es (sccoo~ almoM) of the liver may be treated utihzing 25 dutiog stcrage prior to use. embolhtioo timapy. BridJy. a catheter is inserted via the EhboJkdoo therapy may be utiJized in at least three fcmcd oc brad&d artery and advanced mto the hcpatic prindpaJ ways to assist JII the manage~ot of o&asmsz (1) artuy by steeting it through the art&al system under &iinitive lrcattocot of tumors (USWQ bcuign); (2) for fluoroscopic guidance. The catheter is advanced into the embohxapxpemtive emboJizatioo; and (3) for paJJJative hepatic attfdal tree as far as necessary to aJJow cowlem tioo. Briefly, benign tumors may sometimes be succc&tdJy 30 blockage of the blood vessds supplying the tumor(s), whiJe treated by embdization therapy ,alooc. ExampJes of such spat&g as many of the artuiaJ branches supplying ttormaJ tumors include simple tumors of vascular origin (e.g.. structrnw aspossible. WalJy this will be a segmental branch hacmangiomas). endocrine tumors such as parathyroid of the hspatic artuy. but it couJd be that the entire hcpatic adenomas. and benign bone tumors. 35 artery distal to the origin of the gasuoduodenaJartcry or even multiple separate arteries. will need to be bJockzd For other tumors. (e.g.. renal adenocsrcinoma), preoJx!radepending on the &cot of tumor and its individual blood tivc embolizatioo may be employed hours or days before supply. Once the d&red catheter position is achieved, the surgical nzxctioo in ox-da to reduce operative blood loss. axtuy is cmboJizcd by injecting anti-angiogenic corn@shorten the duration of the operation, and reduce the risk of catheter uotil dissunination of viable malignant cells by surgicaJman@- 40 tiotls (as desaibed above) through the attd flow in the artery to be MocJmd ceases.preferably even after Jation of the tumor. Many tumors may he SOC~MUJI~ observation for 5 miuutcs. OcclusJoo of the attay may be cmbolizcd prcopcrativeJy. iocJudiog fee cxam~rJenasophacoofirmed by JnjcqtJug m&opaque contrast Uuough the ryngcal ttmmrs. gJomus jug&u tumors. merdogiomas. cathcta and demonstrating by fiuoroscopy or X-ray fihn that chexnodectomas.and vagaJ neuromas. E?mboJJm,tioomayakobcutihxedasaprimatymodcof 4s the vessel which previously %IJcd with contrast no longer docsso.Thcsameproce&cmaybcrcpcatedwJthcach beatmat for JnopcrabJt tuahgnancks. in c&r to extend the feuiingatterytobcoccludcd sumivaJ time of patients with advanced disease.HmboJJmAs ooted above, both benign and malignant tumors may tion may produce a marked itnpverntnt in the quality of be cmbolizul utiJi2iogkolnpositions of the present inveoJife of patients with ruahgoant tumors by alleviating unpksaut symptoms such as bJccdhrg. veuous obstruction 50 tion. Rcpmscotative exan@es of benign hcpatic tumors include Hepatocellular Adcnoma. Cavernous and tracheaJcom~xcssJon.The greatest benefit from paJiiaHacmangJoma, and Focal Nodular Hypu@asia. Other tive tumor cmboiization. however. may be seen iu patients benign tumors, which arc more ram and often do not have suffering from the humcraJ effects of mahgnant &ocrinc clioical maoifcstatiws. may also be tawcd. These include tumors. whaein mctastascs from carchtoid tumcrs and other endocrine ncopJasms such as JnsuJinomasand gJucagooo- 55 Bile JIuct Adcnomas, BJJe Duct Cystadcnomas, J%romas. Lipomas, Lciomyomas, Mesotheliomas. Teratomas. mas may k slow growing. and yet cause great distress by Myxomas, and Nodular Jtegeocxative Hypgplasia. vi!tue of the endocaine syncJromeswhich they product. Maligoaot Hcpatic Tumas arc gcncraJJysubdivkicd into In general. uoboJizati00 thempy utiwog anti-aogio@ic tw0catcgosJes:gKimaryandsccotldary.primarytumcrsarlsc compositioos of the present inveodon is typicnuy paformad , in a sindar manner. regardless of the site. BrJcBy. attgJog- 6(1 dinxtJy from the tissue Jn which they are found. Thus, a JKimalylivtrtumaisdaivcxJoriginaltyfromthculls rapJ~y(a road map of the blood vesscJs) of the area to be wbicb male up the liver tissue (such as hepatocytes and embolizcd is first perfarmed by injecting radiopaque coobiliary cdJs). Rc~rcacntatJve el‘ ramples of pimary kpatJc trast through a catheter inserted into 00 attay (rr veio maligoands wJti& may be treated by art&J embokatJoo (depcndingoothesitctobecmbdized)Isrmx-Myistalen. The carbeta may be insuted citha pacutaneousJy or by 63 include HepatocellularcarcJnoma. ChoJangiocarcinoma. AogJosarcoma. Cystadcoocarcinoma, Squamous CclJ surgery. The blood vessel is then embohxed by mfhming Carcinoma. and HcpatobJastoma. anti-aogiogeoic compositions of the present invention

5,716,981 21 22
A secondarytumor. or metastasis.is a tumor which USE OF ANTI-ANGIOGENK COMPGSITIONS originatedelsewhere in the body but has now spreadto a AS COATINGS FOR SIENIS distant organ.The commonroutesfor metastasis dlred are As noted above. the present invention also provides growth into adjacentstructures, spread throughthe Vascular a lymphatic systems.and trackingalong tissueplanesand 5 steots. comprising a generally tubular structure (which the body spaces (peritoneal fluid. cerebrospinal fluid. etc.). iocludeafor example.spiral shapes). surfaceof which is Secondatyhepatic tumors m one of the most common coatedwitb a compositionas desuii above. Briefly, a steotis a scaffolding.usuallycylindrical in shape.that may causes deathIn the cancerpatientandareby far andaway of be insettedinto a body passageway (e.g., bile ducts) or a the most commonform of llver tumor. Although virtually which has been narrowed. any malignancycan metastasize the liver. tumorswhich 1o portion Of a body passageway, to irregularly c~ntured.obstrucZe& occludedby a disease or are most likely to spreadto the liver Include:cancerof the process(e.g.. ingrowth by a tumor) in order to prevent stomach,colon. and pancreas;melanoma:tumors of the closureorreclosureof the passageway. Stcntsact by physilung. oropharynx. and bladder; Hodgkin’ and nons into Hodgkin’ lymphoma; tumors of the breast, ovary. and ally holding openthe walls of the bodypassage which s prostate.Each one of the above-named primarytumorshas rJ they arc kc&d. numerousdifferent tumor types which may be treatedby A variety of stcntsmay be utilixed within the contextof arterial embolization(for example, therearc over 32 difkrthe presentinvcntio~. including for uample. esophageal em types of ovarian cancer). sttnts. vascularstems.biliaty stems.pancreaticstems.ureteric and urethral stents. lacrimal stents.Eustachiantube As noted above, embolization therapy utilhing antisteots. auglogenlccompositionsof the presentinventionmay also M stems.fallopian tube stentsand tracheaYtxonchial be~pr;led to a variety of otherclinical situations whereit is Stats miy be readily obtained from commacial sources. &sired to occludeblood vessels. Within one aspectof the or constructed accordance in with well-known techniques. present invention. attuiovenous malformati00 may be. RepreseMative samples of stemsincludethosedesaii in treated by administrationof one of the above-descrii U.S. Pat No. 4.768523.entitled“HydrogelAdhesive:” U.S. compositions. Briefly. arteriovenous malformations 25 Pat.No. 4.776337,entitledTxpandable IntraluminalGraft, (vascular malformations) refers to a group of diseases and Method andApparatusfor Implanting and Expandable wherein at least one (and most typically. many)abnormal SnWuminalGraft;” U.S.Pat.No. 5.041.126 entitled“Endocommunications betweenarteries veins occur,resulting vascularStem and INkay and System:”U.S. Pat. No. 5.052, in a local tumor-likemasscomposed predominantly blood of 998 entitled ‘ %dwelIing Stent and Method of Use;” U.S. vessels.Suchdisease may be eithercongenital acquired.sc pat No. 5.064.435 entitled “Self-Ekpanding Prosthesis or HavWithin one embodimentof the invention. an arterio ing StableAxial Length:”U.S. Pat. No. 5.089606. entitled venousmalformationmay be treatedby insertinga catheter ‘ Water-insol&Ie Polysaccbaride HydrogelFoam for Mcdivia the femoral or brachial attay. and advancing into tbe it CalAppIkations;”U.S. Pat.No. 5.147370.entitled “Nitinoi feeding artery underfluoroscopicguidance. catbeta is The Stat for Hollow Body Conduits;” U.S. Pat No. 5176.626. Feferably advanced far as necessary allow complete 35 entit3ed”Ipdweliing Stent;” as t5 U.S. Pat No. 5.213580.entitied blockage of the blood vessels supplying the vascular “Biodegradable polymeric EndoIuminal Sealing Process;” maIformation. while sparingasmanyof thearterialbra&es and U.S. Pat. No. 5328.471. entitled“Method andApparasupplyingnormalstructuresaspossible(ideallythis will be tus for Treatmeat of Focal Diseasein Hollow Tubular a single artery.but most oftenmultiple separate xrteriea may Orgaosand Other‘ IIssueLumens.” needto be occluded, depending the extent of the vascular 40 Stats may be coatedwith anti-angiogeaic on compositions malformation sod its individual blood supply). Once the or anti-angiogenicfadccs of the present invention in a desired catheterposition is achieved,eachartery may be varidy of manners.iocluding for example:(a) by directly embolized utilixing anti-angiogeniccompositionsof the af6xingto the stentan anti-angiogenic composition(e.g.,by grescntinvention. either sprayingthe stent with a polymer/drug Elm. or by Within anotkr aspectof theinventioo.embol&tion may 45 dippiog the stent into a polymer/drug solution). (b) by tc accomphshd in order to treat conditionsof excessive coating the stcnt with a substance such as a hydrogelwhich bleeding. For example. menorrhagia (excessivebkeding will in tmn absorb the anti-angiopic composition (or with mcmtmatioo) may be readily treatedby embolization anti-aogiogeoicfactor above), (c) by interweaving antiof uterine ties. Briefly. the uterine arteries are branches aogiogedccompositioncoatedthread (a the polymer itself of the iotemaI iliac artuies bihaally. Within one embodi- 50 formed into a thread)into the stentstructure. by iamting (d) ment of the invention, a catheter may be insuted via the the stent iDto a sleeveor mesh which is comprisedof or femoral cr bra&al arkxy. and advanced into each utaine coated with an anti-angiogeniccomposition. or (e) conattery by steering it through the ark&l system unda structingthe stem itself with an anti-angiogenic composifluoroscopicguidance. cathetershould be advanced as The tion. Within preferred cadmdhents of the invention, the far as necessary allow completeblockageof the blood 5s composition should&rnly adhereto the steotduring storage to vessels the utcxus. to while sparfog manyart&al branches and at the time of insertion, and should not be dislodged as that arise from the uterine attery and supply normalstruc- from the stent when the diameteris expandedfrom its lures aspossible.SdealIya singleuterinearteryon eachside collapsedsizeto its full expansion The anti-angiogenic sire. may be unbolixed,but occasionally multiple separate atter- composition should also preferably not degrade during ies may need to be blocked dependingon the individual 60 storage.pior to imetion. or when warmed to body temblood supply.Oncethe desiredcatheter Positionis achieved pasture after expansioninside the body. In addition, it each artuy may be embolizedby administrationof the should preferably coat the stent smoothly and evenly. with anti-angiogenic compositionsas desuibedabove. a uniform dishibution of angiogcnesis inhibitor. while not In a like manner,arterial embolizationmay be accom- changing the stat contour. Within preferred embodiments plished in a variety of other conditions. including for 6S of the invention, the anti-angiogenic composition should provide a uoifum. predictable,prolonged releaseof the example. for acute bleeding. vasahr abacmalities. central
nervous system disorders. and hypeqlenism. anti-angiogenic f&&or into the tissue surrounding the stent

5716,981 23
onceit hasbeendeployedFor vascularstents. additionto in the abve pputies. the compositionshouldnot renderthe sum thrombogcnic (causingblood clots to fcam). or cause significantturbulencein blood flow (mire than the stent 5 itself would be expected causeif it was uncoated). to Within amther aspectof the presentinvention. methods areprovidedfor expanding lumenof a body passageway, the cmprising insating a stentinto the passageway. stent the having a generally tubular structure.the surface of the stsucturc. being coaufl with all Mti-MgiogeRic composition 10 (or. an antioangiogcnic factor alone),spchthat the passageway is expanded. variety of emb&ments are described A belowwhereinthelumenof a body passageway is expanded eliminate a biliary. esophageal.tracbeaU in order to bronchial,urethrai or vasculx obsimction. Yn addition. a IS representative example describedin more &tail belowin is

24

affect the bib duct (e.g.. adenomaof the biliary system). and.in rxc cases. squamous carcinomas thebile duct cell of and adenocarcinomas the gallbladder.may also cause of compression thebiliary treesod therefore. of result in bilk-y obstruction. Compaession the biliary bee is most commonly due to of tumus of the liver and pancreaswhich compressand tbaefcre obstrud the ducts. Most of the tumors from the pancreas arisefrom cells of the pancreaticduds This is a highly fatal form of 4Xllccz of au cancerdeaths; (5% 26,ooo new cases ymr in the U.S.) with an average 6 months per of survivd and a 1 yearsurvival rate of onIy I#%. When these tumus are located in the bead of the pancreasthey tit+ quentiy causebiliary obstruc#ion. this detractssign& and caatiyfromtbcqualityoflifeofth:patient.\krhile~typcs of panczeatic tauncas genaally rekred to as “car&oma are of the pancreas” tbae are histologic subtypesincluding: adenocarcin~ adenosquamous carcinoma cystadenoEwRple 7. uuchoma. tmd acinx cell aminoma. Hcpatic tumors.as Generally. stentsareinse.rted a similar fashionregardin discussalabove.may also causecompessiooof the biliacy less of the site or the disease bdng treated Brieffy. a ducts. preinsertion examination. usually a diagnostic imaging 20 tree. and thereforecauseobatnrctionof the bii Within oneembcdimcntof the invention.a biiiary stentis pmtdure. endoscopy. direct visualimtion at the time of or iirst insertedinfo a b&try passageway ooe of several in surgay,isgenaallyfirst~~inff&todctamiaethc ways: from the top end by inscztinaa needlethrouahthe apjqdse positioning fa stent inscztion.A guidewireis ttbdominalwall aoil lbrou& the liv& (a pacutaneou~ tranthen advanced through the lesion or proposed site of sheuatic cholanpiaaram or insution. and over this is passeda deIivety catheterwhich 2s cafdating thebiTe dud TK”): from the bottomendbv throngh an endoscope inserteh allows a stent in its coilapscd form to be insertedQpicaliy. tbmgb the anouh.smd. ar$ duodenum CRdoscopiC (an stelltsarc capableof beiugcomprzssed. that they can be so retrograde cfiolangiogram “ERCP”); n by directincision oc inscrtcdthroughtiny cavities via small cathtiffs. and then during a surgicalpmccxbe. A mscrtion examination. expamled a larger diameteronce they are at the desired FTC. ERCl? oc direct visualization at Uie time of surgay to locatioa. Once expanded the stent physicaLlyfcsces the 30 shouldgendy be paformed to determiae appropriatt the wails of thepassageway andholdsthan open.As such. position for stud insertion.A guidewire is then advanced apart theyarecapableof insertionvia a smallopening.andyet arc through the lesion. andover this ad&very catheteris passed still able to hold opena largedknetcz cavity ocpassageway. to allow the stentto be insertedin its collapsed form. If the The stent may be self-expanding(e.g.. the W&tent and diagnosticexam was a ETC. the guidewire and delivery Gimurco sum). balloonexpandable (e.g..the Palmazstcnt 35 catheteris in+tai via the abdominalwail. while if the in andStredterstent).or implantedby a change temature otigiml cxam,wasan ERcPthe swlt maybe placedvia the (e.g.,the Nitinol stmt). mouth. The stent is then positioned under radiologic. Stentsare typically maneuvered place underradiointo cndosmpic. or direct visual control taking particular care to logic or clireu visual como~ taking pxtiallar arc to place 40 placeit preciselyaauss the uarrowingin the biie duct.. The the stentprecisely acrossthe natTowing the organbeing in delivay catheteris thenremovedleaving the stentstanding treated. d&my caQUa is then remove& leaving the The as a sc&olding which holds the bile duct open.A further stent s-cling on its own as a scaffold.A post insertion cholaagbgram be pafamed to document the stent may that examination. usually an x-ray. is often utilized to confirm is appmp&ely positioned. appropriate positioniDg. widlin yet Imother embodiment the invention.nztbcds of IS Within a prefured emkdiment of theinvention,methods areprovidedfor e&min&lg esophageal obstructions. comiireprovidedfor eliminatingbilirpy obstructions, comprising prisiig ins* an esopbagedstentinto an esophagus, the iwuting a biliary stentinto a bitbuy passageway, stent stenthaving a genaally tubular structure.the surfaat of the the having a generally tubular sbuctum. the surface of the Structure being coatedwith an anti-angiogcnic composition structure being coated with a composition as &scribed so as desaibedabove,such that the tsophag~ obi5trwtionis . * above, suchthat thebiliary ObsmctioD e4imbtd Bray. is alumnat& BrMy. the esophaguiis the hollow tubewhich tumor overgmvth of the common bile dud resultsin protransports food and liquids from the mouth to the stomach. gressivecbolestaticjaundice which is incompatible with Cancerof the esophagus invasion by canax arising in or life. Gtnerally.tbe bihy systemwhich drainsbile from the adjacent organs(e.g..cancerof the stomach lung) results or Jiveri#otheduodenumismo.stoftenobstruuedby(l)a ss in the inability to swabw food u saliva. Within this tumorcomposcd of bile duct ceils (ch0lMgioaucin0mii). (2) embodimat, a preinsertionexamination. usually a barium a tumor which invades the bile duct (e.g., pancreatic swallow or endoqy should generally be performedin carcinoma). or (3) a tumor wblch cxms extrinsic pressure orderto determhe the appropiate position for stentinsffandcompresses bile dud (e.g.. enlarged the lymph nodes). tiOR. A catbCtt2 ff CnC&ScOpe may thaw k: p~&iODtd the mouth, Ed a guidcwirc is advancui through the 60 uirough Both#naatybiliarytumcrs,aswellasothertnmors which causecompressiwof the biliary tree may be treated blockage. A stent delivay catbeta is passedover the guide-wireunder radiologic or cndosoopic cmlrol, and a lltwng the stem demibcd berein. one examge of pristentis placedprecisdy aaoas the narrowingin the esopha,(whidt are also mxybiliary~areadenoarrcinomss gus.Apost insatiou cxxninatio~ usuallya bariumswallow died KM&in tumorswhenfound at the bifurcation of the llpppiate positioning. commonbcpatic duct).Thesetumors arealso mfeued to as 6.5 x-ray, may be tlbifircd to coWa other anbodimentsof the invention.methodsare bilhy c.aminomas. cboiedorbolMgiocardRomas. acknoor provided for elimkting tmcheaYbiQnchial obstructions, carcinomasof the biliary system Benign tumors which

5716,981
25

26

comprisinginsting a traciteal/bronchial steminto the trainto the appropriate blood vesselby steeringit through the chea or bronchi, the stent having a generally tubular vascularsystem u&x fluoroscopicguidance.A stent may structure. the surface of which is coated with an antithen be positioned across the vascular stenosis.A post angiogeniccompositionas descriid above.such that the insertionanglogram also be utilized in orderto con&m may tracheal/bronchial obstnrction is eliminated. Brkfly. the 5 -opriate posidoningV trachea bronchiaretubeswhich carry air fromthe mouth and USE OF ANTI-ANGIOGENIC COhSPOSITIONS and nose to the lungs. Blockageof the tracheaby cancer, IN SURGICALPRC~CED~RE~ invasionby cancer arisingin adjacent organs(e.g..cancer of As noted above, anti-angiogeniccompositionsmay be the lung), or collapse of the tracheaor bronchi due to chondromalacia (weakening the cartilagerings) resultsin 10 utilized in a wide variety of surgical procedures.For of inability to breathe. Within this embodiieot of the example.within one aspect of the present invention an invention. preinsertion examinatio5. usually an edosccpy. anti-mgiogeniccompositions the fotm of. for example. (in should generallybe performedin order to determinethe asprayorfilm)maybeutilixedtoccatorsprayanareaprior appropriakposition for stentinsertion.A cathcta n endo- to removalof a tumor.in orderto isolatenormalsurrounding scopeis thenpositioned throughthe mouth.and a guidewire ts tissuesfrommalignanttissue.andlorto prwent the spreadof advanced throughthe blockage. delivery catheteris then A diseaseto suxoundingtissues.Within other aspectsof the passed the guidewirein orderto allow a collqsed stent present over invention.anti-arigiogenic Compositions (e.g.,in the to be inserted The stent is placed under radiologic or form of a spray) may be deliveredvia endoscopicproceendoscopic control in order to placeit preciselyacrossthe dmes in orda to coat tumors. or inhibit angiogenesis a in narrowing. ‘ delivery cathetermay then be nmoved 20 desired locale. Within yet other aspectsof the Fesent Ihe leaving the stentstandingas a scaffoldon its owm A post invention. surgical mesheswhich have been coated with insextionexamination. usually a bronchoscopy be utimay anti-angiogenic campositions the presentinvention may af lized to confine apprcpriate positioning. be utibd in any procedurewhereina surgicalmeshmight be utilized. For example.within one embodimentof the Within another embodiment the invention.methods of are mthraf obstru&ons.ccmpi&g 25 invention a s~rgifal mesh ladenedwith an anti-angiogenic m&d for ewhg composition be utilized during abdominal may cancerresecinsertinga urethralstem into a urethra.the stcnt having a to generallytubularstructure. surface thestructure the of being tion surgery(eg., subsequent colon resection)in cederto provide supportto the stnrctme.and to releasean amountof coated with an anti-angiogenic compositionas descrlki factor. above, such that the urethral obstruction is eliminated. the anti-angiogenic Briefly. the urethrais the tube which drains the bladder 30 Within further aspects the presentinvention.methods of through the penis. Extrinsic nanowing of the urethraas it are provided for treating tumor excision sites.comprising passesthrough the prostate.due to hypatrophy of the administing an anti-angiogenic compositionas described prostate. occurs virtually everymanovertheageof 60 and in above to the reseuion margins of a tumor subsequent to causesprogressivediiliarlty with urination. Within this excision, such that the local recurrenceof cancerand the embodiment. preinsertionexamination, a usuallyan endos- 35 formation of new blood vessels at the site is inhibited. copy or urethrogram shouldgenerallyfirst be performedin Within one embodiment of the invention. the antiorder to determine the appropriate position for stent angiogenic composition(s) (or anti-angiogenicfactor(s) insertion.which is abovetheexternalurinarysphincter the at alone) are administered direU.ly to the tumor excision site lower end. and close to flush with the bladderneck at the (e.g.. appliedby swabbing.brushing or otherwisecoating upper end. An endoscope catheteris thee positioned *o the resectionmatglnsof the tumor with the anti-angiogenic or throughthe penile openingand a guidewireadvanced into composition(s) or factor(s)). Alternatively, the antithe bladder.A &livery catheteris then gassedover the angiogenic composition(s) factor(s)may be incorporated or guidewire in order to allow stent insertion.The delivery into known surgicalpastesprior to administration. Within the into catheter thenremoved.and, stentexpanded place. particularly prefmed embodimentsof the invention. the is tim. oh A post insertionexamina usualiy endoscopy r&o- 45 anti-angiogeniccompositions are applied after hepatic gradeurethrogralumay be utilized to confirm appropriate reseetions malignancy,and after nwosurgicaJ operafar position. tions. Wlthln another embodiment the invention.methods of are Within one aspect of the present invention. antiprovidedfor eliminatingvascularobstructions, comprising ~giogcnic compositions (as described above) may be insertinga vascularstentinto a bloodvessel,the stenthaving M administered the resectionmargin of a wide variety of to a generallytubular structure.the surfaceof the sttucture tumors, in&ding for example; breast colon. brain and being coated with an anti-angiogenic composition as hepaticbunens. example,within oneembodhent of the F%r desaihedabove.suchthat the vascularobsttuctionis elimiinvention, anti-angiogeniccompositionsmay be adminisWed. Briefly. stemsmay beplacedin a wide arrayof blood tered to the site of a ncuroIogical tumor subsequent to vessels, both arteries veins,to &xevent and remrent stenosis55 excision,suchthat the formationof newblood vesselsat the at the site of failed angioplasties. treat natrowingsthat to site are inhibited. Briefly. the brain is highly functionally would likely fail if treated with angioj&asty, to tmatpost and localiwi; i.e., eachspecificanatomical tegion is specialized surgicalnauowings(e.g..dialysisgraft stenosis). Repento cany out a specltk function. Thereforeit is the location tative examples suitablesitesincludethe iliac, ret& and of of brainpathology thatis ofkenmoreimporU& thanthe type. coronary Meries. the superiorvena cava, and in dialysis 60 A relatively small lesion in a hey area can be far man devastating than a much larger lesion in a less important grafts. Within one embodiment. angiographyis ti performedin orderto localizethe sitefor placement the stent. arei. Similarty, a lesion on the surfaceof the brain may bc of This is typically accomplished injecting radiopque conby easytares& surgically,while the sametumor locateddeep trastthroughacathctcr~scrtcdiotoananayavcin~s in the kin may not (one would have to art through too x-ray is taken. A catbeta may then be inserted eitha 65 my vital structures reachit). Also. evenbenigntumors to ~bedangcrousfarseverslnasons:theymaygrowiaa percutaneously by surgeryinto the femoral artery,bracc &id artery. femoral vein. or brachial vein, and advanced key are-n causesignificant damage;even though they and

5,716.981 27
would be cured by surgical resectionthis may not be possible; and firrally, if left uncheckedthey can cause iaueascd iatracmaial pressure.The skall is an endosed spaceincapableof expansion.Therefore.if somethingis growlag in one kation, somcthiogelse must be being 5 compressed another location-the result is increased in pressureio the skull or maeasedintracranialpressure.If such a condition is left untmated.vital Structures can be comprcssaLnsultiog in death. The incidence of CNS IO (centralnervoussystem)malignancies g-16 per 100.0tK~. is The prognosisof primary maligoancyof the brain is dismal. with a mediansurvivalof lessthanoneyear.evenfollowing surgical resection.These tumors. especially ghomas.are pz&minantly a local diseasewhich recur within 2 ceatimeters of the original focus of diseaseafter surgical 15 removal Represeotative examplesof brain tumors which may be treatedutiliring the compositionsand methods descrfbed herein include Glial Tumors (such as Anaplastic Astrocytoma. Gliobiastoma Multiform. Pilocytic 20 A%%ncycoma. oligodcndmglioma. EpMdymomat Myxopapillary Epcodymoma, Subependymoma. Choroid Plexus Papilloma); Neuron Tumors (e.g.. Neuroblastoma. Gaaglioneuroblastoma. Ganglioneuroma. and Mcdulloblastoma~ Pincai Gland TIunors(e.g.. Pinoobias- 25 toma and Piacocytoma); Mcnigtal Tumors (e.g., Menio@oma, Meaingeal Hemaogiopcricytoma. Meoiagcal Sarcoma); Tumors of Nerve SheathCells (e.g., Schwannoma (Neurolemmoma) Ncurofibroma);Lrltomas and (e.g..Hodgkin’ and Non-Hodgkin’ Lymphoma(ineluding 30 s s MaIfanumerous subtypes. both primary and secondary); mative Tumors (e.g.. Craniopbaryogioma.Epidermoid Cysts.Dermoid Cysts and Colloid Cysts): and Metastatic Tumors(which an be derivedfrom virtually any tumor.the most coamoo be& from lung, bm4sLmclaaoma.kidney. 35 and gastrointestinal tumors). tract lnhmmatory arthritis is a serioushealth problems in developed countries,partia&uly giventhe increasingoum- 40 bcr of agedindividuals.For example,ooc form of infiammatoryarthritis.meumatoidarthritis (RA) is a multisystem dKooic, relapsiag.ioflammatorydisease uokoowocause. of Although many organsCM be aifcctcd, RA is basically a severeform of chronic synovitis that sometimesleads to 45 d&raction aad ankylosis of affectedjoiots (taken from Robbins Patfrolopical Basis of Discare. bv R. S. Cotma. V. l&oar, aod S. ‘ Robbins.*W.B. Saunkrs Co.. 1989). i. Pathologicallythe diseaseis charauaizcd by a marked thickeniogof tbc synovial membrane wbidt forms vikus 50 jxojccrioas that extend into the joint space, multiiayuing of the synoviocyteliaiog (syaoviccytc proliferation),ittfiltration of the synovial memkane with white blood cells (roauophagcs.lymphocytes, plasma celts, and lymphoid follicles; called an “iniiammatapysyuovitis”), and deposi- 55 tion of fibrin with cellular necrosis within the syaovimn Ihe tissueformedas a result of this process calledpannusand is eveotuallythe pannus grows to fill the joint splcc. The paoousdevelops extensivenetworkaboew blood vessels an throughthe process aagiogellesia of which is csscntiaito the 60 cvolntion of the synovitis. Releaseof digestive enzymes {matrix lnetall~s (e.g.. collagcnase, stromeiysia)l and other mediate of the i&unxnatory vss (e.g., hydrogenperoxide, snpaoxides. lysosomalenzymes,and gwxiw%s arachadonic metabolism)firornthe cells of 65 of acid the paanustissueleadsto the progmive dcstructlooof the cartilagetissue.Tbc panuusinvadesthe art&&r cartilage

28
leadiagto erosionsandfragmentation the caMagetissue. of Eventually t&e is exosionof tie subchondtalbone with fibrous ankylosis and ultimately bony ankylosis. of the involved joint believed.but not conclusivelyproven.that lt is geoadly RA is an autoimmunedisease.and that many different arthriogCoicstimuli activate the immune respoosein the immuaogenetically susceptible host.Both exogenous infectious agents (Ebstcin-Barr Virus. Rubella virus. Cytome@z$rus. Hapes Viis, FItman T-cell Lymphotropit Vi& Mycopksma. and others)and endogcaous pm teins (collageo. protooglycans.altered immunoglobulins) have betto implicatedas the causative agentwhich triggers aa inamnopriate host immune response. Regardless the of incitiag agent.autoimmunityplays a role in the progression of the disease. particular.the relevantantigenis ingested Ja by antigen-presenting (ma~ophages dendriticceils cells or in the synovial mcmbmnc).processedand presented T to lymphocytes. T cells initiate a cellularimmuneresponse The and stimulate the prolifcratioo and difFerentiation B of lymphocytesiato plasma cells. The end result is the production of an excessiveinqxopriate immune response directedagainstthe host tissues [e.g.. anti&odiesdirect& againstm II collagen.antiies directedagainstthe Fc portion of autologcusIgG (called “Rheumatoid Facta”)]. This fiather amplifiesthe immuneresponse hastens and the dcstruUi00 of the autilage tissue. Once this cascadeis ioitiated nnmmus mcdiatas of cartilage desrruction are responsible the progressionof rheumatoidartkitis. fcr Thus. within one asped of the present invention.methods arefrovidedfatreatingor~cnt.ingintIammatory arthritis (e.g.,rheumatoidarthritis) comprisingthe stepof administaiog to a patienta tbera~euticaby effective amountof an aoti-angiogeoic factor or anti-angiogeuic compositionto a joint. Witbin a pefcrrcd embo&nent of the invention, anti-angiogenic factors (including anti-aogiogenic compositions.as described above) may be administered dimctly by intra-articularinjecsioo.asa surgicalpaste.a as an oral agent (e.g.. containing the anti-angiogenic factor thalidanidc). Onerepresentative exampleof sucha method is set forth in mcxc detail below in Example 19. As utilized within the contextof tbe presentinvention.it should be understood efficatiousadmkshation of the that aoti-aogiogenic factors and compositionsdascriid herein may be asssstd io several ways, including: (1) by prcvcntiog a lesscaing the pathological and/or dioical symptoms associated with rheumatoidarthritis: (2) by dowureguiating

the.white blood cell ruponse which initiates the iofiammatory cascadeand results in synovitis. swelhng.pain. and tissuedestmtioa; (3) by fnhibiting the “tumor-likenproliferation of synoviocytesthat leads to the development a of locally invasive and destructive pannus tissue: (4) by
decreasing the production/aczivity of matrix mcta&jmtcinases produced by white blood cells. syooviocytes, chmdrocytes,and codothelialcells, which degrade the car-

tilage matrix and result in iucversible destructiouof the articdar cartihge; and (5) by inhibiting blood vesselfamation which providesthe framewak and nutrientsnecessary for the growth and development the pannustissue. of Furthermore.the anti-angiogcnicfactors cc compositions should not be toxic to normal cbondrocytes tbcsapeutic at lCXl%EWhOftheSCSpU3SWiUbedisCIJ~edinmoFtde~ below. k Inflammatay Response Neutmphilsarefound in abundance in the synovial fluid, bat only ia small numbersin the syuoti membrane itself.

5,716,981 29
h is estimatedthat mom than 1 billioo neutrophilsentera moderately inflamed rheumatoid knee joint each day (Holingswath et al.. 1%7) and remain there because no pathwayexistsby which they canleavetbejoint Thesecells releasereactivefke radicalsand lys~somalenzymes wbid, 5 degra& the cattllagetissue.Other PMN productssuch as pstagladins and leukotrienes augmentthe inflammatory response n&t more inflammatoryozlls into thejoint and
tiSSW.

30
growth of a suppcrkg Vasculature angi0genesi.s). (i.e. AIJ these tidings are suggestive a tissue in which DO& of growth regulationas beenlost. Witbin one embodknt. inhibition of synovkyte prolifaation may be determined fff example.analysisof by, SH-tbymidineincarporation into synoviocytes.(it in vitro synovioqte proliferation. Sudh methodsarc illustratedin mue detail below in Example23. c. Ma&ix Metallopoteinases (MMP) Irreparable degradationof the cartilage extracellular mat& is beIievedto be largely dueto the enzymaticaction Of matrix metaU0pr0t&aseson the componentsof the cartilage matrix. Although mmur~us other enzymesare likely iW0lVed in the developmentof RA. coliagenase CAMP-1)and strometysi~~ -3) play an importantrole (MM’ (Vhxdti U al., 1994)in disease progression. Theseenxymes arecapable degrading 11collagen and proteoglycans of type respecdvel~ the 2 major exfncellular components cartiof lage tissue.Qtokines suchas IL-l. cpidermalgrowthfactor (E@). platelet-derived growshfactor. and tuma necrosis aad facta areall potentstimulatorsof collagenase str~melysin production.As descrii ab0ve.numerOus types celi found in the arthritic joint (white blood ceils, synovi~cytes. endothdial cells. and cbondr~cytes) capableof syntheare
sizing and seeming MMP!S.

Lymphocytes.panicularly T cells. are present~JIabun- IO dance in the diseasedsynovial tissue. Activated T cells producea varietyof lymphokinesandcoOpuate B ceils with to pduce autoantibodies. ceils productsresult in the T activationmauopbages. cell which is thoughtto havean a importat role in the patbokgy of the disease. The mac- I.5 raphages producea variety destructivelysosomalenzymes, ~osfaglaodins. and moaokines and are also capableob stimulating angiogenesis.One of the more iq0rUnt monokim secreted macrophages Cl. Briefiy. It1 is by is known to: stimulatesyathesis releaseOf collagenase 20 and by synovicqtes and synovial fibroblasts,inhibit prOteOglycan synthesis by drondmcytes. activate osteoclasts,induce changesin the endotbeliumof the syn~vial vasculatute [stimulationof endothelial productionof plasminogen activator ande~lonysh’ mulating factor, expression leukocyte 23 of adhesionmolecules,promOtion of pr0c0agulantactivity (Wider et al.. 1991)]. and act as a chemoattractant fa lymphocytes and neutrophils. WW one embodiment.d0wnregulationof the white blood cell response,or inhibition of the intlammat~ry 30 response. be assessed determination the effectOf may by of the anti-angiogenic factahor anti-angiogenic comp0sition on the response neutr~phils of stimulated with ~psonixed CPPD crystals or opsonized zyr~san.Such methodsare illustrated 95 in more detail below in Example22.

In p&iferatiog rheumatoidsynovial tissue. collageoase and stromeJysin becomethe major gene productsof the paanusandmay compriseas much as 2% of the messenger RNAs produced the synovialfibroblasts by (Brinkerboffand Auble. 1990).Increased levels of collagenase stromeland yain arepresentin the cartilageOfpatientswith RA and the level of cnxymeactivity in the joint correlates with the wetl Severityof the lesion (Mattel-Pelletieret al.. 1993;Walakovitis et al.. 1992). Because these enzymes are fundamental to the pathology of RA and result in irreversrblecartilage

B. SynoviocyteHype&&a damage.many thaapeutic strategies have beendevisedto inhibit their dfects. During the development RA, the synovial lining cells Of Numerousnaturallypresentinhibitors of MMP activity become activated by products of intlammarionOr through 40 havebeenidentifiedandnamed‘ TlMPS” far Xssue Lnhibiphagoeytosisof Immune complexes.Several subtypesof tars of Metalloproteinases. Many of theseproteininhibitors synovial Lining cells have beenidentified and all Of them combecomeintensely activatedand undeqo excessive byper- bind with the active MhiPs to f0rm 1:l noncovalent plexeswhich inactivatethe MMP enxymes. TlMPs are The plasia and growth when stimulated.As the .synovial tissue _ and organizesto form a pannus,the number of synovi~cytes.45 producedlocally by dtondrOcytes synovial fibroblasts and are likely responsibleffp the normal regulation of blood vessels. connective tissueelements, i&unmatory and conneUive tissue degradation. It is thought that much of tire cells increases form a mass100 timeaits original size.In to many ways. the synovitls in rlmmatold artbiitis behaves damaget0tbecarGagemaMxisduet0al0calimbaIance between MMP and TR@ activity. This is probablydue to much like a 10cakd neoplasia(Harris. 1990). In fact while culturedrheumatoidsynovial cells developthe pbenotypic y) inueasedproduction of metaLtoprOteinases the production of TiMPs remainsat a normal a c0nstantlevel cbarauerlstics of anchorage-independent gr~wtlr usually associatedwith ne0plasticcells if they given suflicient (Vincetti et al.. 1994).To overcomethis. thaapeutic stratto TIMPs (e.g..the plat&tde.rived growth factor (L@atis et al., 1989). Xn egieshavebeendesigned addexogenous cltanially modified tetracyclinemolearles.collagensub additioa the synoviocytesalso produce large amountsof eollagenase, stromelysin. prostagbdins, and Interl&in.l. 5s strate analogucs) or to upregulate TLMP production The tumor-like prolifaation of tbe cells of the synovial (retin~ids, transf0uning growth faaa 0, E-6. IL 1 1. oncostatin in an elfcatto restorethe enxymaticbalaace. M) connectivetissuestroma(synoviocytes. fibrcblast-lilrecells Howeverthis approach yet to translateinto signiksnt has and ncovascular tissue) produces a pannus with maay feahues of a 10caliud maliguancy.SuppOrtingthis tumcs clirdud results. appoacbis to inhibit Or downregulate the analogy are several findings: the paunus expresses higb 40 An alternative levels of oncaproteins such as c-myc and c-fos, produces production of the MbXPsto restore a ncnmal balanceof metatloproseinasesfacilitate surrwnding tissueinvasion, activity. Naturally okxlming compouruisfrNF& all-tram to retinoic acid) and synthetic compounds (retin~ids,glucoc~rexpresscytoskeletdmarkerscharactaistic of poorly diffahave beettdemonstrated inlubit MMP ta entiatedmesencbymaf tissue(e.g.,vimentin); synoviocytes ticoid hormones) in vitro grow rapidly. do not cOntact inhibit, fwm fOei and 65 activity by suppressing tmscri#ion and synthesis these Of pmteins. A post-transcriptional methodof blocking MME’ can be grown under anch0rag4ndependent conditionsin releasecOuldalso be expected result in a decrease the to in soft agarose;and pannustissueis capableof inducing the

5,716,981 31
amount of MAE’ produced and an improved balance betweenhfMP and 7XMP activity ia the joint Within one embodiment a decrease the productionoc in activity of MMP’ may be determinedby. for utanlple. S analysisof IL1 induced coJlagenase expression. such One methodis illustratedin more &tail be10win Example24. D. Angiogenesis The development an extensivenetwark of new blood 10 of vesselsis essentialto the developmentof the synovitis pescnt in rheumatoid arthritis (Harris,1990:Folkmanet al.. 1989;Sano et ah. 1990). Severallocal mediatorssuch as piatdctdexived growth factor (PDGF),TGF-j3.andfibroblsst growthfacta 0 are likely responsible the induction 1S fcr andpapetuation neovascuM&ion within the synovium. of Pamus tissnt composedof new capillaries and syaovial connectivetissueinvades and destroysthe attic&r cartikge. The migradngangiogenicvessels themselves prodncc and secreteinaeased levels of metaUoprot&amssuch as 20 cdage~~c and stromelysin capableof degradingthe cartilage matrix (Caseet al. 1989).The newly formedvessels are also quite Ye&y” with gaps present between the miaova6ada.rendothelialcelts. This facilitates the exudation of plasmapcteins into the synovium(which increases75 swelling). cnhanccs WBCs movementfrom the c+culation into the pannustissue (which increases inflammation).and kads to the paivascursr accumulationof mononuclear i.nfiammatory (wilda et aL. 1991). cells In sltmmaty, etldo#dial tissle plays an impomintrole 30 the in the development this disease expressing neec+ of by the say surfacereceptorsto atlow intlammatciy cells to Leave the circulation and enter the developingpannus.seucting pteolytic enzymes cap&k of degradingthe cartilage matrix, and proliferating to form the new vessels 35 (angiogenesis) required for the pannustissueto inueasein size and invadeadjacenttissues. Within one embodiment,inhihitiw of uew blood vessel formation may he readily determined a variety of asays. in including the CAh4 assay describedabove and within 40 J&ample2. NEOVASCUL.ARDISEASESOFTTiE EYE As noted above. the present invention also provides 45 methods treatingneovaseutar fcr diseases the eye,in&dof ing for example.comeal neovas&ar%Gon, neovascuisr glaucoma proliferative diabetic retinopathy. retrolental fibrdlasia andmacular degenaation. Briefly, corneaI neovasadadzadon a resultof injury to so as the antaia segmentis a significant causeOf deapscd visual acuity and blindness. and a majar risk factor frr rejectionof comealallografts.As described Burga et ah. by Lab. Invest. 48:16%180. 1983, corneai angiogenesis Involves three phases:a lxe-vascularlatent period. active 55 neovascuMMcm, and vascularmaturationandregression The identity andme&a&m of variousangiogenicfactarr, induding elements the inffammstoryresponse. of such as leukocytes. platelets.cytokine5,andeicosanoids, unidena tified plasmaconstituentshave yet to bt revealed. 60 cuualtiy lm clinically 6atisfactorytherapy exists fu inhibition of comeal neovaaarlarimtioncr regressionof existingcorncalnew vessels.Topicalcorticostesoids appear to havesomedinical utility, presumably limiting stromal by inaammatic4l. 65 Thus, within one aspectof the presentinventice methods are provided fa; treating neovascuiar diseasesof the eye neovasc&rirdtion). comprisingthe stepof administering to a patient a therapeutically eifeetive amount of an antianglogenic composidoIl(as dtsaibut above)to the cornea sudt that theformationof bioodvesselsis inhibit& Briefly, the corneais a time which MlrmauyIadts blcmdvessels. In certain pathological conditions bowever, capillaries may extendinto the corneafrom the pericorncalvascularplexus of thehmbus.Whenthe cornea becomes vasculsrixed. also it bea-mes doudbd resulting in a dcdine in the patient’ s visual acuity.Visualloss may hecome completeif the cornea completelyopacimm. Blood vessels enterthe corneain a variety of patterns can and depths. depending upon the poccss which incites the newaac&&&on. These patternshave beentraditionally defmedby *thdmologisu in the following types:pannus trachomatosus. pannus leprosus.pannus phyictenulosus. pannusdegeneradvus. glaucomatous and pannus.The ccroealstromamay alsobe invadedby branches the anterior of ciliary artery (called interstidal vascularization) which causesseveraldistinct clinical lesions: tamiual loops. a Yxush-iike”pattern.an umbelform. a lattice form inters&
tial arcades (from cpisderal vessels), and aberrant irregular vessds.

32 SW23 corned as aeovasadarization (illcludiag corneal gratl

5

A wi& variety of disorders can result in corneal newascukrization. including for cxamp~e.cormal i&ctions (e.g. trachoma. herpessimplexkeratitis.leishmaniasis and onchoces&sis).immunological processes (e.g.. graft rejection and Stevens-Johnson’ syndrome).alkali bums. s trauma i&mmadon (of any cause).toxic and nutritional &ficiency states, as a complicationof wearing contact and lenses. While the catweof comeal neovascularixation vary, may tire response the corneatu the insult and the subsequent of vascltkr ingrowthis similar regxdltss of Thecause.Briefly. thefocatioDoftheinjuryappuuYtobeofimpatanceasonty thoselesionssituatedwithin a critical distanceof the limbus will incite an angiogenic response. is likely due to the This fact that the angiogenic factas responsi& for eliciting the vascularinvasion are createdat the site of the lesion. and must diffuse to the site of the nearestblood vessels(the limbus) in orderto exut their effect.Pasta certain distance from the limbus. this would no longer be possibleand the limbic endothelium would not be induced to grow into the cornea Scvaal angiogenicfacta are likely involved in this prccess.many of which xc productsof the infiammatay response. Indeed.neovasadadzadon of the canea appears to only occur in as6ociadonwith an inflammatory cell infiltrate. and the degreeof angiogermisis proportional to the Went of the iaflanunatmy reaction. Corncal edema fttrtha facilit&s blood vessel ingrowth by looseningthe corn4 stromal frtunewwk and px&ling a pathway of “least resistance” through which the capillariescan grow. Following the initial intlammatq reaction, capillary growthiutothecomeaprocce&inthesamemannaasit occursin othertissues.The ncumaliyquiescent endothelial celts of the limbic capilkrks andvenulesare &m&ted to divide andmigrate.The endc4heJial pjeet away from cells their vesselsof &gin. digest the surroundingbasement membrane the tissuethroughwhich they will travel. and and migratetowardsthe sourceof the angiogenicstimulus.The blind endedsproutsacquire a huuen and theu anastomose toget@ to form c.apiUy loops. The end result is the estabWtment of a vascular pkxus within the anneal stroma. #mti-angiogcnic faucrs and compositionsof the present invention are useful by blocking the stimulatoryeffectsof

5716,981 33
angiogenesis promoters. reducing endothelial cell division,
decreasing endotbelial cell
migration.

and impairing

the

activity of the proteolytic enzymessecreted the endotby helium. Within particularly preferred embodiments of the invention. an anti-angiogenicfactor may be preparedfor and carotid CBV~~OUS fistulas. XII its early stages. neovastopical administrationin saline(combiuedwith any of the culat glaucoma nay be diagnosed by high magnification presnvaiivta and anthnicrob4al agentsCoJnmonIy in used slitbmp biomiuoscopy where it reveals small. dilated. ocularprepmtions). andadministered eyedrop in form The dimrgahtd capillarits (which leak fluorescein) on the anti-angiogcnic factor sohrtionor suspension be prcmay surfsc of the iris. Later gonioscopy demonstrates progrespared in its pure form and administered several times daily. sive oblit~ation of the anterior chamba angle by fibrovasAlternatively. anti-angiogeniccompositions.preparedas cnlar bands. While the anttrioc dmmb~ angle is still open. dtscrii above.may also be administered directly to the However.once comea.within preferredembodhnents, anti-angiogenic consuvative therapies may be of assistaoce. the the angleclosessmgicalinttrvtotion is requiredin order to composition is preparedwith a muco-adhesive polym~ alltviatt the pressure. which binds to cornea Within further embodiments, the Therefcxe. within one embodiment the invention antiof anti-angiogenicfactors or anti-angiogeniccompositions may be utilized as an adjunct to conventionalsteroid angiogtnic factors (either alone cr in an anti-angiogenic composition. as &scaibed above) may be administered rn-PY. topicahy to the eye in order to treat early forms of neovasTopical therapy may also be useful prophyWkaUy in culat glaucoma mrneal lesions which are known to havea high probability Within other embodiments of the invention, antiof inducing an angiogenic response(such as chemical bums).In theseinstances treatment. the li$eiy in combina- angiogenicannpositionsmay be implantedby injection of the compoeEtion Ute m&m of the anterim chamber into tion with steroids.may be instiMed immediatelyto help angle.This pvides a sustained locahxedincreaseof antipreventsubsequent complications. factor,andpevents blood vessel growth into the Within Otha embodiments. anti-angiogenic the composi- angiogenic atta llnplaPted or injected anti-angbgtnic compositions tions describedabove may be injected directly into the comeal stroma by an ophthalmologistwdcr microsoopic which are placedbetweenthe advancingcapillaries of the iris and the anteriorchamberangle can “defend” the open guidance. preferredsite of injection may vary with the Tht anglefkom mvasc~on. As capillarieswill not grow morphology of the individual lesion. but the goal of the administrationwould be to place the cmnposition at the s” within a significant radius of the anti-angiogenic advanciag front of the vasculahue (i.e.. interspersed compositioa patency of the angle amld be maintained Within 0thK anbodimcets.the anti-angiogeaic composition between the blood vesselsasrdthe normal cornea). most In may also bt placed in any location such that the anticasesthis would involve perilimbic cornealinjection to factor is continuoustY released the aqueous into “protect”the cornta from the advancing bloodvessels. This ?= angiogenic the faaor conmethodmay alsobt utilized shottlyaft~ a comealinsult in =-I httmor.This would increase anti-angiogenic withirt thebwna. which in Uri-n bathesthe surface a&r to prcphylauiczdlyprevent corneal neovascularira- certhation of the iris and its abnormal @lbuiess. tbtreby providing tion. In this situationthe materialcould be injectedin tbe aootha mechanismby which to deliv~ the medication. pedimhic cane.43 inte~-~ptrsed betweentht corneallesion aadits Undesired potentialhmbic blood supply.Suchme& 4. These*erapeutic modalitiesmaY alSobe UsefulPophYhctically and in combinationwith existing treatments. cdsmayalsobeutihxedinasimilarfasbiontoprevent capillary invasion of transphUned corneas.Ia a sustained. within amber aspwl of the presentinvention, methods release forminjections might only bertquirtd 2-3 timaspa are providedfa; treating proliferative diabetic retinopatby. year.Asteroid could also be added the injection solution ccmqxisingthe step of administeringto a patient a themto co-ito reducehflamnation resultingfrom the injection its&. ,5 gxmticallyefkU.ive amountof an anti-angiogenic Within anotheraspectof the presentinvention,methods tion to tht eyes,such that the formation of blood vesselsis tffective amoUntof an anti-angiogenic compositionto the bt similar to thatdescribed abovefor neovascular glaucoma. blood vesselsis inhibited. ;u) In pattiadat, backgromrddiabeti~ retinopathyis Meved to diabulc Jeutlopatllylmdtr the iJJfluBriefly, neovasc~lar glaucoma a pathological is condition convat to pJ-olifKauve wherein new capillaries develop in the iris of the eye. ‘ Ihe enct of retinal hypoxia. Generaliy. neovasarlar tissue sproutafrom the optic mxvc (usually within 10 mm of the a&genesis Usuallycaiginates from vesselslocatedat the tdgt), and fkwn the surface of the rttina ia regions what pupillaq margin.and progresses aaoss the root of the his and into the trabecu3a.r meshwork FJtihsts and O&K 55 t&UC pafuSiOn is pear. tidy the C%piEarieS grow connectivetissueelementsare associated the capiuary bttweta the inner limiting znembmne the rttina and the with of growth and a tibrovascular membrartedevelops which posmior surface of tht vitrtous. Evenmally. the vessels !paia aaossthe antaior surface thehia. EventuallyU&s grow into tht vitteous and throughthe inner limiting memof tissuertaches the anttrior chamberangle whae it forms braoe.As the vitttous cormacts.traction is applied to the synechiae. Thesesync&& ia ttxn coalesce, scar.and con- 60 vease&,oftenresulting in Waring of the vesselstmd blindtract to uhimattly close off cbeanteria cbambcr at&. Tbt ing of thevitreousdueto hemorrhage. Fibroustractionfrom scar formation prevents adequate drainage of aqueous stxrrhg in the rttina may alsoprodUceretinaldefacbmertt humor throughthe angleand into the trabectrlar meshwork The conventionnlthtxapy of choice ia pametinalphotoresulting in an increasein intraoa.darpresmre that may coagulatiar~ dtatast retinal tissue, andtherebydecrease to result in blindness. 65 rttiml oxygendemands. AlthoUghinitially effectivt. thereis Neovasculat glaucoma generally occursasa complication a high relapst rate wllh ntw ltsions faming in othexparts of diseasesio which retinal ischemiais predominant. In of the retina Complications of this therapy include a
eye. such that tb.e formation of

particular,aboutone thhd of the patientswith this disord~ have diabeticretinopathyand28% havecentratretinal vein occlusion.0th~ causes in&de chronic retinal detachment. end-stage glaucoma.carotid artery obstructive disease. retroltntal fitroplasia. sicldecell anemia.intraoatlar tumors.

34

are provided for treating neovascuk glaucoma. comprising inbibi@i the step of administeringto a patient a thhbapoutically Briegy, tirepathologyof diabeticretinopathyis thoughtto

5.716,981
35
deerease peaipheralvision of up to 50% of patients in in orderto thesapcutieally treat a cancer or tumor. For mechanical abrasions the cxmlca.laser-induced of catarau example,anti-angiogenic factas or compositions described formation. acute glaucoma.and stimuMion of subretiual llmin may be fcXml&ed for topical delivery. in order to ncovascular growth (which can result in loss of vision). As treat cancerssuch as skin cancer.head and neck tumas. a result. this prccedureis performedonly when severalrisk 5 breast tumors. and Kaposi’ sarcoma.Within yet 0th~~ s factas are present.and the risk-benefitratio is clearly in aspects.tbc anti-aagiogeoicfactors a compositionspro favor of intervention. vi&d haeiu may be utilized to treat superficialforms of bladdercancerby, for example.intravesicaladministration. Tllaefae. witlliu particularly preferredcmbodhents of the inventioll. pliferative diabetic lctinopatlly may bc IO addition to cancer, however, nmcrous etbcr nontreatedby injection Of an anti-angiogenic factor(s)(or auti- 10 tumor&& angiogenesis-dependent diseaseswhich are angiogeniccomposition)into the aqueoushumor a the cbmcteked by the abnormalgrowth of blood vessels may vitrcolls, in adcr to blaease the Iocal coIlaoaation of also be mated with Ihe aoti-angiogenic factors or compoanti-angiogeaic facta in tbe retiua Preferably.this treatsitionsof the present invention. Rqxesenuuiveexamples of ment shouldbe initiated Mor to the acquisitionof severe such non-tumorigenic angiogenesis-dependent diseases &case rquiring photoabagulation. WIthin other emhodi- 1s indude byput+& scarsaud k&ids. proliferativediabetic meritsof the invention.&erics whirh feed the neovascular retinopathy (discussed above). rheumatoid arthritis lesions may be embolized (utilizing anti-angiogenic (diawssedabove).artaioveoousmalfcrmations(discussed compositions, desdi above) as above), atherosdrrotic plaques. delayed wound healing. Within anotheraspectof the presentinvention. methods hemophilic joints, nonunion fractures. Osier-Weber areprovidedfa treatingretrolentalfibrobiasia.comprising 2~ syndrome, psoriasis. pyogenic granuloma. sclemderma. m~nanhagia (di~~ussd &we) and vascularadhethe step of administeringto a patient a tbempeuticaby ~IWOIM. sions. dfcetive amount of a0 anti-fmgiogcuicfactor (a antiangiogcIliccomposition) the eye, suchthat tbc fcxmation to Fa example,within one aspect of the presentinvention of blood vesselsis iubibited. methodsare provided for treating hypertrophicscars and keloids. commisiag the step of administuing one of the Bridfy. retrolentali&oblasia is a conditionocaming in premature infants who receive oxygentherapy. periph- abovedescrii anti-angiogeniccorm~~itionsto a &perThe ml retillal v-. particularly 011 tempcral side, trophic scar a keloid. the does not lxmme fully formed until the end of fetal life. Briefly. healingof wounds and scarformation occursin Excessive oxygen(evenlevels which would be physiologic tbrce pbascs:inflammation. proliferation. and maturation. at term) and the formation of oxygen free radicals are The first phase. inflammation, occurs in responseto an thought to be importmt by causing damageto the blood injury which is severe enoughto hmak the skin. During this vesselsof the immatumretina.Thesevesselsconstrict,and phase.which lasts 3 to 4 days. blood and tissue fluid form tbcn become smluumlly oblita7!tcdon exposutcto oxygtu. II adhesive coagulum fibrinousnetworkwhich serves and to As a result. the peripheralretina fails to vascu.la&eand bind the wound surfaoes together.This is then followed by rctiual ischemia ensues. re:sponse the iscbemia. In to neovas- a proliferativepbascin which thereis ingrowthof capillaries culari&on is inducedat the junction of the namaJ andthe and conncdivetissuefrom the woundedges.andclosureof isdmnic retiua. the skin defect, l%tally, onw capillary and fibroblastic III 7.5%of the eases tbcsc vcssclsregressspoutancously. proliferation has cursed, the maturation process begins However.in the remaiGg 25% thereis continuedcapillary whereinthe scar contractsand becomesless cellular. less vascular,and appears and white. This fmal phasemay flat growth, cxxmction of the fibrovasatlar component.and take between 6 and 12 xnontbs. traction oo both the vcssclsand the retina.This resuhsin vitreous hemorrhage and/or retinal detachment which can If too much COMC&VC tissueis producedandthe wound ’* lead to blindness. Neovascular anglwzlosurc glaucoma is rcmaiaspcmistcntlycdlular. the scarmay becomered and ~ also a c~mplicatioa of this condition raiscd.IfthescarnmaiEIh witbin the bo~darics of the ~woullditisrdaredtoasahypntrophicscar,butif As it is often impossibleto determinewhich caseswill beyondthe original scaraudinto the stxounding spont~ccusly resolve and which will progressin severity, it extends tissue. the lesion is refured to as a keloid. Hypatro@ conventionaltreatment(i.e.. surgery)is generallyinitiated scarsandkel~ids am producedduring the secondand third only in patieotswith established disease a well devcland cped pathology.This “wait and see”approachprecludes phasesof scar fcrmation. Several woundsare particularly proneto excessive endotbelialand t&mblasticproliferation, early intavention, andallows the progression disease of in induding buns, open wounds. and infected wounds.With the25% who follow a complicated course. Therefore,within hypatrophic scars,some degreeof matmtioo oocursand one cmboddent of tbe invention, toplcal administmtiooof grsdualimprovement occurs.In the caseof keloidshoweva. anti-aogiogeoic factors(or anti-angiogcnic compositions. as an actualtumor is producedwhich filIl becomequite large. described above) may kc accomplished in infants which are Spoutaneous imgovemcnt in SW&eases rarely occurs. at high ri*fa developing this condition in an attemptto cut down on the incidenceof progressionof ntroiental fibrol%crefaG withio one cndmdbcnt of the peaent iuvenplasia within otbff embodhats. inhavitruws injections tioa eitherauti-augiogenic factas alone.a anti-aagiogenic an&or intmoadar implantsof an anti-angiogenic composi- compodions as &scribed above.arc directly injcctcd into a tion may hc lItaid. such me#ods arc patticularlypreferred bypertropaicscara keioid, in ader to preventthe progmsin cases established of disease, crda to reducethe need sion of these lesions. Tlke frequency of injections will in for surgery. depend upon the rckasc kinuics of the pOrrma used (if present).and the clinical response. This therapyis of parUrHEiR -c USES OF AmIticular value in the prophylactic trcatmnt of conditions ANOIOOB’ IC N’ COMPOSITlONS wbicb are known to result io tke dcvclopmcntof hype Anti-angiogcnicfactors and compositions the present tropbic scars and keloids (e.g., burns), and is preferably of inventionmay be utilized in a variety of additionalmethods initiated after the proliferative phase has bad time to

36

5,716,981 37 38
progress (apgcoximately d8YS after the initial injury). but 14 intraocufarly. intranasaJJy.Jntradermahy.sublingually, beforehypertrophicscar or keJoiddeveJqmtent. oraJJy.topically. iotravesicaJJy. intrathecaJJy.topically. intracranially, As notedabove.wJthJn anotheraspectof the present intravenously, intraperitoneally. yet JntramnscuJarJy. SubartaneousJy. even directly into a or invention. vascular grafts are provided comprisJng syna site. tbetic tube. the surfaceof which Js coated~4th an anti- 5 tuma or disease O&r rgesentative routesof admin&XP andcoionoscopy. which angiogenic compositionas desaibedabove.Briefly, vascu- istrationindude gastmscopy. do not re@re fnU operatingprocedurtsandhospitaJizatioa lar grafts are synthetictubes. usuaIJymade of Ihcsmn or of personnel. Gokex. hated surgically to bypass attcriat blockages, but may require the presence medJc.aJ mastfrquently from theaortato thefemoraJ. the femocaJ cc The anti-angiogenicfactors. anti-angiogenk composito the poplitcaJattery.A majccproblemwhich partJc&arly t* tiom andpharmacautJcaJ composJtions providedhereinmay complicates femoral-popJiteaJ bypassgraftsis the famation be placedwidtin containers. alongwith packagingmat&d of a subendothelial scar-Jike reactionin the bloodvesselwaJJ which providesinstructionsregardhrg useof suchmatethe called aeoiraimal hyperplasia.which natrows the lumen rials. GeneraLly, such instructionswiJJ include a tangible within and adjacentto eitherendof the graf&andwhich can expression describingthe reagentconcentration. weJJas as be progressive.A graft coated with a containing ant& ts within certain embodiments. rdadve asnounts excipient of angiogenicfactors (or anti-angiogenk compositions,as ingredfentscx dfJuents(e.g.. water. saJ.ine PBS) which or desuibedabove)may be utilJzedto limit the fcnmatJon of may be necessary reconstitute anti-angiogenic to the factor. anti-angiogenic composition. or pJuumaceuticaJ composJneointimalhypexpJasia either end of the gratt. The graft at may tben be surgicaflyplacedby conventional bypasste&don. 20 The following examples offeredby way of illustration. niques. are Anti-angiogeniccompositionsof the presentJnvention and not by way of Jimitation. may ako be utilized in a variety of ctJtczmanners.For example. they may be incorporated sllrgicaJsuturesin into ader to preventstitch granulomas. implantedJn the uterus Example1 (in the same manner as an IUD) for the treatment of 25 t&orrhagia ox as a form of female birth control. admitusPreparation Anti-InvasiveFactor of tered as either a paitooeal Iavage fluid or for perJtc+teaJ The shonldcrgirdle and skuJifrom a dogfishis excised JmpJamation the h‘ in eatment endometriosis. of attached a to wJtJt in monodonalantibodydirected against activated endo&eJJaJ thenscraped a scalpe1 orda to removeall muscleand cottnectiVe tissuefrom the cartilage. cartilage The celts as a form of systemicchemotherapy. utiJizedJn 30 associated or by diagnostic imagingwhenattached.to radioactivelyJabeled is thenbomogenizcd with a tissuegrinda. and extracted a stirring at room temperature 2 to 5 daysin a for monocloaalantibodywhichrcoogaizesadivatodendothelial continuous solution contaJnJng 2.OM guanhlhtm hydrochloride and CdlS. o.mM ME!s at pH 6.0. 33 KRMtTLtU’ IONANDAD-ON Afta 2 to 5 days.the cartiJage extractis passed through gauzenettingin orderto removethe Jarger constituents. The As noted above. anti-angiogeniccouqositions of the filkak is then passed throughanAmicon u.ltratJJtratJon unit Hesentinvention may be formuJated a variety of forms in which utilizes spiral-woundcartsidges.with a molecular (e.g.. miaosphaes. pastes.tihns cc sprays).Fur&r. the The &rate (containJ.ng proteins compositions the present of invention may be fonnuJated 40 weight cutofTof 100.000. to wiUi a moJecuJar weight of lessthan100.000 drdtons) then is containmore than one anti-angiogenic factor, to contaJn a variety of additional compounds, have cataJa physical diabzed anainst 0.02M MES buffer I& 6) with an Amicon to UJ&ti%n ,unJtwhich retains~xo&s ‘ a molecular titJt pperties (e.g.. ehstidty, a pariictdar meJtingpoJnt or a Weight Of greater than 3.000 daJtons. UtiJixingthis method, specifiedreleaserate). WsUdncertain emJ3odJments the of low molecular weight proteins and constituents are Jnvention.compositionsmay be combined Jn order to amountsof guanbiiumHCL achievea &sired effca (e.g..sevemJ preparations mJab ” removed.as well as excessive of 9 The diaiysate is concentrated a JinaJcmcenkation to spbaeamay be combinedin orderto achieveboth a quick WeJ. aad a slow or prolonged release of one or mare antiangiogenic factor). ExampJe 2 Anti-angiogenicfactors and compositions tJtepresent so of Analysis of VariousAgentsfor Anti-Angiogenic inventionmay be admJnJstered alone.cr in combkuteJther Activity tion with phatmaceuticaUy physiologicahyacceptabJe or carrier. cxcipJent.sor d&tents. GeneraJJy. such carxders A. CbJckCborJoaJJantoJc Membrane (“cam”) shouldbe nontoxicto recipientsat the dosages concenand Assays kationsemployed.GrdJnatiJy. preparation suchcom- 55 the of positions eataS comJ%aing therapeutic agent with the Fatilizcd domesticchick embryoswae incubatedfor 3 buffas. antioxidantssuch as ascorbicacid, low molectdar dayspiar to she&lessarlturing. In this pcedsxe. the egg weight(less than about 10 residues)poJyJqtides, wins. contents were emptkd by removmgthe shelllocatedaround amino acids. carbohydrates including ghzcose.sucroseor the air space. iatuior sheJJ The membrane then severed was dexkitts. d~elatingagentssuch as EMi% glutathioneand 60 andthe oppositeendof the sheJJ perforated allow the was to o&r stabilizersand excipients.NeutraJbnffued saJJne bt contents the egg to gentty slideout from the bluntedend. of dine mixed with nonspecificserumaJbumJn exen&ary are The egg contentswerecmptie.d round-bottom into stuihacd appriqdte diluents. glassbowls and coveredw&Jrpetri dish covers.‘ These were As notedabove,anti-angiogenic fauors. anti-angiogenic thenplacedinto an incubatorat 90% relative humidity and compositions.or phanm~ceutical compositionsprovhled 65 3% CO, and incubatedfcr 3 days. hereJn may be preparedfm admJnJstration a variety of by Pa&axe1 {Sigma St. Louis, Mich) was mixed at condifferent routes. JncludJng for example intrart.icuJarly, centrationsof 1, 5. 10. 30 ug per JO mJ aJicJuot 0.5% of

5.716981 39
aqueousmc#yIcchulose. since pa&tax& is insolublem wata, glassbeadswere usedto poduce fine par&& Ten miaolita aliquotsof this sohltionwa dried on parafhmfor 1hourformingdisks2mmindiamucr.Thcdricddisks containing pachtaxel wen then CarefuRyplaced at the 5 growing edgeof ea& CAM at day 6 of incubation Controls wae Wed by placing paclitaxcl-free methylcellulose disksontbeCAMsovn~esamctimtcanst.Aftna2biy exposure(day 8 of incubation)the vas&ahnc was examined with the aid of a stcreomicroscopc. Liposyn II. a white 10 visibility of the vascular details. The vasculature of unstained. living embryoswae imagedusing a zdss stem omiffoscopc which was intcrfaoxl with a video camera @age-MTJ Inc., Michigan City. Ind). These video signals were thendisplayed 160timesmagnificationandcaptmcd 1s at using an image analysis system(Vidas, Kontrun: Etching.
Gummy). Image negatives were tbca made on a graphics recorder (MO&I 3ooo; Matrix Instlumen~. orangeburg. opaque solution, was iajaztcd into the CAM to inmasc the

40
dermallayercontaining capillaries which lie subjaant to the ectodum adventitialcek and an inna. singk endcell layer (FIG. ID). At the clcctronmicroscopiclevel. the typical structuraldetailsof the CAM capillariesare dcmonstrated. T&&xlly, thesevesselslie in close assockdion with theinnacclilayaofcccodam(FlG. lE) Alla 48 hoursc~posttrcto pachtaxelat concwatrations of 0.25,O.!il,5.~0. Q 30 ug, eachCAM was examined under living c0nditbns with a storeomicroscopc equippedwith a Vidodco3npUU hltcrfacc in otdcr to evaluate the cfkts on angiogemsis. imaging setupwas usedat a magnificaThis tion of 160timeswhich permittedthe &ct visualizationof bloodcellswithin the capillaries:therebybloodflow in areas of interestcould be easily assessed recorded,For this and study. the inhibition of angiogencais d&ted as an area was of the CAM lacking a capUary network and vascularblood flow. Throughoutthe expcimtllrs. avascularzones wue assessed a 4 point avascular 011 gradient(TableI). This scale represults the degreeof ovcrau inhibition with maximal inhibition rqxesentedas a 3 on the avascular gradientscale Paclitaxel was vay consistent aud induced a maximal avasculsrzone(6 m m in diameter or a 3 on the avascuhtrc gradientscale)within 48 hours depending its concentraon

N.Y.).

The membranes of the 8 day-old shell-lessembryowere 20 flcnnicd with 2% glutmld&yde in O.lM Na cacodylatc buffer; additional fixative was injected under the CAhf. Afta 10 minutesin situ. the CAM wasremovedandplaced into fresh fixative for 2 hcurs at room temprrat~. The tiO0. tissue was then washed overnight in cacodylate buffer 25 containing6% suaose.The areasof i&rest WEXC postfixed TABLE I in 1% osmiumtetroxidefor 1.5 hoursat 4“ C. The tissues wcre thendehydrated a gradedseriesof ethanols. in solvent AVASCULAR GJtADm exchangedwith propyleneoxide, and embedded Spun in o-ncwnfdvpnrhrity resin. Thin sectionswere cut with a diamondknife. plaocd 30 on coppergrids. stained.and examinedin a Joel 12OODI dwtron miao5copc. Similarly. 0.5 mm sedlons wac cut and stainedwith toluencblue for fight microscopy. At day 11 of development. chick embryoswere usedfor The dose-dependent. experimentaldata of the CSects of the ccuosion casting technique.Macox resin (Ted P&a. 35 paciitaxelat dW@ent concentrations shownin Table IL are IJK.. R&ding. Calif.) wasinjectedinto the CAM wscuktm using a 3O-gauge hypodumic needle.The castingmaterial TABLE I[ consistedof 2.5 gramsof Mctcox CL-2B polymer and0.05 gramsof catalyst(55% benzoylpaoxide) having a 5 minute pcdymaizationtime. Afta injection.theplasticwasallowed to sit iu situ for au hour at room temperature then and overnightin an oveuat 65’C. The CAM was thenplacedin 50% aqueoussolution of sodium hydroxide to digestall crganic components. The plastic castswere washed cxtensiv&y in distilled wata. air-dried, coated with gold/ paLl~x&&&mand&wed with the Philips SOlB scanning

Resrjs of the abovecxpgimentsareshownin FIGS.1-4. Briefly. the genaal featuresof the namal chick shell-less egg culture are shownin FIG. IA. At day 6 of incubation, so the embryo is centrally positioacdto a radial& expanding networkof blood vcsscLs; CAM develops the adjacent the to embryo.Thesegrowing vesselslie dose to the surfaceand are readily visible making this system an ideahzcd modelfor ttle study of angiogenesis. Living, uustainedcap&ty netwarks of the CAM can be imaged nouinvaaivclywith a sterwmiuoscope.FIG. lB illustrateaswb a vascularareain
which the cellular blood ckmcnts within ca@hrics wcxc

TABLE III

xtxaded with the USC a videofcompuuzinterface.The of 3-dimcusionalzr&tecturc of suchCAM capillary networks is shownby the corrusionca5ung methodand vkwed ill the scanning electron microscope(FIG. 1C). These castings revealedunderlyingvesselswhich projecttoward tbc CAM qpii p8cmaxd-tr~ CAMS are also shown with Ibe surface what they form a single layer of anastomotic transparent mdbylcclUosc disk antrally positioned ova capiharies. 65 the avascular ZOI~C measuring mm in diam&r. At a slightly 6 Transversesectionsthrough the CAM show an outer highermagnificati~ the per&by of aucb avascular zones cctodam consistingof a doublecell layer. a broadermeso- is dearly evident (FTG. ZC): the SulTwnding ftmuiooal

5,716,98 1 41
vesselswere often tcdirected away from the source of naclitaxel(PIGS. ZC and 2D). SU& aneular redirect&z of blood flow was nevu observedundK;ormal conditi&. Anotherfeatureof the effectsof paclitaxelwasthe ftxmation of Moodislandswithin the avascularzonerepresenting S the aggregation blood cells. of The associated morphological altorations of the paclitK.cl-treated CAM arereadily anuatent both thelight at &d eledron microscopic levels: Fb; the convenience-of presentation. threedistinct phases genKaltransitionfmm 10 of the normaI to the avascular state are shown. Near the puiphery of the avascular ZODC CAM is hallmarked the by an abundance mitotic cells within all three germlayers of (FIGS.3A and4A). This enhanced mitotic division wasalso a consistentckrvatioo for capillary cndothelial cells, 15 Howeva. the endothelialcells remained junctioWy intact with no extravasation of blood cells. With further degradatioathe CAM is CharactKized the tIrcak&wn by and dissolution of capillaries (PIGS. 3B and 4B). The presumptive end&dial cells. typically arrested mitosis, 20 in still maintain a closespatialrelationship with bloodcellsand lie subjacent the ectode?m, to howeva. thesecells am not junctionaIIyLink& The most centralportionof the avascuIar zonewas characterlizbd a thickenedecM~mal and by endodermal layer (PIGS.X and 4C).Althoughtheselayers 25 were thickened.the cellular junctions remainedintact and the layersmaintainedtheir structuralcharacnuistics. within the mesaterm. scatteredmitotically arrestedcells were abundant; these celk did not exhii the endothelialall pohrization observed the formK phase. in Also, throughout30 this avascularregion. degenaatingcells were commona5 noted by the electron dense vacuolesand cellular debris (FIG. 4C). In summaty,this study demonstrated 48 hoursaftea that paclitaxclapplicationto the CAM. angicgenesis inhib- 35 was ited- The blood vesselinhibition formedan avascular zone which wasrepresented threetransitional by phases pacliof taxel’ elfect.The ceniral. most affectedarea of the avass cular zonecontaineddisruptedcapillarieswith extravasated red blood cells: this indicated that intercellularjunctions 40 betweenendotbelial cells were absent.The cells of the endcderm ectodermmaintainedtheir intercelIuhu and junctions and therefore these germ layers remained intact; however.they were slightly thiclrcocd.As the nomudvascular amawas approached. blood vessels the retained tbelr 45 junctioaal complexesaad therefore also remained intad. At the paiphcry of the paclitaxel-treated zone,futthcr blood vessel growth was inhibited which was evident by the typed direchg or “elbowing”effectof the bloodvessels (FIG. 2D). xl Paclitaxel-treated avascularwns alsorevealedan abundanceof cells arrested mitosis in all threegerm layersof in the CAM; this was uniqueto paclitaxel since no prwious study has illustrated such an eveat. By being arrestedin mitosis. cndothelial cells could not undergotheir normal s5 metabolic functions involved in angiogeoesis. In comparison.the avascular zone fcxmed by suramin and cortisoneacetate not producemitotically arrest& cells in do the CAM; they only preventedfurtha blood vesselgrowth into the treated area.Therefore,eventhoughtheseagents 60 are anti-MgIogenic,there are many points in which the an@ genesis psncess may be targeted. The effectsof paditaxel OVK the 48 hour durationwere also ObsCrVCd. During this paid of obseaw.tion was it noticedthat inhibition of angiogenesis occursas e&y as 9 65 hours aftet application. Histological sectionsrevealed a similarmorphologyas seenin thelirst transitionphase the of

42
avascular zone at 48 hours illustratedin PIGS. 3A and 4A. Also, we observed the revasculatization in processinto the avascularwne previously observedIt has beenfound that the avascularwne formed by heparinand angiostaticsteroids becamerevascukiud 60 hours after application.In one study.pacIitaxel-treated avascuiartones did not revaswlarize for at least7 days&a applicationimplying a more potent long-termeffect Example3
3Wapsulation of Suxamin

One milliliter of 5% EXVAX (poly(ethylene-vinyl acetate)cross-linked with 5% vinyl acetate)in did&. romethane(“m is mixed with a fixed weight of submiaon groundsodiumsuramln. mixtureis injectedinto This 5 ml of 5% polyvinyl Alcohol (‘ TVA”) in waterin a 30 ml Batbottomedtesttube.TubescontainingdiffKent weightsof the drug arethen suspended a multi-sample in water bath at 4o”for 90 minuteswith automated stirring.The mixturesare removed and microsphere samplestakenfor size analysis. Tubes are centrifuged at 10tM g for 5 min. The PVA supernatant is removed and saved for analysis (n~encapsulated drug).The tnicrosphexes then washed are (vatexed) in 5 ml ofwata andrecenttifuged.The ml wash 5 is savedfar analysis(surfacebounddntg). Microspheres are then welted in 50 ul.of methanol,andktexed ih 1 ml of DCM to dissoIvethe ELVAX. Ilte microsuheres thea are warmedto 40”C.. and 5 ml of 50”C water& slowly added with Stirring. This pimcahm resultsin the immediateevaporation of DCM, thereby causing the releaseof sodium sttraminintotheSmlofwater All sampleswere assayed drug contentby quantififor cation of ff~orcscencc. Briefly. sodium sutamin absorbs uvh-is with a lambda mar. of 312 nm. This absorptionis hnearintheOto 100ug/mlrangeiabothwata8nd5%WA. Sodiumsuramb ako ffuoresces strongly with an excitation maximumat312muandemissionmaximumat400mu This flucreswnceis quantitiable the 0 to 2.5uglml range. in is The resultsof theseexperiments shownin PIGS. 5-11. Resultsare shownin FIGS. 5-10. Briefly. the size distritution of mkzospheres number(PIG. 5) a by weight (PIG. by 6) appears be unaifectedby inclusion of the drug in the to’ DCM. Goodyieldsof microspheres the 20 to 6Opmrange in may be obtaiqq3. The encapsulation suramktis very low (~1%)(seeFlG. of 8). Howeveras She weight of drug is increased the DCM in thetotal amountof drug empdatad increased althoughthe 8 eacapdation deueascd. is showain PIG. ?,50 ug of As is shownin PIG. 9 (size distriibntionby weigh& Encapsulation of sodim suraminin 5% PVA containinn10% NaCl is shownin PIGS.10 and 11 (size distributiou cwcight, and number.respectively). To assesssuratnin and cc&one acetate as potential anti-*ogenic agents,each agent was mixed with 0.5% methylcelluIose applied the dried disks containingthe and agentonto the developingbIood vesselsof the 6-&y old CAM. A combioationtreatmentof suramin (70 pg) with cmtiswe acetate fig) was successful inhibiig angio(20 in genesis whentestedon the CAh4for 48 hours.The resulting avasctdar region measured mm in diametK and revealed 6 an absence blood flow and the appcamnce sparse of of blood isIan& (FIGS. 28A and 28B). Example4 BncapsuIation Paclitaxel of Five hundredmiczograms eitherpa&axe1 or bacc&n of (a paclitaxel analog.avaiIablehpm Inflazymephatmaceu-

5,716,981 43
ticals Inc., Vancower. British Columbia. Cenada) art dissowed in 1 ml of a SO50 ELVAX:poly-l-lactic acid mixture in dcm Micmspheres are then prepared in a dissolution machine (Six-spindle dissolution tester, VaaderKaap. vaa Kell LadustriesInc.. U.S.A.) in triplicate at 200 rpm, 42’ C.. for 3 hours. ?vlicrospheresso patepared washed twice in art wata aad sired on the micrcscope. Determiaation of pa&axe1 encapsutafion is undertaken in a uv/vis assay (uv/vis lambda max. at 237 mm. fluorescence assayat excitation 237, emission at 325 mn; Pluorescence results are presented in square brackets [ 1). Utilixiag the procednres dcscxibcd above. 58 pg (+I-12 pp) 175 pg (+/-25 pg)] of pa&axe1 may be encapsulated from a total SO0 pg of starting material. This represents 12% (M-2.4%) [15% (t&S%)] of the orijJinai weight. cf 1.2% (+-I-&.25%) [IS% (+/-OS%)J by weight of the polymer. After 18 hours of htmbliag in aa oven et 37’ C., 103% (+/-lo%) [6% (+/-5.6%)] of the total peclitaxel had heen released from the microsphaes. For baccatin. NO+/-15 pg [83-M-231 & of bacc&ia caa be encapsuiatedfrom a total of 500 w start@ matmial.This repseats 1 20% (d-3%) [17% (i/-S%) of the origiaal weight of baccatia. and 2% (+03%) [1.7% (+/-OS%)] by weight of the polymer. Afta 18 hours of tnmblitrg ia an oven at 37O C.. 55% (M-136) 160% (if-23%)] of the baczatia is released &om the microspheres.

44

The abdcminal wound is closed with 6.0 resorptible suture with skin clips. and the anesthetic terminated The rat is returned to the animal care facility to have a staadard diet for 14 days, at which time each tumor &posit will measure 1 an in &meter. The same procdure is repeated using Westar rats and a Colon Cancer cell line (Radiologic Oncology Lab. M. D. Aadersoa Houston. Tex.). Ia this instance, 3 weeks are required post-injection for the tumor deposits to measure lCXtilldiamacrCa& i%ftK 2 w 3 We&S, depending on the rat species, the same general anesthetic prooedme is followed and a midline abdominal iadsion ls pesfonaed. The duodenum is flipped and the gastroduodeaal artery is identified aad mobilized, Ties arc placed above and below a cutdowa site oa the mk#wrtion of the gasiroduodenal artery (GDA). and 0.038 inch polyethylene tubing is iatroduced in a retrograde fat&ion into the artery using an operatiag microscope. The tie below the iasertion point will ligate the artery, while the one above wiil fu the catheter in place. Aagiography is performed by iajectiag 0.5 ml of 60% rediopaque coatrast rnatcriaI through the catheter as an x-ray is taken. The hepatic artery is then embolixed by refluxiag particles measuring 15-200 )un through the gastroduodeaal artery catheter until flow. obsaved via the ope~atiag miaosccpe. is seen to cease for at least 30 seconds. Occlusion of the hepatic artery is coaiirmed by repeating an angiogram through the GDA catheter. Utikiag this procedure. one-half Fxample 5 of the rats receive U-200 pm particks of polymer alone. and the 0th~ half receive 15-200 p particks of the AMIysis of surgical Paste Coateiaing Aati30 polymer-anti-angiogenic factor composition. The upper Angiogenic Compositions GDAligatureistightenedtooccludctht GDAas thecatheter is withdrawn to ensure hemostasis. aad the hepatic artery Fisk rats weighing approximately 300 grams arc (although cmbdized) is left intact. The atxlomen is closed anestbelimi, aad a 1 cm transverse upper abdominal iaciwith 6.0 absorbable suture and surgical clips. Sian is made.Tw*tenths of a milliliter of saline coataiaiag Tbc rats are subsequently sacrificed at 2.7.14.21 and 84 lx 106 live 9 L gliosarcoma cells (eluted immediately prior days post-emboliration in order to determine efscacy of the to use from tissue culture) ere injected into 2 of the 5 hepatic aati-angiogenic factor. Briefly. genemf anesthetic is given, Iobts by piercing a 27 gauge needte 1 cm through the liver aad utihxiag aseptic precantions. a midline incision percapsule. The abdominal wound is closai with 6.0 nsorptible formed- Tbe GDA is mobilized again. and after placing a suture end skin clips aad the GA tumiaated. ligature near the junction of the GDA and the hepatic attuy After 2 we&s, the tumor deposits will measure approxi(i.e.. well above the site of the previous cutdown). a 0.038matciylemAtthistime.bothhepatichuwrsarereseckd inch polyethylene tubing is inserted via cutdown of the and the bare matgin of the liver is packed with a hemostatic vessel aad augiography is perfamed The rat is thea euthaagent. The rats are divided into two grcnrps:half is admianixed by inject@ Euthanyi iuto the dorsal vein of the tail. istued polymeric canier alone. aad the other haif receives Once euthanasia is confirm&, the liver is removed en bloc an anti-angiogeuic composition. aIoag with the stomach. spleen md both lungs. Rats KC sacrificed 2.7.14.21 aad 84 days post hepatic Histologic analysis is performed on a prepam slide resection. la particular. the rats are cuthanized by i@ecting stained with bematoxylia and eosia (“H aad E”) staia. IWhanyl into the dorsal vein of the t&L The Iha, splcea. Briefly, the luags am sectioned nt 1 cm intervals to assess and both lungs are removed. and histologic analysis is passage of embolic materhI through the hepatic veins and performediaordert.ostudythetumcesforevideaceof into the right side of ckufatioa. The stomach and spleen arc anti-angiogenic activity. also sectioned in crdex to assessiaadvc&at immobiioa fromrellux of particles into the C&X accessof the collaterel Ejrample 6 circulatloR Embolia3tion of F&t Artf!ries Example 7 Fii rats weighing approximately 300 grams are ants-

thetized Utilixiag aseptic proadures, a 1 cm transverse lkaasplaatatioa of Bitiery Steats ia Bats abdomdl iacision is made. aad the liver identified 60 General anesthetic is administ~ed to 300 gram Fkha !iC&tths of a milEEta of saline containing 1 million live rats. A 1 cm traasvcrse iacision is thea made ia the upper 9 L gliosarcome cells (ehlted imme&mly prior fromtissue abdomen, aad the liver ideatificd In the most supafwial culture) is injected each of the 5 hcpatic lobes by into lobe,01mlafsalineoon~gl~oa~sof9L piacing a 27 gauge needle 1 cm throogh the liver capsalt. One-tenth of a millilk of acamel saliae is injected iato the glloarcoma c&s (elutcd thm tissue culture immedkkiy needle as it is withdrawn to ensure that tbKC is DO spillage 65 priortouse)isinjccltdviaangaugc~etoadepmof 1 cm iato the liver capsule. Hemostasis is achieved 8ftK of celb into the pcrltoneal cavity. A pledger of g&ram is placed on each of the puncture sites to ensure hemostasis. removaI of the aeedle by placiag a pledger of gelfoam at the