AU2361499A - Targeted delivery via biodegradable polymers - Google Patents

Description

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AUSTRALIA

Patents Act 1990 12** 1

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FOCAL, INC.

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Targeted delivery via biodegradable polymers The following statement is a full description of this invention including the best method of performing it known to us:r-; El 1Ale

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b- I- I 2 SBackground of the Invention This invention is generally in the area of drug delivery and gene therapy devices and more specifically in the area of delivery of drugs and gene transfer via polymeric microparcicles, including liposomes in a polymeric matrix.

A variety of materials have been developed for delivery of drugs, nucleic acids, and biologics. Examples include microspheres, microcansules, and microparticles formed of biodegradable or non-biodegradable polymers which release the incorporated material over time or following exposure to specific conditions.

Targeting of the materials, other than through direct administration at the targeted site, has been very difficult. Most are administered systemically if multiple release sites are recuired.

20 More recently, polymeric gels or films i have been utilized for drug delivery and gene therapy, especially of small oligonucleotides such as antisense. Liposomes have also been utilized for delivery of genetic material, with varying degrees of success, primarily due to the inherent instability and short half-lives of the liposomes.

Gene therapy is typically used to refer to delivery of nucleic acid molecules which control expression of a particular endogenous gene, or to delivery and expression of an exogenous gene, which functions in addition to, or in place of, a defective or missing endogenous gene.

Three methodologies have been developed as the principal mechanisms for gene therapy: delivery via cationic lipids, for example, in the form of liposomes or vesicles, molecular conjugates, and recombinant viral vectors. These 3 methods were recently reviewed by Morgan, PA-

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ioche., 62:11 (2153, Mulligan, Science 260:926 (1993n, and Tolstoshev, Ann. Rev. Pharm. Toxicl., 32:573 (1993).

Although the three major groups of aene transduction methodology are relatively efficient, the percentage of targeted cells that can be transduced in vivo remains relatively low. To treat conditions reqruirng a higher percentage of gene transduction, new technologies for increasing the percentage of transduced cells would be very useful.

~Furthermore, it is very difficult to targer cells for delivery of genes, other than through local administration or through selection of viral vectors which infect only certain types of cells, such as replicating cells. Local delivery has advantages in that the effective local concentration is much higher than can normally be achieved by systemic administration, while the A S;systemic concentration remains very low, thereby J avoiding serious side effects. There are few methods available, however, which allow one to target scattered areas throughout the body, to l 25 achieve local release without systemic involvement.

The ability to express recombinant genes in the blood vessel wall has raised prospects for gene therapy of vascular disease. Two general approaches to introducing genes into the vessel wall have been studied. In one approach, referred to as direct gene transfer, target cells are first isolated and gene transfer is accomplished in vitro; cells that express the recombinant gene product are then selected and transplanted into the host vessel wall. In the second approach, genes are delivered "in situ" to cells within the vessel wall; this direct, in vivo approach to delivery of 4 cenes is a ractiv as a therapeutic modalicy since t mizi-=aes the need to remove vascular cells from the oatient. Since direct gene transfer precludes the opportunity to select for positive cransfectants, however, it is essential that an adecuate amount of DNA be introduced and expressed by the taraer tissue. Vascular smooth muscle cells may be suitable targets for the direct gene transfer approach because of their proximity to the lumen surface and abundance in the vessel wall.

Furthermore, abnormal accumulation of smooth muscle cells is a feature of atherosclerosis and of certain accelerated forms of vascular disease, such as restenosis following balloon angioplasty.

15 One notential means of transfecting smooth muscle cells within the vessel wall is through the use of cationic liposomes. Liposomemediated gene transfer is a convenient method of transferring recombinant DNA into cells and has 20 been used to directly transfect the arterial wall of live animals. The efficiency of successful gene transfer using cationic liposomes, however, is variable and highly dependent on the cell type.

Most in vitro experience to date has been with S 25 continuous/immortal animal cells lines. The results studied using these types of cells, however, have uncertain implications for the Slikelihood success of direct arterial gene transfer in patients.

Local delivery of growth factors has been attempted in several ways. Takeshita, et al., J.

Clin. Invest., 93:662-670, (1994), delivered a bolus of a transforming vector for the growth factor VEGF to rabbits. Unfortunately, delivery was not limited to a local area. U.S. Patent No.

5,238,470 Nabel et al discloses administering S transforming vectors to arteries via a 1 double-balloon catheter. A major limitation to this method is that the genetic material is administered all at once, resulting in inefficient transduction. A further limitation is that Nabel requires a substantial instillation time, approximately 30 minutes, resulting in prolonged arterial blockage.

There is therefore a need for an improved, specific delivery means for nucleic acid molecules and other drugs, or bioactive molecules.

Summary of Invention In a first aspect, the present invention consists in a method of delivery of biologically active molecules to a targeted site in an animal where treatment is needed, the method including: S* selecting niicroparticles having a diameter between 1 and 100 microns, said microparticles comprising a synthetic biodegradable polymer and S.:1 biologically active molecules; and 15 administering the microparticles to the animal so that the microparticles selectively lodge at the targeted site within the animal where release is desired, for a sufficient amount of time to permit controlled release of a therapeutically effective amount of the biologically active molecules, wherein the biologically active molecules are selected from the group consisting of growth factors, cytokines, angiogenesis factors, interferons, interleukins, colony-stimulating factors, immunosuppressant molecules, clotdissolving agents, peptide fragments thereof, and nucleic acid constructs capable of synthesizing these compounds; and wherein the synthetic polymer is biodegradable, polymerizable 25 macromer comprising at least one water soluble region, at least one degradable region which is hydrolysable underin vivo conditions, and at least one polymerizable group on said macromer having the ability to form additional covalent bonds resulting in macromer interlinking, wherein the polymerizable groups are separated from each other by at least one f 30 degradable region.

Preferably, the microparticles are administered into the circulation and release the biologically active molecule after the microparticle selectively lodges. The targeted site is preferably an area where vascularization ori revascularization is needed, and wherein the biologically active molecule is a 4 35 growth factor selected from the group consisting of vascular endothelial growth factor (VEGF), endothelial cell growth factor (ECGF), basic fibroblast 'i.

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6 growth factor (bFGF), bone morphogenic protein (BMP), platelet derived growth factor (PDGF), active peptides thereof, and nucleic acid constructs capable of expressing the growth factors.

The microparticles can be administered in a pharmaceutically acceptable carrier by a route of administration selected from the group consisting of intravenous, intraarterial, percutaneous, intramuscular, subcutaneous, direct lavage, aerosol, and via surgical incision. Preferably, the route of administration is intravascular.

Preferably, the targeted site is an area of a blood vessel where abnormal accumulation of smooth muscle cells has occurred. Examples include an area of a blood vessel where restenosis has occurred after angioplasty, an area where atherosclerosis has occurred, particularly where the atherosclerosis is present in the lower limbs causing claudication.

The targeted site is also an area where ischaemia has occurred.

S 15 The microparticles are preferably administered into a region or organ so as to lodge or adhere at a locus within the organ.

In one form, the microparticles are polymeric microspheres in the form of a hydrogel, preferably the hydrogel is composed predominantly of a Spolyalkylene oxide. The hydrogel can be made by the polymerization of a S 20 water-soluble macromer, said macromer including a water-soluble backbone, .:one or more degradable linking groups, and one or more reactive groups covalentlv linked to said degradable linking groups.

In a further preferred form, the targeted region is the lung.

In a second aspect, the present invention consists in a method of 25 increasing the vascularization of a tissue, the method including: selecting at least one biologically active molecule which is a deliverable growth factor equivalent, said equivalent comprising at least one of a protein and a nucleic acid encoding a protein, characterized in that said protein stimulates the growth of blood vessels; mixing said equivalent with a biodegradable, biocompatible polymeric material capable of being polymerized to form a delivery vehicle for said deliverable growth factor equivalent; conveying said mixture to a tissue in need of increased vascularization: and polymerizing said polymeric material: I

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7 whereby a depot is formed which locally releases deliverable growth factor equivalent to said tissue.

In one form, the polymer is polymerized after it is conveyed to said tissue. In another form, the polymer is polymerized before it is conveyed to said tissue.

The polymeric material is preferably in the form of microspheres.

Preferably, the growth factor equivalent is entrapped in a material limiting its diffusion rate before being mixed into said polymerizable material. The diffusion-limiting material is preferably selected from liposomes and polymeric microspheres.

The deliverable growth factor equivalent is preferably selected from Sthe group consisting of growth factors, cytokines, interferons, interleukins, colony-stimulating factors, angiogenesis factors, immunosuppressant molecules, peptide fragments thereof, and nucleic acid constructs capable of 15 expressing these equivalents.

The deliverable growth factor equivalent is preferably selected from the group consisting of vascular endothelial growth factor (VEGF), endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF], bone morphogenic protein (BMP), platelet derived growth factor (PDGF), peptides thereof, and nucleic acid constructs capable of expressing the factors.

In a third aspect, the present invention consists in a method of increasing the effective vascularization of a tissue, the method including: selecting at least one biologically active molecule which is a clot- 25 dissolving material, said material comprising at least one of a protein and a nucleic acid encoding a protein, characterized in that said protein accelerates the removal of clots or prevents clot formation; mixing said biologically active molecule with a biodegradable, biocompatible polymeric material capable of being polymerized to form a delivery vehicle for said deliverable biologically active molecule; I[c) conveying said mixture to a tissue in need of increased vascular permeability; and polymerizing said polymeric material; whereby a depot is formed which locally releases said biologically 35 active molecule to said tissue.

Sfy 8 i Preferably, the biologically active molecule is at least one of tissue plasminogen activator, streptokinase, urokinase, or heparin.

In the three aspects according to the present invention, the degradable region preferably contains at least one linkage from the group consisting of hydroxy acids, amino acids, peptides, anhydrides, phosphoesters, orthoesters. and carbonates.

In the first two aspects according to the present invention, the targeted organ, tissue or region is preferably selected from the group consisting of the urinary tract (including ureters and urethras), bronchi, biliary and pancreatic ducts, the gastrointestinal tract (including the gut or intestines, and the stomach), nasolachrimal ducts, sinus cavities, the eye, eustacian tubes, spermatic tubes, fallopian tubes, the lung, the heart, bone. cartilage, lymph nodes, the skin, and organs bathed by the cerebrospinal fluid.

In a fourth aspect, the present invention consists in the use of the 15 method according to the first aspect of the present invention for inhibiting the expression of a gene at a site in a vessel.

Detailed Description of the Invention i Targeted, enhanced delivery of biologically active molecules is enhanced by the use of polymeric carriers for targeting of the molecules to specific areas. In one embodiment, the polymeric carrier is a hydrogel which serves to immobilize the bioactive molecules at the site of release. In another embodiment, the polymeric carrier is in the form of microparticles that are i r g targeted by size and degradation and release properties to particular regions S. 25 of the body, especially the alveoli and capillaries.

Polymeric Carriers Selection of Polymeric Material Polymeric carriers should be biodegradable, sufficiently porous to permit efflux of the biologically active molecules, and sufficiently m U1 i L A LO 11 o 0. (D t 1 -A p, to A *4 .4 .4 IUj 0 (D I) 0 11 1 A lJ H I- H 141a 1 0 (1 '1 Iii I- (P 01 1

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It l~ Ilcal aC. Z=_CL Lrcmt Scuc-- S=1 ar' Sigm Cc- Ol or el- sv__helzea -ronm rncmr clbtalrieQ r V=ad chr ah l m n-c~~tmarS an dearaQ~~ga ::cc ncn~-toxic coTTnetsTS are _e;obo~n~ a-r c'nem-1 ,aily ccnua-ihl w~z-h -nfri SUTaS-._ to he d-'-era aff C~end not tc anature -vie aczv Sszazics; are Sttlc~~t' ucousto ail-_w ra co--Zorazoi o biclo07a 4 cIV aCtiVz -ojecules annr ei une-an c; r-O.i zz=. O1-ver by =rrusion, erosion or a comLornaiion. rrtereoz; ar abl orial h a!)P!a .Lioatoo by adherence or by qeo~rer__C 'facts, SUCn as my beiana rorm~en 7n O~ace or Lz tLedc and subseOuecSly molded or formed into micrnatale 'lncn are tracea at: a desired.

locaclon; are cacan-e of neingadelivered by tec-im'iaas or, rtj-n--rmamr -nvasivifty, such as by 8cheela-paroscone or enaoscome Tyoces orf C. -monamers, macromers, and ocaiiers that can- be used are descrinbd -in more deta__ below.

Hyd rogei Hvdronaels are oreferred. emboaM'ears r applicaton to a cissue tFor direct deliVvy since 3 S they intrias-ically have most of: these desiable lorooerties. Particularlv o'e-trred are ge I w-ich are comnosed Dredominan' 1 yl of ~oyt-y n lycol.

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an- iniiaccr, bVy chemical Po~lvmerizacion with a Ce-x-no bY "inl-rerfaciall" pnoooivm-ization, with a dye adsorbed to the Lissue toC be coated, as described below.

Exampnles of these macromers are PEGoligolacn-yl-acrylates, as described by Hill-West, e: al-,Po NatI'. Acad. Sci. US 15 -5971, 1994; Obsc. Gynecol1 83:59-64 1994. The choice of approprilate end cans permits rapid photopolymerization and gelation; acrvlates can be polymnerized using several initiating systems, e.g., an eosin dve, by brief exposure to ultraviolet or 1'iil 14-ihz Poiv(echyleneglycol) or a PEG central szructural unit (core) is useful on the basis its high hydrophilicity and water solubility, accompanied by excellent o'ocompatibility. A short poly(a-hydroxy acid), such as -Dolyalycolic acid, is a preferred chain extension. because it rapidly degrades by hydrolysis of the ester linkage into glycolic acid, a harmless metabolite. Although highly crystalline polyglycolic acid is insoluble in water and most common organic solvents, the entire macrorner is wiater-soluble and can be rapidly gelled into a biodegradable network while in contact with aqueous tissue fluids. Such networks can be used to entraD ndhomogeneously disperse water-soluble drugs and enzymes and to deliver them at a controlled rate.

other p~referred chain extensi-ons are polvlactic acid, polycaprolactone, polyorthoesters, and polyanhydri'des. Polyo~ides may also be used.

These materials are particularly useful for controlled delivery, especially of hydrophilic materials, since the water soluble regions of the polymer enable access of water to the materials entzrappDed within the polymer- Moreover, it is ncss-Isr-- cclxmerize temacromer ==-ai'nC z~n mareril z beenzrazued w ztncut_ exccsing Lhe mat a to orca"'c -its. izelease may occur by oaz~usion of the mat~erial fr1om tohe zolymer prior toD dearada _ic and/or by diffusion of the material from thne uolvrer as i'degrades, denendazng uuon t-he nora sizes wzh.; the coly-mer, whic'k is cotroll ed by the molecular weichro between crosslinks and the crcsslink density. Deactivation of' thle entranned material is reduced due to the immobilizina and oraoecoive effect of the crel and cauas"IrOcfl1 burst effects associated with-' other cocb-e-ees systems are avoided. W~hen the entracned material -is an enzyme, the enzvme can be exrosed to substrate while the enzyme is entraoped, provided the gel proport_--ons are chosen to allow thie substrate to permeate the gel- Degradation or the oolymer- facilitates eventual controlled release cr free macromolecules in vivo by gradual hydrolysis of the terminal ester linkages.

An advanzage of these macromers are that they carn be polyrner.tzed rapidly in an aqueous surrounding. Precisely conforming, semi-permeable, biodcradblefilms or membranes can thus be formed on tissue in situ to serve as biodegradable barriers, as carriers for living cells or other biologically active materials, and as surgical adhesives. Inz a zarticularly preferred embodiment, the macromers are aDolie-d to tissue having a uohotoinitiator bound thereto, and lojvmerized to form an ultrathin coating. This is especialily useful in forming c oatinas on the inside of tissue lumens such as blood vessels where there is a concern regardin~g restenosis, and in forming tissue barriJert during surgery which thereby prevent adhesions from forming.

Seneral terms, the macrcmers are oolymers chat are soluble in acueous solutions, or nearly aqueous solutions, such as water with added dimethylsulfoxide. They have three components including a biodegradable region, preferably hydrolyzable under in vivo conditions, a water soluble region, and at least two photopolymerizable regions. The term "at least substantially water soluble" is indicative that the solubility should be at least about 5 g/100 ml of aqueous solution.

The term "polymerizable" means that the regions have the capacity to form additional covalent bonds i resulting in macromer interlinking, for example, carbon-carbon double bonds of acrylate-type 15 molecules. Such polymerization is characteristically initiated by free-radical formation resulting from photon absorption of S' certain dyes and chemical compounds to ultimately produce free-radicals.

In a oreferred embodiment, a hydrogel is formed from a biodegradable, polymerizable, macromer including a core, an extension on each end of the core, and an end cap on each extension. The core is a hvdrophilic polymer or oligomer; each 25 extension is a biodegradable oligomer; and each end cap is an oligomer, dimer or monomer capable of cross-linking the macromers. In a particularly preferred embodiment, the core includes hydrophilic poly(ethylene glycol) cligomers of molecular weight between about 400 and 30,000 Da; each extension includes biodegradable poly (a-hydroxy acid) oligomers of molecular weight between about 200 and 1200 Da; and each end cap includes an acrylate-type monomer or oligomer containing carbon-carbon double bonds) of molecular weight between about and 200 Da which are capable of cross-linking and polymerization between copolymers. More soe~~: =refe a rre emboQdime. Inccrnrazas a corn cf. Vo1V~ethlen=~ ci__"cO, olia'omers o:mecula' waig.!= about- 10,000 Da; exzelsiofls conz Orri o L oo vlcoljc acid) oligomers or S molecular weight about 250 Da; and end cap:s conis~ngacrylate moieties of about 100 Da molecular weiafltxamri-es of suitable materials fruea the core vrater soluble region are Doly(ethylele glycol), poiy(ethylele oxide), Qolyv(viflyl alcohol), poly(vinv1Pyrroliole), poly(ethyloxazol1ne)f poly Cethylene oxide) -co-pool (-oropyleneoxide) block cooolymers, voysaccharides or carbohydrates such as hvaiuronic acid, dextran, he-oarin sulfate, chondroitinf sulfate, heparin, or alginate, or proteins such as gelatin, collagen, albumin, or ovalbumin.

Biodegradable regions can be constructed from Polymers or monomers using linkages suscertible to biodegradation, such as ester, oentide, anhydrde orthoester, and phosphoester AExamples of biodegradable components which are hydrolyzable are Polymers and oligomers of glycolide, lactide, e-cap~rolactone, other ahydroxy acids, and other biologically degradable polymers that yield materials that are non-toxic or Present as normal metabolites in the bony.

Pref erred poly(a-hydrxy acid)s are poly(glycolic acid), poly(DL-lactic acid) and noly(L-laCtic acid). other useful materials include poly~atnino acids), poly(aflhydrides), poly(orthoesters), and poly(phosphoesters). Polylactones such as poly(Ecaprolactole), pOly(E-caprolacton1e), poly (6and pojy(gamma-butyrclactone), for example, are also useful.

4A 16 orereao~r o_-Vruer~ize"u usiLng f-ree rdcl gene-fated by a ohotoinitiator. The ohotojinitiator Dreferably uses the visible cr long wavelenath ultraviolet radiation. Preferred ;polymeriLzable regions are acryliates, diacrylates, oligoacrylates, z~encyaes, oligomethoacrylates, or bioloaic= 1 1y accentable vhotouolvmerizable cqrouus.

A -oref erred tertiary am~re is triethanol amine.

Useful cho Coini tiators are those which initiate free radical polymerization of the *macromers wiLthout cytotoxicity and within a short time frame, m.inutes at most and preferably seconds.

*6 Preferred initiators for Initiation using long wavelengrth ultraviolet photoradiation are ethyl :eosin, 2,2-dimethoxy-2-phenyl acetoohenone, other acetophenone derivatives, and campDhororuinone. in all cases, crosslinking and polymerization are initiated among copolymrers by a light-activated free-radical polymerization initiator such as 2,2dimTethoxy-2-ohenylacetoohenone or a combinauion of ethyl eosin (10-4-io- mM) and triethanol amine (0.001 to 0.1 for example.

In another embodiment, the process is carried out by providing a material that is conformable, at least temporarily, at body temperature, yet which may be rendered nonconformable upon completion of the deposition process, such as a poloxamer' (a polyethylene oxide-polyethylene glycol block copolymer).

Poloxamer' can be selected which are icud room temperature and solid at body temperature.

Pavixngs ox Films in other embodiments, such as that described in U.S. Patent No. 5,231,580 to Slepianl, polymeric pavrings or films are applied to the tissi.- using a catheter, endoscope or laparoscope.

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Znolycanrolactone, id oport~nOesterS, and one r ma-erlals commonly used for imol~antaz:_Jn or e Dsc reauairements fo h material to ne usea in the process are biocotnoatibilitv and the carpacity to be chemically or physically C) recon~igured under condit ions which can be achieved in vivo. such reconfiguration condit-oI's preferably involve uhnotopolymeriZation, but can involve heating, cooling, mechanical. deformation, as srret~cn_-ng) or chemical reactions such as uolymerization or crossinking.

Tn their conformable state, the coating materials may exhibit a wide variety of forms.

mhecanmer orcombinais thlyereo, mantainedsa Thcabe oresembntins tolyerso, mn inrs, a solutions, suspensions, or dispersions.

Microvarticles *In a preferred embodiment, the microparticle has a diameter which is selected to 4lodge in particular regions of the body. Use of microspheres that lodge within organs. or regions is known in studies of blood flow (Flaim et al, J Pharmacol. Meth. 11:1-39, 1984; Hleymann et al, Prog. Cardiovaso. Dis- 20:55-79, 1977), but not in delivery of active materi.als.' For examnle, a micronartiLcle selected to lodge in a caopillaiI will tynically have A diameter of between 10 to 25, most breferably 15 to 20 microns. Numerous methods are known for preparing microparticles of any Particular size range. in the various auolications of the present invention, the sizes may range from 0.2 micron up to 100 microns. synthetic methods ~~~for gel micropartileS, or for iirnrilsfo a

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kniown, and znclude C in zC em",lI-n in_ szra"ec aros, and 4- separazed' phases. For solid materils or creformed gels, known meth-ods include wet. or cry millingr or grinding, pulverization., classification by air Jet or sieve, and the like.

Microinarticles can be iaz=ricaued fromn da l'r i c-r~ olymers using a-variety of di fferent_ methods known to those skilled in the art.

Exemolary mrethoas include those Set forzh below.

Polylactic aci-d blank microrarticles were fabricated usina the methods: solvent evarorat-ion, as described by E. MathiJowitz, et J.Scannina Microsconv, 4, 329 (90;LR ek 15 et al., Fertil. Steril., 31, 5455 (11979); and S.

Benita, et al., J. ?harm. Sci., 73, 1721 (1984); hot-melt microencapsulation, as described by E.

Mathiowitz, et al., Reactive Polymers, 6, 275 (1987); and spray drying. Polyanhydrides made of bis-carboxyohen0xypro-al and sebacic acid with molar ratio of 20:80 P(CPP-SA) (20;80-j (Mw 20,000) were prepared by hot-melt microencapsulation.

Poly(fumaric-co-sebacic) (20:80) (1Mw 15,000) blank microDarticles were prepared by hot-melt microencapsulat-0fl. Polystyrene microparticles were prepared by solvent evaporation.

FHydrogel microparticles were prepared by dripping a polymer solution from a reservoir though microdroulet forming device into a stirred ionic bath. The specific conditions for alginate, chitosai, alginate/polyethyleninide (PEI) and carboxymethyl cellulose (0MC) are listed in Table a. Solvent Evaoration. In this method the polymer is dissolved in a volatile organic solvent, such as methylene chloride. The drug (either soluble or dispersed as fine particles) is

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Ii- 19 added o t-he solution-, a-nd the mixzure is suspended _n a- a,-L7ueous so1utlof tinat contains a suroace ac-iv= agn uh as po ly(vinlyl alcohol) the '-e-ulig emulsion is stz-rred until m-ost of th_,e oraanic solvent evaporated, leaving solid microoarticles. Several different bolymer concentrations were used: 0.05-0.20 g/ml. The solution is loaded with a drugr and suspended in 200 loo)(im).Atrml of vigorously stirred distilled water containing hours of st41ring, the organic solvent evaporates fromthe zolvmer, and the resulting mic roparticles are washed with water and dried overnight in a lyoohilizer. Micronarticles with-1 different sizes 1s (1-1000 mi crons) and morohologies can be obtai4ned *bv this method. This method is useful for relatively stable polymers like polyesters and pOolystyrene.

However, labile polymers, such as polyanhydrides, may degrade during the fabricationA process due to the presence of water. For these polymers, the following two methods, which are performed in completely anhydrous organic solvents, are more useful.

b. Hot Melt Microencausulatiol. in this method, the polymer is first melted and then mixed with the solid narticles of dye or drug that have been sieved to less than 50 microns. The mixture is suspended in a non-miscible solvent_ (like silicon 310 oil), and, with continuous stirring, heated to SOC above the melting point of the polymer. Once the emulsion is stabilized, it is cooled until the polymer particles solidify. The resulting microbarticles are wgashed by decantation with poetroleum ether to give a refloigpowder.

Microvarticles with sizes between one to 1000 microns are obtained with this method. The -Ij o prp r 4 zrocenure zs used tr trnae lrofaces m~acie of ann de :o-nh owever, thll z to 'o0, ers wl-I mo-icd between 1000-:)0,000c. lva Removal. Thzstec2q s r- ay deiunedor polyan'ydrid I-r. tniS method, t-he drug is dispersed or dissolved in a i 0 solution of thne Selected polymer in a volatile organic solvenzt like met~n.yl ne chnloride. This mixture Is supeded by stirig in an oranc oil (cnas silicon oi)t om emfu slo.U! solvent evapora2Jorn, tlSmethlod cant be used to *make mi,4cropart.;c _es frm olvners with 11,igh melt ing points and different molecular weights- Mice-roparticles; that range between 1-300 micronS can be obtained by this procedure. The external moIolg of spheres producedwt hstcri~ ow 20 is highly dependent on the type of polymer usea.

d SurcivrDrvil Tn this metnod, the polymer is dissolved in methylenle chloride (0.04 viL) A nwn amount of the active drug is Suspended (insoluble drugs) or cc-dissolivea (soluble drugs) in the polymer solution. The solution or the dispersion is then spray-dried.

t -ITypical process parameters for a m~-L 5Dsray drier (Buchi) are as follows: polymer concentration 0.04 g/mL, nettemperature -241C, outlet temperature 13-15 oc, aspirator setting Dump setting 10 maiL/mitlute, spray flo 0 Nl/hr, and nozzle diameter =0.5 mm.

Microartiloles ranuging between 11-10 microns are tpo olerue. This method is Primarily 4 used for preparing microparticles designed to

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improve imaging- of the intestinal tract-, since ror -a -"UI -4 ,re nr I~ n-.e areJ oue raog a -mieu -cuin mie i zh has: suj I at~e or some bloaO::ive a-ent, and then extrua=en Zterouan a microndroule rct i~ng aevi-ce, which in cc ome instances ernnloV5 a IF-_ow nlrrcoan aas to break to f t-h dronlet. slo wI st read (ano-roxiazely 100-170 RPM) ionic hardenilna bath- is D OS~ tiond e~ow thne extrudnr.g aev-ce t.o catch the :ormlt rM.-croroilers The micronarticles are to incubate in the bath For twenty to thirzy minutes -n order to allow sufficient time for aelation to occur. Microoarto' rarticle size is controlled by usinar various size extruders or varying either tenatrogen gas or no±vmcr so.luciof flow rates.

The matrix Is vreferabilv In the form oicronartiLe such as a microspnere (where te biLoloaica_,llv active molecules are da-Isversed .oughout a soL:an.ye~ matrix or microcausule (where the biologically active -molecue ar nasulated in the core ofI a no v.-er-ic shell). The size and cCmnositlon of the noDvmerilc device is selected to result iz n.avorable release kinetics irn tissue. The siz is also I309 selected according to the method of delivery which 3 Mis to be used, typically ijconInto a ti--ssue or administration of a siisnensicn bv aerosol into the nasal and/or pulmonary areas, and where approriate, entrapament at tres where rel ase is desired. The matrix comoSit4 on can be selece -to hot only have favorable deggradation rates, but tbeformed of a material which is bodeivt 22 a- muzosaL Surzace,' or SIc sta not tio den-rade iti 1 ybur- to- vS b difusion Ov.er an eyxtenaec. period.r L-Dcsomels are avai.lale cteCa 2 vZ~ a variety o-F EU-CO-1 erS. Alternativelv-, liPcosomeCs can be Drenared accordina to ttto~known to cnose skilled e art-, fcr example, as described in TJ.S- Paten-- No- 4,522,811 (wihIs incorporated byrt~~flC i -is enti~rty) Fo7r example lizoomfl formulatiofS may be prepared by dissolving aPrOrat 'pd(s) (such-- as szearV _-osphatidly, ethancla-mi-ne, stearoy. phosnfla:JId7,I Choline, aracn--ad.oVI- -ohsphanidyl co2nand cno---tercl) i n an iogicsolvent thats then evaoraced, leavin .g behind a. thin film J-or-.d ii o h Furace of the container- natCU oUino the active compound or its monoohosflflate, diohosphate, and/or triphaszhate derivatives are then introduced nt the container. container is then swirl ed bv hand to free linid material from the sides of the container and to disnerse li"pid agrgregateSr tereby forming the liposomlal suspensionl.

Biol.ogically Active Molecules BioOg4Cally active roeue nhic can be incorporated into the polytmeric carrler--, directly and/or indirectlYr w. wthn miroarties whi ch are iTmob- 1 zdi h carrier, include oroteins, nuc acid-. m-OIe- carbohvcrates, lpdand combinations thereot.

Examples of proteins include cytokifles such as inter ferons and interleukifls, poet ifl, an cooysimlta factorS, growth factors, -0io fid -Factors and fragments th-rof Exa-=plas of nucleic acid molIecul1es include grenes and cDNAs

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I V '1 U a -V a 14 't" c- -c rc rre r. v C. r7C n r ~aa '3 a 1 ~d v d= otnt o Visib1~~~~~~ aa~iou asoue o n~xnnrrztaonLOW r~rn~st a __-soS ax~s -o-cr-ccls ierzr-o rQatA n~asa uo s Iornc Lasr Ott a sore aoe trasorn e crad cu::er 2 s ootros as cn se conds, i o a ver sm Fowre- loaca 0w2aveo aV 30-UV em/v nnorse-rv V~art~ sou os -1 lasXr -'SO t:V' na tm=rr~zatno aniirq (0.01Sz~ to c sn. r ex--c at Cell raa s a.ic*-e7 iun anarcon ro c0r~cnan as Daer -rmnsod&'- Usn: Oliun so-C. S -,ower r0 W/oiitt and zre--ro scanne re (0.0on1 zoccr d n. as =a:natruiary ar arerr-o aser oAr cans atran, n uicsratr actta to te srrae o a rsstemacst; 905eaov 11tm-'I Q 1 a uz a zower Of5~L Zr r photciniiator is applied to the surface of the tissue, allowed to react and bond to tissue, the unbound photoinitiator is removed by dilution or rinsing, and the macromer solution is applied and polymerized. This method is capable of creating uniform nolymeric coating of between 10 and 500 microns in thickness, more typically 50 to 200 microns, which does not evoke thrombosis or localized inflammation.

Apolication of a Pavina or Coatina Local administration of a polymeric material can be performed by loading the composition in a balloon catheter, and then apDlving the composition directly to the inside of a tissue lumen within a zone occluded by the catheter balloons. The tissue surface may be an internal or external surface, and can include the .interior of a tissue lumen or hollow space whether naturally occurring or occurring as a result of surgery, percutaneous techniaues, trauma or disease. The polymeric material can then be reconfigured to form a coating or "paving" layer in intimate and conforming contact with the surface.

The resulting paving layer optionally can have a sealing function. As used herein, the term "'sealing" or "seal" means a coating of sufficiently low porosity that the coating provides a barrier function. The term "paving" refers to coatings in general wherein the coatings are porous or perforated or are of a low porosity "sealing" variety. The coating preferably has a thickness on the tissue surface on the order of 0.001-1.0 mm, however, coatings having a thickness outside that range may be used as well. By appropriate 4 35 selection of the material employed and of the configuration of the paving material, the process 27 can be tailored to satisfy a wide variety of oobiocical or clinical situations.

The monomers, macromers and polymers that can be used for this application are selected from the same group as described above for formation of microparticles- Preferably, the polymeric material has a variable degree of conformability in response to a stimulus. The material is preferably substantially non-conformable in vivo upon comoletion of the coating process. The material, in its conformable form, can be positioned in contact with a tissue or cellular surface to be coated and then stimulated to render it nonconformable. The material is preferably rendered S 15 non-conformable by applying photochemical stimulus, but can optionally be achieved solely by chemical or thermal stimulus. The material is positioned at S' either an internal or external treatment location S" and contacted with the tissue or cellular surface 20 to be paved or sealed, and the material is then converted into a non-conformable state to form a biocomoatible coating on the tissue surface.

The coating can be applied using a catheter, such as a modified PTCA catheter. The material is preferably applied using a single catheter with single or multiple balloons and lumens. The catheter should be of relatively low cross-sectional area. A long thin tubular catheter manipulated using fiuoroscopic guidance is preferred for providing access to the interior of organ or vascular areas. The material can also be applied to the surface to be coated by spraying, extruding or otherwise internally delivering the material in a conformable form via a long flexible tubular-device having single or multiple lumens.

During the step of positioning the o material at the desired location, the location may c sf: _28 be accessed by either invasive surgical techniques or by relatively non-invasive techniques such as laparoscopic procedures or percutaneous transluminal procedures. The process of fixing the shape of the material can be accomplished in several ways, depending on the character of the original material. For example, in its conformable state the material can be formed using the balloon nortion of a balloon catheter after which the conditions are adjusted such that the material is rendered non-conformable. In the preferred embodiment, gels are rendered non-conformable at S" the treatment site by photopolymerization using infrared, visible, UV, or ultrasonic radiation to the material. Optionally, the polymers can be rendered non-conformable by localized heating or by chemical means. Thermal control can be provided, for example, using a fluid flow through or into the balloon, or using a partially perforated balloon such that temperature control fluid passes through the balloon into the lumen. Thermal control can also be provided using electrical resistance i heating via a wire running along the length of the catheter body in contact with resistive heating elements. This type of heating element can make use of DC or radio frequency (RF) current or external RF or microwave radiation. Other methods of achievina temperature control can also be used, including light-induced heating using an internal optical fiber (naked or lensed).

In one embodiment, the step in which the conformable material is contacted with the tissue surface may be considered as a "molding" procedure in which the conformable material is molded into 35 substantially conforming contact with the body tissue before rendering it non-conformable. It is noted that the transition of the material from a 29 conformable to a non-tonformable sta-e may involve a phase change in the material, however, such a phase change is not necessary. For example, in certain embodiments, the terms "conformable" and "non-conformable" are primarily relative descriptions of a material which undergoes a significant change in viscosity and flowabilizy without undergoing an actual phase change.

Alternatively, the transition of the material between its conformable and non-conformable states may be the result of an actual phase change in the material resulting either from the addition or removal of energy from the material The polymeric materials may be aDplied in 15 custom designs, with varying thicknesses, lengths, and three-dimensional geometries spot, stellate, linear, cylindrical, arcuate, spiral) to achieve varying finished geometries. Further, the Sprocess may be used to apply material to the inner 20 surfaces of hollow, cavernous, or tubular biological structures (whether natural or artificially formed) in either single or multilayer configurations. The process may also be used, when appropriate, to occlude a tissue lumen completely.

The paving coating may be applied as a continuous layer either with or without perforations. In the case in which the paving coating is applied without perforations, it is 0 30 referred to as a "seal" to act as a barrier layer .on the surface of the tissue. The coating can also be applied to cellular surfaces, for example, to coat or encapsulate individual or multiple cells.

Administration of MicroDarticles The injectable microparticles can be .jS administered to a patient intravenously, intramuscularly, or subcutaneously or in other j~~n known ways appropriate to the therapeutic effect desired, including as an aerosol or sprav for lungs or by direct lavage through orifices. The narticles can be lyophilized and then formulated into an aqueous suspension in a range of microgram/ml to 100 mg/ml prior to use.

The desired concentration of biologically active molecules in the polymeric carrier will depend on absorption, inactivation, and excretion rates of the drug as well as the delivery rate of the molecules from the carrier. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be l further understood that for any particular subject, specific dosage regimens should be adjusted over Stime according to the individual need and the professional judgment of the person administering "S or supervising the administration of the compositions.

20 The microparticles can be administered once, or may be divided into a number of smaller doses to be administered at varying intervals of time, depending on the release rate of the particle, and the desired dosage.

Solutions or suspensions used for intravenous, intramuscular, or topical application, or other delivery route can include any of the following components, as required: a sterile diluent such as water for injection, saline 30 solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as Sascorbic acid or sodium bisulfite; chelating agents 35 such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium 31 chloride or dextrose. The parenal preparation can be enclosed in amocules, disposable syringes or multiple dose vials made of glass or plastic. If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline

(PBS).

Catheters can be made of any known material, including metals, such as steel, and thermoplastic polymers. Occluding balloons can be made from compliant materials such as latex or silicone, or non-compliant materials such as polyethylene terephthalate (PET). The expansible 1 member is preferably made from non-compliant macerials such as PET, (PVC), polyethylene or 15 nylon. If used, the balloon catheter portion of a dilatation may optionally be coated with materials such as silicones, polytetrafluoroethylene (PTFE), S. hydrophilic materials like hydrated hydrogels and other lubricous materials to aid in separation of 20 the polymer coating.

In addition to blood vessels, the process may be utilized for other applications such as coating the interior of veins, ureters, urethras, bronchi, biliary and pancreatic duct systems, the 25 gut, nasolacrimal ducts, sinus cavities, the eye, and eustachian, spermatic and fallopian tubes.

Likewise the process can be used to provide a 4 paving layer in the context of transhepatic portosystemic shunting, dialysis grafts, arterio- 30 venous fistulae, and aortic and other arterial aneurysms- The paving and sealing material of the process can also be used in other direct clinical applications even at the coronary level. These .include but are not limited to the treatment of 35 abrupt vessel reclosure.post PCTA, the "patching" 6f significant vessel dissection, the sealing of Svessel wall "flaps" either secondary to catheter i 32 Snjury cr spontaneously occurring, or the sealing of aneurysmal coronary dila-ions associated with various arteritidies. Further, the method provides intraoperative uses such as sealing of vessel anastomoses during coronary artery bypass grafting and the ability to provide a "bandaged" smooth polymer surface.

Treatment of Specific Disorders Vascularization 1 10 A common problem in aging is atherosclerosis affecting the arteries of the lower limbs. This can cause claudication, or sharp pain S. when walking. This disease can be treated by inducing the creation of additional collateral 1; 15 circulation in the affected region (in this case, the leg) by introducing a growth factor such as VEGF (vascular endothelial growth factor), or a DNA which can express it. The growth factor or DNA can be delivered either by creating a thin coating o20 containing the factor inside an artery leading to the region, or by injecting microparticles containing the factor into the artery feeding the affected limb or region. In the latter case, the microparticles are preferably at least 15 microns in diameter, preferably 20 microns or more, to cause the delivery particles to localize predominantly in the region. (It should be noted that some microparticles will probably exit the treated region, and lodge in the lungs or elsewhere; this effect must be accounted for in treatment nlanningc.) Another application includes revascularization in cardiac tissue including the V 'myocardium, and revascularization after stroke or ischemia. _i 1 i 'p 33 Reaeneration or Repair of Tissues Yet another application is in regeneration or repair of particular organs.

Delivery of various bone morphogenetic proteins can be useful for controlled remodeling of bone, or de novo bone or cartilage formation, in which it is critical that the developmental or morphogenetic effects be strictly confined to the target site, and not exhibited throughout the organism. Local deposition of biologically active molecules can be useful in repairing bone in areas such as the nasal passages and sinuses, where precise control of positioning is required.

Examples of other tissues which can be 15 treated in this manner include the stomach and Yi 'intestines, where growth factors help accelerate repair of ulceration, repair of external ulceration of skin, and general wound repair.

Other organ systems susceptible to 20 treatment include any organ system in which material flows through the organ from a source, so that a factor can be administered, either in a coating or as particles, for instillation into the :p organ to be treated by flow. Exemplary organs include lymph nodes, the bile duct, the urinary tract, the lungs, the space occupied by the cerebro-soinal fluid, and the like.

SThe present invention will be further understood by reference to the following nonlimiting examples. Example 1: Gene delivery from a gel in vitro.

l Previous work on gene transfer into arteries has involved administration of DNA in a liquid vehicle, including pressing the liquid into artery walls with a balloon catheter- For more efficient local delivery, a thin, locally-deposited l 34 gel-,whcb DNA can di ffEuse into the target tissue, was ult-±ilzed.

Tans~e Procedure Positively-charged linosomes Transfection-reagent, Boehringrer Mannheim) containing the cationic lipid analogue 1,2dioleovl1oxy-3 -(trimethvlamrnmonium) oroDane (DOTAP) were used fOR transfections. Plasmid DNA was purified by centrifugation through a cesium chloride gradient- Five gg of DNA was mixed with gg of liposomes in 200.gL of Hanks Balanced Salt Solution (HBSS, Gibco).

Expression VectorYs and Analysis of Recombinant Gene Expression The luciterase expression vector employed was DoRSVLUC (gift of Dr. A. Brasier, Galveston, TX, to Dr. Jeffrey M. Isner, St. Elizabeth's Hospital, Boston, MA, who provided the vector). This contains a 5' deletion of firefly luciferase cDNA, with transcription under the control of the Rous sarcoma virus long terminal repeat promoter. Use

A

of this reporter gene allows f or cuantificaticn of gene expression in cell lysates. Cells were washed 3 times with calcium-free HBSS and extracts prepared using a cell lysis reagent (Promega, Madison WI) containing 11A Triton-Xl001. Half of Athe extract was taken for analysis of total protein content, performed using the Biorad Microassay procedure. Bovine serum albumen (Img/ml) was added to the other half as a carrier protein and luciferase activity was measured. For this, a g1 aliquot was mixed at room temperature with 100= p1 of lucif erase assay reagent (Promega) containing beetle luciferin. Emission of Light, integrated 'Alternative nomenclature: l-(2,3- Dioleoyloxy)propyl]-N,N,N, trimethyl-ammonium methyl sulf ate.

over 10 seconds, was measured us 4 n'g a 711miorete fTure- DeSia_:ns, Moe 20Ge, Surnnwrale CA-) Hesults=, read as liurht units, were within the ange *of the Qetecticrn systemr as evaluated using serial dilutions of anonmount o Iucl~erase (Sia, Product No. Lh-9009)_ Backaround acti-:vityv, measured using phosohate-buffered saline or lysat~es ofr ncptransfecned cel -is, was coflSistentt v zer~o.

niasmid DRSVL?-C, obtained from the laboratory of Dr. Jeff-rey isner of St. Elizabeth's haoital in Boston, which encodes the enzyme luifeaseunder the control of a S1140 promoter, was dissolved In a gelling preo-vrner. The 15~ Drenoolvmer had a core of polyethylene glycol, MWV8000, with about S lactate residues at each end, capoed by an acrvlate group at each end, .synthesized according to the teachings of PCT US 93/17669 by Hubbell et -hereby incorporated by reference. The polymer concentration was 10W. The a-Dlication method was otherwise essentiallyZ identical to that described in Hill-West et a!, Proc. Nat. Acad. Sci. USA 91:5967-5971f 1994.

Tissue surfaces were stained by application of- 1 mM Bosin Y, and washed. The Dolymer solution contaiing the DNA, and also 100 m.M ethanolamine and 0.15%- n-vinyl1 oyrrolidone, and was polymerized essentially as in Hill-West. The amount of ulasmid in the delivery vehicle was between 0.02 and 2 microarams. Lioomes as described abo-ve were ootionallv included. The lumainal surface of rabbit ar-erial strips, which were maintained in tissue culture essentially as described by Takeshita et al-, j. Cin. invest-, 93:652-661, (1994), were stained with a Dhotoinitiatingr dye, Bosin Y, according to PCT US 93/01776 by Hubbell et and washed in medium. Preoolymer solution C23-. wt 36 Prezolvmer solu:ti;on in salin~e) DNA,474 or wicncut- adcoeci Ir-7,somes (at a rat_ or carzs bv weiahz o- li oscrnes Der oart of DINA), was aur-,ed as a soot to stained art-rial StrIoDs. The S solution was chotonolymerized wi reen lig-ht to foarm a hyvdrogel. As controls, artery scrizs were treated wt DINIM/oreoolvmer Solict'o, wrh-ich was not alled by cnttltia~f.After 7 days in culture, luciferase ex-oression was measured 4nl '0 Turner Lig'ht Units per gram of tissue by standard methods.

At the occimal level of DNA application nicrograms/dose) controls had 3.3 ~3.3 TLU -Jg; tissue treated gels cotrlgDNA not encapsulated within liposomes had lU.7j3 G TLU/g; *and tissue treated with gels containing D-NAP encansulated withi n livosomes had 20.8 1-7 TLU/g.

The results demonstrate that gene *transfer and expression can be ac-omolished by delivery with'a gel, with or without linosomes, and ~that the efficiency of delivery is significantly 'Higher than when the DNA is merely applied to the tissue sur-face. However, the results also inaricace that the efficacy of transfer can be greatly increased by inooration ofL the DNA into liposomes prior to immobilization- Exammle 2: In vivo Luciferase Gene Delivery via Photopolymerizable Hydrogel.

in vrivo gene de livery was demnonstrated using the rat carotid artery model- interracially polymerized gels, prepared as described in exatnole 1, were formed in the right carotid artery of rats.

The left side was untreated and servied as contro' Gels contained 25 micrograms DNA per m! or prepolymer Solution containing 101i w/v prenolymer encapsulated in 100 microgramis liposomes/mi of prepolymer, and otherwise were deposited as.

described by 'Hill-West et al. Arteries were 37 n-A W C-re= z CC7 Z= n h ick. After 3 days, razs were =acrzce-- and ztissue exam-ned zcr lucf as No gene exoressi-on was evident i-r control (utae)arreri'es, while arae ar eS had 8 62 T--U/g.

As additional ccnnrois, D-NA/macrorner soluti-ns were arnlied either to tn-e acaventlal (out-side' surf ace of arte ries, a-d flus~lld with sa~~-or were aDD-Hea to L-1e interior surface, and non 4lluminae.. Th-e former treatment gave 5_21 Tt,.U/a, anld the latter gave 3.9. 3 .7 Variations and modifications of the claimed invention will be obvious to chose skilled in the art 'rom the foregoing de~a 4 1 d descriotion of the invention. it is intended that all of these 20 variations and modif icat ions be included within the J scope of the aunended claims.

4-S

Claims (40)

1. A method of delivery of biologically active molecules to a targeted site in an animal where treatment is needed, the method including: selecting microparticles having a diameter between 1 and 100 microns, said microparticles comprising a synthetic biodegradable polymer and biologically active molecules; and administering the microparticles to the animal so that the microparticles selectively lodge at the targeted site within the animal where release is desired. for a sufficient amount of time to permit controlled release of a therapeutically effective amount of the biologically active molecules, wherein the biologically active molecules are selected from the group consisting of growth factors, cytokines, angiogenesis factors, interferons. Sinterleukns. colony-stimulating factors, immunosuppressant molecules, clot- dissolving agents, peptide fragments thereof, and nucleic acid constructs 15 capable of synthesizing these compounds; and wherein the synthetic polymer is biodegradable, polymerizable macromer comprising at least one water soluble region, at least one degradable region which is hydrolysable underin vivo conditions, and at S" least one polymerizable group on said macromer having the ability to form 20 additional covalent bonds resulting in macromer interlinking, wherein the polymerizable groups are separated from each other by at least one degradable region.

2. The method of claim 1 wherein the microparticles are administered into the circulation and release the biologically active molecule after the microparticle selectively lodges.

3. The method of claim I wherein the targeted site is an area where vascularization or revascularization is needed, and wherein the biologically 7 active molecule is a growth factor selected from the group consisting of vascular endothelial growth factor (VEGF), endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), bone morphogenic protein (BMP), platelet derived growth factor (PDGF). active peptides thereof, and nucleic acid constructs capable of expressing the growth factors. i 4. The method of claim 1 wherein the microparticles are administered in a pharmaceutically acceptable carrier by a route of administration selected from the group consisting of intravenous, intraarterial, percutaneous, intramuscular, subcutaneous, direct lavage, aerosol, and via surgical incision. I 39 The method of claim 4 wherein the route of administration is intravascular.

6. The method of claim I wherein the targeted site is an area of a blood vessel where abnormal accumulation of smooth muscle cells has occurred.

7. The method of claim 1 wherein the targeted site is an area of a blood vessel where restenosis has occurred after angioplasty-

8. The method of claim 1 wherein the targeted site is an area where atherosclerosis has occurred.

9. The method of claim 8 wherein the atherosclerosis is present in the lower limbs causing claudication. The method of claim 1 wherein the targeted site is an area where ischaemia has occurred. S11. The method of claim 1 wherein the microparticles are administered into a region or organ so as to lodge or adhere at a locus within the organ. 15 12. The method of claim 1 wherein the microparticles are polymeric microspheres in the form of a hydrogel.

13. The method of claim 12, in which the hydrogel is composed predominantly of a polyalkylene oxide. S14_ The method of claim 12, in which the hydrogel is made by the polymerization of a water-soluble macromer, said macromer including a water-soluble backbone, one or more degradable linking groups, and one or more reactive groups covalently linked to said degradable linking groups

15. A method of increasing the vascularization of a tissue, the method including: selecting at least one biologically active molecule which is a deliverable growth factor equivalent, said equivalent comprising at least one of a protein and a nucleic acid encoding a protein, characterized in that said protein stimulates the growth of blood vessels; mixing said equivalent with a biodegradable, biocompatible polymeric material capable of being polymerized to form a delivery vehicle for said deliverable growth factor equivalent; conveying said mixture to a tissue in need of increased vasculrizatian: and polymerizing said polymeric material; whereby a depot is formed which locally releases deliverable growth factor equivalent to said tissue. 77

16. The method of claim 15 in which said polymer is polymerized after it is conveyed to said tissue.

17. The method of claim 15 in which said polymer is polymerized before it is conveyed to said tissue.

18. The method of claim 17 in which said polymeric material is in the form of microspheres.

19. The method of claim 15 in which the growth factor equivalent is entrapped in a material limiting its diffusion rate before being mixed into said polymerizable material.

20. The method of claim 19 in which the diffusion-limiting material is selected from liposomes and polymeric microspheres.

21. The method of claim 15 wherein the deliverable growth factor Sequivalent is selected from the group consisting of growth factors, cytokines, interferons, interleukins, colony-stimulating factors. angiogenesis factors, S 15 inmunosuppressant molecules, peptide fragments thereof, and nucleic acid constructs capable of expressing these equivalents.

22. The method of claim 15 wherein the deliverable growth factor S. equivalent is selected from the group consisting of vascular endothelial Igrowth factor (VEGF), endothelial cell growth factor (ECGF), basic fibroblast 20 growth factor (bFGF], bone morphogenic protein (BMP), platelet derived growth factor (PDGF), peptides thereof, and nucleic acid constructs capable i S;of expressing the factors.

23. A method of increasing the effective vascularization of a tissue, the i method including: selecting at least one biologically active molecule which is a clot- i dissolving material, said material comprising at least one of a protein and a nucleic acid encoding a protein, characterized in that said protein accelerates the removal of clots or prevents clot formation; mixing said biologically active molecule with a biodegradable, biocompatible polymeric material capable of being polymerized to form a delivery vehicle for said deliverable biologically active molecule; S(c) conveying said mixture to a tissue in need of increased vascular permeability; and S(d) polymerizing said polymeric material; whereby a depot is formed which locally releases said biologically-- active molecule to said tissue. 41

24. The method of claim I or claim 23 wherein the biologically active molecule is at least one of tissue plasminogen activator, streptokinase, urokinase, or heparin. The use of the method of claim 1 for inhibiting the expression of a gene at a site in a vessel.

26. The method of claim 1, wherein the targeted region is the lung.

27. The method of claim 1, claim 15 or claim 23, wherein said degradable region contains at least one linkage from the group consisting of hydroxy acids, amino acids, peptides, anhydrides, phosphoesters, orthoesters, and carbonates.

28. The method of claim 1 or 15, wherein the targeted organ, tissue or region is selected from the group consisting of the urinary tract (including .i ureters and urethras), bronchi, biliary and pancreatic ducts, the gastrointestinal tract (including the gut or intestines, and the stomach), 15 nasolachrimal ducts, sinus cavities, the eye, eustacian tubes, spermatic tubes, fallopian tubes, the lung, the heart, bone, cartilage, lymph nodes, the skin, and organs bathed by the cerebrospinal fluid.

29. Use of microparticles having a diameter between 1 and 100 microns, said microparticles comprising a synthetic biodegradable polymer and 20 biologically active molecules for the preparation of a medicament for delivery of biologically active molecules to a targeted site in an animal where treatment is needed; .where upon administration of the microparticles to the animal, the microparticles selectively lodge at the targeted site within the animal where l 25 release is desired, for a sufficient amount of time to permit controlled release of a therapeutically effective amount of the biologically active molecules, wherein the biologically active molecules are selected from the group "i consisting of growth factors, cytokines, angiogenesis factors, interferons, interleukins, colony-stimulating factors, immunosuppressant molecules, clot- dissolving agents, peptide fragments thereof, and nucleic acid constructs capable of synthesizing these compounds; and wherein the synthetic polymer is biodegradable, polymerizable macromer comprising at least one water soluble region, at least one degradable region which is hydrolysable underin vivo conditions, and at 35 least one polymerizable group on said macromer having the ability to form 1 S':I additional covalent bonds resulting in macromer interlinking, wherein the I I ivy. I r r r r r polymerizable groups are separated from each other by at least one degradable region. The use of claim 29 wherein the microparticles are administered into the circulation and release the biologically active molecule after the microparticle selectively lodges.

31. The use of claim 29 wherein the targeted site is an area where vascularization or revascularization is needed, and wherein the biologically active molecule is a growth factor selected from the group consisting of vascular endothelial growth factor (VEGF), endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF, bone morphogenic protein (BMP), platelet derived growth factor (PDGF), active peptides thereof, and nucleic acid constructs capable of expressing the growth factors.

32. The use of claim 29 wherein the inicroparticles are administered in a pharmaceutically acceptable carrier by a route of administration selected 15 from the group consisting of intravenous, iniiaarterial, percutaneous, intramuscular, subcutaneous, direct lavage, aerosol, and via surgical incision.

33. The use of claim 32 wherein the route of administration is intravascular.

34. The use of claim 29 wherein the targeted site is an area of a blood vessel where abnormal accumulation of smooth muscle cells has occurred. The use of claim 29 wherein the targeted site is an area of a blood vessel where restenosis has occurred after angioplasty.

36. The use of claim 29 wherein the targeted site is an area where atherosclerosis has occurred.

37. The use of claim 29 wherein the atherosclerosis is present in the lower limbs causing claudication.

38. The use of claim 29 wherein the targeted site is an area where ischaemia has occurred.

39. The use of claim 29 wherein the microparticles are administered into a region or organ so as to lodge or adhere at a locus within the organ. The use of claim 29 wherein the microparticles are polymeric microspheres in the form of a hydrogel.

41. The use of claim 40, in which the hydrogel is composed predominantly of a polyalkylene oxide. N i 43

42. The use of claim 40, in which the hydrogel is made by the polymerization of a water-soluble macromer, said macromer including a water-soluble backbone, one or more degradable linking groups, and one or Smore reactive groups covalently linked to said degradable linking groups.

43. Use of at least one biologically active molecule which is a deliverable i growth factor equivalent, said equivalent comprising at least one of a protein and a nucleic acid encoding a protein, characterized in that said protein stimulates the growth of blood vessels in the preparation of a medicament for increasing the vascularization of a tissue; wherein said equivalent is mixed with a biodegradable, biocompatible polymeric material capable of being polymerized to form a delivery vehicle i for said deliverable growth factor equivalent: said mixture is conveyed to a tissue in need of increased vascularization: and 15 said polymeric material is polymerized; whereby a depot is formed which locally releases the deliverable growth factor equivalent to said tissue. *44. The use of claim 43 in which said polymer is polymerized after it is 2 conveyed to said tissue.

45. The use of claim 43 in which said polymer is polymerized before it is conveyed to said tissue.

46. The use of claim 45 in which said polymeric material is in the form of microspheres. .47. The use of claim 43 in which the growth factor equivalent is entrapped in a material limiting its diffusion rate before being mixed into said polymerizable material.

48. The use of claim 47 in which the diffusion-limiting material is selected from liposomes and polymeric microspheres.

49. The use of claim 43 wherein the deliverable growth factor equivalent is selected from the group consisting of growth factors, cytokines, interferons, interleukins, colony-stimulating factors, angiogenesis factors, immunosuppressant molecules, peptide fragments thereof, and nucleic acid tj constructs-capable of expressing these equivalents. i 44 The use of claim 43 wherein the deliverable growth factor equivalent is selected from the group consisting of vascular endothelial growth factor (VEGF), endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), bone morphogenic protein (BMP), platelet derived growth factor (PDGF), peptides thereof, and nucleic acid constructs capable of expressing the factors.

51. Use of at least one biologically active molecule which is a clot- dissolving material, said material comprising at least one of a protein and a nucleic acid encoding a protein, characterized in that said protein accelerates the removal of clots or prevents clot formation for the preparation of a medicament for increasing the effective vascularization of a tissue, wherein said biologically active molecule is mixed with a biodegradable. ,biocompatible polymeric material capable of being polymerized to form a delivery vehicle for said deliverable biologically active molecule; 15 said mixture is conveyed to a tissue in need of increased vascular permeability; and said polymeric material is polymerized; whereby a depot is formed which locally releases said biologically active molecule to said tissue. 20 52. The use of claim 29 or claim 43 wherein the biologically active molecule is at least one of tissue plasminogen activator, streptokinase, uroldnase, or heparin.

53. The use of claim 29, wherein the targeted region is the lung.

54. The use of claim 29, claim 43 or claim 51, wherein said degradable region contains at least one linkage from the group consisting of hydroxy acids, amino acids, peptides, anhydrides, phosphoesters, orthoesters, and carbonates. A a- The use of claim 29 or 43, wherein the targeted organ, tissue or region is selected from the group consisting of the urinary tract (including ureters and urethras), bronchi, biliary and pancreatic ducts, the gastrointestinal tract (including the gut or intestines, and the stomach], nasolachrimal ducts, sinus cavities, the eye, eustacian tubes, spermatic tubes, fallopian tubes, the lung, the heart, bone, cartilage, lymph nodes, the skin, and organs bathed by the cerebrospinal fluid. Dated this 1st day of April 1999 FOCAL, INC. Patent Attorneys for the Applicant: S|F B RICE CO f ^y