AU2004200646A1 - Anti-angiogenic compositions and methods of use - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANTS: Angiogenesis Technologies, Inc. AND The University of British Columbia ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Nicholson Street Melbourne, 3000.

INVENTION TITLE: "Anti-angiogenic compositions and methods of use" The following statement is a full description of this invention, including the best method of performing it known to us:

-IA-

Description ANTI-ANGIOGENIC COMPOSITIONS AND METHODS OF USE This is a divisional of Australian patent application No. 71746/00, the entire contents of which are incorporated herein by reference.

Technical Field The present invention relates generally to compositions and methods for treating cancer and other angiogenic-dependent diseases, and more specifically, to compositions comprising anti-angiogenic factors and polymeric carriers, stents which have been coated with such compositions, as well as methods for utilizing these stents and compositions.

Background Of The Invention Cancer is the second leading cause of death in the United States, and accounts for over one fifth of the total mortality. Briefly, cancer is characterized by the uncontrolled division of a population of cells which, most typically, leads to the formation of one or more tumors. Although cancer is generally more readily diagnosed than in the past, many forms, even if detected early, are still incurable.

A variety of methods are presently utilized to treat cancer, including for example various surgical procedures. If treated with surgery alone, however, many patients (particularly those with certain types of cancer, such as breast, brain, colon and hepatic cancer) will experience recurrence of the cancer. In addition to surgery, many cancers are also treated with a combination of therapies involving cyvtotoxic chemotherapeutic drugs vincristine, vinblastine, cisplatin, methotrexate, 5-FU, etc.) and/or radiation therapy. One difficulty with this approach, however, is that radiotherapeutic and chemotherapeutic agents are toxic to normal tissues, and often create lifethreatening side effects. In addition, these approaches often have extremely high failure/remission rates.

In addition to surgical, chemo and radiation therapies, others have attempted to utilize an individual's own immune system in order to eliminate cancerous cells. For example, some have suggested the use of bacterial or viral components as adjuvants in order to stimulate the immune system to destroy tumor cells. (See generally "Principles of Cancer Biotherapy," Oldham Raven Press. New York. 1987.) Such agents have generally been useful as adjuvants and as nonspecific stimulants in animal tumor models, but have not as of vet proved to be generally effective in humans.

Lymphokines have also been utilized in the treatment of cancer.

Briefly, lymphokines are secreted by a variety of cells, and generally have an effect on specific cells in the generation of an immune response. Examples of lymphokines include Interleukins and as well as colony stimulating factors such as G-CSF, GM-CSF, and M-CSF. Recently, one group has utilized IL-2 to stimulate peripheral blood cells in order to expand and produce large quantities of cells which are cytotoxic to tumor cells (Rosenberg et al., N. Engl. J. Med. 313:1485-1492, 1985).

Others have suggested the use of antibodies in the treatment of cancer. Briefly, antibodies may be developed which recognize certain cell surface antigens that are either unique, or more prevalent on cancer cells compared to normal cells. These antibodies, or "magic bullets," may be utilized either alone or conjugated with a toxin in order to specifically target and kill tumor cells (Dillman, "Antibody Therapy," Principles of Cancer Biotherapy, Oldham Raven Press, Ltd., New York, 1987). However, one difficulty is that most monoclonal antibodies are of murine origin, and thus hypersensitivity against the murine antibody may limit its efficacy, particularly after repeated therapies. Common side effects include fever, sweats and chills, skin rashes, arthritis, and nerve palsies.

One additional difficulty of present methods is that local recurrence and local disease control remains a major challenge in the treatment of malignancy. In particular, a total of 630,000 patients annually (in the U.S.) have localized disease (no evidence of distant metastatic spread) at the time of presentation; this represents 64% of all those patients diagnosed with malignancy (this does not include nonmelanoma skin cancer or carcinoma in situ). For the vast majority of these patients, surgical resection of the disease represents the greatest chance for a cure and indeed 428,000 will be cured after the initial treatment 428,000. Unfortunately, 202,000 (or 32% of all patients with localized disease) will relapse after the initial treatment. Of those who relapse, th'e number who will relapse due to local recurrence of the disease amounts to 133,000 patients annually (or 21% of all those with localized disease). The number who will relapse due to distant metastases of the disease is 68,000 patients annually (11% of all those with localized disease). Another 102,139 patients annually will die as a direct result of an inability to control the local growth of the disease.

Nowhere is this problem more evident than in breast cancer, which affects 186.000 women annually in the U.S. and whose mortality rate has remained unchanged for 50 years. Surgical resection of the disease through radical mastectomy, modified radical mastectomy, or lumpectomy remains the mainstay of treatment for this condition. Unfortunately, 39% of those treated with lumpectomy alone will develop a local recurrence of the disease, and surprisingly, so will 25% of those in which the resection margin is found to be clear of tumor histologically. As many as 90% of these local recurrences will occur within 2 cm of the previous excision site.

Similarly, in 1991, over 113,000 deaths and 238,600 new cases of liver metastasis were reported in North America alone. The mean survival time for patients with liver metastases is only 6.6 months once liver lesions have developed. Non-surgical treatment for hepatic metastases include systemic chemotherapy, radiation, chemoembolization, hepatic arterial chemotherapy, and intraarterial radiation. However, despite evidence that such treatments can transiently decrease the size of the hepatic lesions systemic chemotherapy and hepatic arterial chemotherapy initially reduces lesions in 15-20%, and of patients, respectively), the lesions invariably reoccur. Surgical resection of liver metastases represents the only possibility for a cure, but such a procedure is possible in only 5% of patients with metastases, and in only 15-20% of patients with primary hepatic cancer.

One method that has been attempted for the treatment of tumors with limited success is therapeutic embolization. Briefly, blood vessels which nourish a tumor are deliberately blocked by injection of an embolic material into the vessels. A variety of materials have been attempted in this regard, including autologous substances such as fat, blood clot, and chopped muscle fragments, as well as artificial materials such as wool, cotton, steel balls, plastic or glass beads, tantalum powder, silicone compounds, radioactive particles, sterile absorbable gelatin sponge (Sterispon, Gelfoam), oxidized cellulose (Oxycel), steel coils, alcohol, lyophilized human dura mater (Lyodura), microfibrillar collagen (Avitene), collagen fibrils (Tachotop), polyvinyl alcohol sponge (PVA; Ivalon), Barium-impregnated silicon spheres (Biss) and detachable balloons. The size of liver metastases may be temporarily decreased utilizing such methods, but tumors typically respond by causing the growth of new blood vessels into the tumor.

A related problem to tumor formation is the development of cancerous blockages which inhibit the flow of material through body passageways, such as the bile ducts, trachea, esophagus, vasculature and urethra.

One device, the stent, has been developed in order to hold open passageways which have been blocked by tumors or other substances. Representative examples of common stents include the Wallstent, Strecker stent, Gianturco stent and the Palmaz stent. The major problem with stents, however, is that they do not prevent the ingrowth of tumor or inflammatory material through the interstices of the stent. If this material reaches the inside of a stent and compromises the stent lumen, it may result in blockage of the body passageway into which it has been inserted. In addition, presence of a stent in the body may induce reactive or inflammatory tissue blood vessels, fibroblasts, white blood cells) to enter the stent lumen, resulting in partial or complete closure of the stent.

The present invention provides compositions and methods suitable for treating cancers and other angiogenesis-dependent diseases which address the problems associated with the procedures discussed above, and further provides other related advantages.

Summary of the Invention Briefly stated, the present invention provides anti-angiogenic compositions, as well as methods and devices which utilize such compositions for the treatment of cancer and other angiogenesis-dependent diseases. Within one aspect of the present invention, compositions are provided (hereinafter referred to as "anti-angiogenic compositions") comprising an anti-angiogenic factor and a polymeric carrier. A wide variety of molecules may be utilized within the scope of the present invention as anti-angiogenic factors, including for example Anti-Invasive Factor, retinoic acids and their derivatives, taxol, taxol analogues and taxol derivatives, and members of the group consisting of Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1 and Plasminogen Activator Inhibitor-2. Similarly, a wide variety of polymeric carriers may be utilized, representative examples of which include poly(ethylene-vinyl acetate) crosslinked with 40% vinyl acetate, poly (lactic-co-glycolic acid), polycaprolactone polylactic acid, copolymers of poly(ethylene-vinyl acetate) crosslinked with 40% vinyl acetate and polylactic acid, and copolymers of polylactic acid and polycaprolactnne. Within one embodiment of the invention, the composition has an average size of 15 to 200 um.

Within another aspect of the present invention methods for embolizing a blood vessel are provided, comprising the step of delivering into the vessel a therapeutically effective amount of an anti-angiogenic composition (as described above), such that the blood vessel is effectively occluded. Within one embodiment, the anti-angiogenic composition is delivered to a blood vessel which nourishes a tumor.

Within yet another aspect of the present invention, stents are provided comprising a generally tubular structure, the surface being coated with one or more anti-angiogenic compositions. Within other aspects of the present invention, methods are provided for expanding the lumen of a body passageway, comprising inserting a stent into the passageway, the stent having a generally tubular structure, the surface of the structure being coated with an antiangiogenic composition as described above, such that the passageway is expanded. Within various embodiments of the invention, methods are provided for eliminating biliary obstructions. comprising inserting a biliary stent into a biliary passageway: for eliminating urethral obstructions. comprising inserting a urethral stent into a urethra; for eliminating esophageal obstructions, comprising inserting an esophageal stent into an esophagus; and for eliminating tracheal/bronchial obstructions, comprising inserting a tracheal/bronchial stent into the trachea or bronchi. In each of .iese embodiments, the stent has a generally tubular structure, the surface of which is coated with an antiangiogenic composition as described above.

Within another aspect of the present invention, methods are provided for treating tumor excision sites, comprising administering an antiangiogenic composition as described above to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within yet another aspect of the invention, methods for treating corneal neovascularization are provided, comprising the step of administering a therapeutically effective amount of an anti-angiogenic composition as described above to the cornea, such that the formation of blood vessels is inhibited. Within one embodiment, the anti-angiogenic composition further comprises a topical corticosteroid.

Within another aspect of the present invention, methods are provided for inhibiting angiogenesis in patients with non-tumorigenic, angiogenesis-dependent diseases, comprising administering a therapeutically effective amount of a composition comprising taxol to a patient with a nontumorigenic angiogenesis-dependent disease, such that the formation of new blood vessels is inhibited. Within other aspects, methods are provided for embolizing blood vessels in non-tumorigenic. angiogenesis-dependent diseases, comprising delivering to the vessel a therapeutically effective amount of a composition comprising taxol, such that the blood vessel is effectively occluded.

Within yet other aspects of the present invention, methods are provided for expanding the lumen of a body passageway, comprising inserting a stent into the passageway, the stent having a generally tubular structure, the surface of the structure being coated with a composition comprising taxol, such that the passageway is expanded. Within various embodiments of the invention, methods are provided for eliminating biliary obstructions, comprising inserting a biliary stent into a biliary passageway: for eliminating urethral obstructions, comprising inserting a urethral stent into a urethra; for eliminating esophageal obstructions, comprising inserting an esophageal stent into an esophagus; and for eliminating tracheal/bronchial obstructions, comprising inserting a tracheal/bronchial stent into the trachea or bronchi. Within each of these embodiments the stent has a generally tubular structure, the surface of the structure being coated with a composition comprising taxol.

Within another aspect of the present invention, methods are provided for treating a tumor excision site, comprising administering a composition comprising taxol to the resection margin of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within other aspects, methods are provided for treating corneal neovascularization, comprising administering a therapeutically effective amount of a composition comprising taxol to the cornea, such that the formation of new. vessels is inhibited.

Within yet another aspect of the invention, pharmaceutical products are provided, comprising taxol, in a container, and a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the taxol, for human or veterinary administration to treat non-tumorigenic angiogenesis-dependent diseases.

Briefly, Federal Law requires that the use of a pharmaceutical agent in the therapy of humans he approved by an agency of the Federal governrent.

Responsibility for enforcement (in the United States) is with the Food and Drug Administration. which issues appropriate regulations for securing such approval, detailed in 21 U.S.C. 301-392. Regulation for biological materials comprising products made from the tissues of animals, is also provided under 42 U.S.C.

262. Similar approval is required by most countries, although, regulations may vary from country to country.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions, and are incorporated by reference in their entirety.

Brief Description of the Drawings Figure 1A is a photograph which shows a shell-less egg culture on day 6. Figure 1B is a digitized computer-displayed image taken with a stereomicroscope of living, unstained capillaries (1040x). Figure IC is a corrosion casting which shows CAM microvasculature that are fed by larger, underlying vessels (arrows; 1300x). Figure ID depicts a 0.5 mm thick plastic section cut transversely through the CAM. and recorded at the light microscope level. This photograph shows the composition of the CAM, including an outer double-layered ectoderm a mesoderm containing capillaries (arrows) and scattered adventia cells, and a single layered endoderm (En) (400x).

Figure IE is a photograph at the electron microscope level (3500x) wherein typical capillary structure is presented showing thin-walled endothelial cells.

(arrowheads) and an associated pericyte.

Figures 2A, 2B, 2C and 2D are a series of digitized images of four different, unstained CAMs taken after a 48 hour exposure to taxol.

Figures 3A, 3B and 3C are a series of photographs of 0.5 mm thick plastic sections transversely cut through a taxol-treated CAM at three different locations within the avascular zone.

Figures 4A, 4B and 4C are series of electron micrographs which were taken from locations similar to that of Figures 3A, 3B and 3C (respectively) above.

Figure 5 is a bar graph which depicts the size distribution of microspheres by number ELVAX with 10 mg sodium suramin into

PVA).

Figure 6 is a bar graph which depicts the size distribution of microspheres by weight ELVAX with 10 mg sodium suramin into.

PVA).

Figure 7 is a line graph which depicts the weight of encapsulation of Sodium Suramin in 1 mi of 5% ELVAX.

Figure 8 is a line graph which depicts the percent of encapsulation of Sodium Suramin in ELVAX.

Figure 9 is a bar graph which depicts the size distribution of ELVAX microspheres containing 10 mg sodium suramin made in 5% PVA containing 10% NaCl.

Figure 10 is a bar graph which depicts the size distribution by weight of 5% PLL microspheres containing 10 mg sodium suramin made in PVA containing 10% NaCI.

Figure 11 is a bar graph which depicts the size distribution by number of 5% PLL microspheres containing 10 mg sodium suramin made in PVA containing 10% NaC1.

Figure 12 is a line graph which depicts the time course of sodium suramin release.

Figure 13 is an illustration of a representative embodiment of hepatic tumor embolization.

Figure 14 is an illustration of the insertion of a representative stent coated with an anti-angiogenic composition of the present invention.

Figure 15A is a graph which shows the effect of the EVA:PLA polymer blend ratio upon aggregation of microspheres. Figure 15B is a scanning electron micrograph which shows the size of "small" microspheres.

Figure 15C is a scanning electron micrograph which shows the size of "large" microspheres. Figure 15D is a graph which depicts the time course of in vitro taxol release from 0.6% w/v taxol-loaded 50:50 EVA:PLA polymer blend microspheres into phosphate buffered saline (pH 7.4) at 37C. Open circles are "small" sized microspheres, and closed circles are "large" sized microspheres.

Figure 15E is a photograph of a CAM which shows the results of taxol release by microspheres Figure 15F is a photograph similar to that of 15E at increased magnification.

Figure 16 is a graph which shows release rate profiles from polycaprolactone microspheres containing 5% or 10% taxol into phosphate buffered saline at 37C. Figure 16B is a photograph which shows a CAM treated with control microspheres. Figure 16C is a photograph -which shows a CAM treated with 5% taxol loaded microspheres.

Figures 17A and 17B, respectively, are two graphs which show the release of taxol from EVA films, and the percent taxol remaining in those same films over time. Figure 17C is a graph which shows the swelling of EVA/F127 films with no taxol over time. Figure 17D is a graph which shows the swelling of EVA/Span 80 films with no taxol over time. Figure 17E is a graph which depicts a stress vs. strain curve for various EVA/Fl27 blends.

Figures 18A and 18B are two graphs which show the melting point of PCL/MePEG polymer blends as a function of MePEG in the formulation (18A), and the percent increase in time needed for PCL paste at 60 0 C to begin to solidify as a function of the amount of MePEG in the formulation (18B).

Figure 18C is a graph which depicts the brittleness of varying PCL/MePEG polymer blends. Figure 18D is a graph which shows the percent weight change over time for polymer blends of various MePEG concentrations. Figure 18E is a graph which depicts the rate of taxol release over time from various polymer blends loaded with 1% taxol. Figures 18F and 18G are graphs which depict the effect of varying quantities of taxol on the total amount of taxol released from a blend. Figure 18H is a graph which depicts the effect of MePEG on the tensile strength of a MePEG/PCL polymer.

Figure 19A is a photograph which shows control (unloaded) thermopaste on a CAM. Figure 19B is a photograph of 20% taxol-loaded thermopaste on a CAM.

Figures 20A and 20B are two photographs of a CAM having a tumor treated with control (unloaded) thermopaste. Figures 20C and 20D are two photographs of a CAM having a tumor treated with taxol-loaded thermopaste.

Figure 21A is a graph which shows the effect of taxol/PCL on tumor growth. Figures 21B and 21C are two photographs which show the effect of control, and 20% taxol-loaded thermopaste on tumor growth.

Figure 22A is a photograph of synovium from a PBS injected joint.

Figure 22B is a photograph of synovium from a microsphere injected joint.

Figure 22C is a photograph of cartilage from joints injected with PBS, and Figure 22D is a photograph of cartilage from joints injected with microspheres.

Detailed Description of the Invention As noted above, the present invention provides methods and compositions which utilize anti-angiogenic factors. Briefly, within the context of the present invention, anti-angiogenic factors should be understood to include any protein, peptide, chemical or other molecule which acts to inhibit vascular growth. A variety of methods may be readily utilized to determine the antiangiogenic activity of a given factor, including for example. chick chorioallantoic membrane assays. Briefly, as described in more detail below in Examples 2A and 2C. a portion of the shell from a freshly fertilized chicken egg is removed, and a methyl cellulose disk containing a sample of the antiangiogenic factor to be tested is placed on the membrane. After several days 48 hours), inhibition of vascular growth by the sample to be tested may be readily determined by visualization of the chick chorioallantoic membrane in the region surrounding the methyl cellulose disk. Inhibition of vascular growth may also be determined quantitatively, for example, by determining the number and size of blood vessels surrounding the methyl cellulose disk, as compared to a control methyl cellulose disk. Particularly preferred anti-angiogenic factors suitable for use within the present invention completely inhibit the formation of new blood vessels in the assay described above.

A variety of assays may also be utilized to determine the efficacy of anti-an2iogenic factors in vivo, including for example, mouse models which have been developed for this purpose (see Roberston et al., Cancer. Res.

51:1339-1344, 1991). In addition, a variety of representative in vivo assays relating to various aspects of the inventions described herein have been described in more detail below in Examples 5 to 7, and 17 to 19.

As noted above, the present invention provides compositions comprising an anti-angiogenic factor and a polymeric carrier. Briefly, a wide variety of anti-angiogenic factors may be readily utilized within the context of the present invention. Representative examples include Anti-Invasive Factor, retinoic acid and derivatives thereof, taxol, and members of the group consisting of Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1 and Plasminogen Activator Inhibitor-2. These and other anti-angiogenic factors will be discussed in more detail below.

Briefly, Anti-Invasive Factor, or "AIF' which is prepared from extracts of cartilage, is known to contain constituents which are responsible for inhibiting the growth of new blood vessels. These constituents comprise a family of 7 low molecular weight proteins (<50,000 daltons) (Kuettner and Pauli, "Inhibition of neovascularization by a cartilage factor" in Developmtent of the Vascular System, Pitman Books (Ciba Foundation Symposium 100), pp. 163- 173, 1983), including a variety of proteins which have inhibitory effects against a variety of proteases (Eisentein et al. Am. J. Padhol. 81:337-346, 1975; Langer et al., Science 193:70-72, 1976; and Horton et al., Science 199:1342-1345, 1978).

AIF suitable for use within the present invention may be readily prepared utilizing techniques known in the art Eisentein et al, supra; Kuettner and Pauli, supra: and Langer et al., supra). Purified constituents of AIF such as P.'OPER\MKR\SPECI\71746-00.040 doc.- 1/02/04 11 Cartilage-Derived Inhibitor (see Moses et al., Science 248:1408-1410, 1990) may also be readily prepared and utilized within the context of the present invention.

Retinoic acids alter the metabolism of extracellular matrix components, resulting in the inhibition of angiogenesis. Addition of proline analogs, angiostatic steroids, or heparin may be utilized in order to synergistically increase the anti-angiogenic effect of transretinoic acid. Retinoic acid, as well as derivatives thereof which may also be utilized in the context of the present invention, may be readily obtained from commercial sources, including for example, Sigma Chemical Co. R2625).

Taxol is a highly derivatized diterpenoid (Wani et al., J. Am. Chem. Soc. 93:2325, 1971) which has been obtained from the harvested and dried bark of Taxus brevifolia (Pacific Yew) and Taxomyces Andreanae and Endophytic Fungus of the Pacific Yew.

(Stierle et al., Science 60:214-216, 1993). Generally, taxol.acts to stabilize microtubular structures by binding tubulin to form abnormal mitotic spindles. "Taxol" (which should be understood herein to include analogues and derivatives of taxol such as, for example, baccatin and taxotere) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076, U.S. Patent Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,526 and EP 590267) or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Missouri (T7402 from Taxus brevifolia).

In the context in which the term "taxol" is used throughout this specification, the compound known as "taxol" is equivalent to the compound known as "paclitaxel".

Suramin is a polysulfonated naphthylurea compound that is typically used as atrypanocidal agent. Briefly, Suramin blocks the specific cell surface binding of various growth factors such as platelet derived growth factor epidermal growth factor transforming growth factor insulin-like growth factor and fibroblast growth factor Suramin may be prepared in accordance with known techniques, or readily obtained from a variety of commercial sources, including for example Mobay Chemical Co., New York, (see Gagliardi et al., Cancer Res. 52:5073- 5075, 1992; and Coffey, Jr., et al., J. of Cell. Phys. 132:143-148, 1987).

P:OPER\MKR\SPECl\71746.00-040.doc.IS/02/04 11A- Tissue Inhibitor of Metalloproteinase-1 ("TIMP") is secreted by endothelial cells which also secrete MTPases. TIMP is glycosylated and has a molecular weight of 28.5 kDa. TIMP-1 regulates angiogenesis by binding to activated metalloproteinases, thereby suppressing the invasion of blood vessels into the extracellular matrix. Tissue Inhibitor of Metalloproteinases-2 ("TIMP- 12 may also be utilized to inhibit angiogenesis. Briefly. TIMP-2 is a 21 kDa nonglycosylated protein which binds to metalloproteinases in both the active and latent, proenzyme forms. Both TIMP-1 and TIMP-2 may be obtained from commercial sources such as Synergen, Boulder, Colorado.

Plasminogen Activator Inhibitor 1 (PA) is a 50 kDa glycoprotein which is present in blood platelets, and can also be synthesized by endothelial cells and muscle cells. PAI-1 inhibits t-PA and urokinase plasminogen activator at the basolateral site of the endothelium, and additionally regulates the fibrinolysis process. Plasminogen Activator Inhibitor-2 (PAI-2) is generally found only in the blood under certain circumstances such as in pregnancy, and in the presence of tumors. Briefly, PAI-2 is a 56 kDa protein which is secreted by monocytes and macrophages. It is believed to regulate fibrinolytic activity, and in particular inhibits urokinase plasminogen activator and tissue plasminogen activator, thereby preventing fibrinolysis.

A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include Platelet Factor 4 (Sigma Chemical Co., #F1385); Protamine Sulphate (Clupeine) (Sigma Chemical Co., #P4505); Sulphated Chitin Derivatives (prepared from queen crab shells), (Sigma Chemical Co., #C3641; Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced. by the presence of steroids such as estrogen and tamoxifen citrate); Staurosporine (Sigma Chemical Co, #S4400); Modulators of Matrix Metabolism. including for example, proline analogs {[(L-azetidine-2-carboxylic acid (LACA) (Sigma Chemical Co., #A0760)), cishydroxyproline, d,L-3,4-dehydroproline (Sigma Chemical Co., #D0265), Thiaproline (Sigma Chemical Co., #T0631)], a,adipyridyl (Sigma Chemical Co., #D7505), $-aminopropionitrile fumarate (Sigma Chemical Co., MDL 27032 4 -propyl-5-(4-pyridinyl)-2(3H)oxazolone; Merion Merrel Dow Research Institute); Methotrexate (Sigma Chemical Co., #A6770; Hirata et al., Arthritis and Rheumatism 32:1065-1073, 1989); Mitoxantrone (Polverini and Novak, Biochemn. Biophys. Res. Comm.

140:901-907); Heparin (Folkman, Bio. Phar. 34:905-909, 1985: Sigma Chemical Co., #P8754); Interferons Sigma Chemical Co., 13265): 2 Macroglobulinserum (Sigma Chemical Co., #M7151); ChIMP-3 (Pavloff et al., J. Bio. Chem.

267:17321-17326, 1992); Chymostatin (Sigma Chemical Co.. #C7268; Tomkinson et al., Bioclhem J. 2S6: 4 75-4S0, 1992); 4-Cyclodextrin Tetradecasulfate (Sigma Chemical Co., #C4767): Epunemycin; Estramustine (available from Sigma; Wang and Stearns Cancer Res. 48:6262-6271, 1988); Fumagillin (Sigma Chemical Co., #F6771; Canadian Patent No. 2,024,306; Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST"; Sigma.G4022; Matsubara and Ziff, Clin. Invest. 79:1440-1446, 1987); (D- Penicillanmine ("CDPT'; Sigma Chemical Co., #P4875 or P5000(HCI)); 0-1anticollagenase-serum; a2-antiplasmin (Sigma Chem. Co.:A0914; Holmes et al., J. BioL Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide, Angiostatic steroid. AGM-1470. carboxyaminolmidazole, metalloproteinase inhibitors such as BB94 and the peptide CDPGYIGSR-NH: (SEQUENCE ID NO. 1) (Iwaki Glass, Tokyo, Japan).

Anti-angiogenic compositions of the present invention may additionally comprise a wide variety of compounds in addition to the antiangiogenic factor and polymeric carrier. For example, anti-angiogenic compositions of the present invention may also, within certain embodiments of the invention, also comprise one or more antibiotics, anti-inflamatories, antiviral agents, anti-fungal agents and/or anti-protozoal agents. Representative examples of antibiotics included within the compositions described herein include: penicillins; cephalosporins such as cefadroxil, cefazolin, cefaclor; aminoglycosides such as gentamycin and tobramycin; sulfonamides such as sulfamethoxazole; and metronidazole. Representative examples of antiinflammatories include: steroids such as prednisone, prednisolone, hydrocortisone, adrenocorticotropic hormone, and sulfasalazine; and nonsteroidal anti-inflammatory drugs ("NSAIDS") such as aspirin, ibuprofen, naproxen, fenoporfen, indomethacin, and phenylbutazone. Representative examples of antiviral agents include acyclovir, ganciclovir, zidovudine.

Representative examples of antifungal agents include: nystatin, ketoconazole, griseofulvin, flucytosine, miconazole, clotrimazole. Representative examples of antiprotozoal agents include: pentamidine isethionate, quinine, chloroquine, and mefloquine.

Anti-angiogenic compositions of the present invention may also contain one or more hormones such as thyroid hormone, estrogen, pr: .sterone, cortisone and/or growth hormone, other biologically active molecules such as insulin, as well as THI Interleukins -12,-and gamma interferon or TH2 Interleukins -4 and -10) cytokines.

Anti-angiogernic compositions of the present invention may also comprise additional ingredients such as surfactants (either hydrophilic or hydrophobic; see Example 13), anti-neoplastic or chemotherapeutic agents vinblastine, doxyrubicin, adriamycin, or tamocifen), radioactive agents Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In- 111, 1-123, 1-125, 1-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212) or toxins ricin, abrin, diptheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A).

As noted above, anti-angiogenic compositions of the present invention comprise an anti-angiogenic factor and a polymeric carrier. In addition to the wide array of anti-angiogenic factors and other compounds discussed above, anti-angiogenic compositions of the present invention may include a wide variety of polymeric carriers, including for example both biodegradable and non-biodegradable compositions. Representative examples of biodegradable compositions include albumin, gelatin, starch, cellulose, dextrans polysaccharides, fibrinogen, poly (d,l lactide), poly (d,l-lactide-coglycolide), poly (glycolide), poly (hydroxybutyrate), poly (alkylcarbonate) and poly (orthoesters) (see generally, Ilium, L, Davids, S.S. (eds.) "Polymers in controlled Drug Delivery" Wright, Bristol, 1987; Arshady, J. Controlled Release 17:1-22, 1991; Pitt, Int. J. Phar. 59:173-196, 1990; Holland et al., J. Controlled Release 4:155-0180. 1986). Representative examples of nondegradable polymers include EVA copolymers, silicone rubber and poly (methylmethacrylate).

Particularly preferred polymeric carriers include EVA copolymer ELVAX poly(ethylene-vinyl acetate) crosslinked with 40% vinyl acetate; DuPont), poly(lactic-co-glycolic acid), polycaprolactone, polylactic acid, copolymers of poly(ethylene-vinyl acetate) crosslinked with 40% vinyl acetate and polylactic acid, and copolymers of polylactic acid and polycaprolactone.

Polymeric carriers may be fashioned in a variety of forms, including for example, as nanospheres or microspheres. rod-shaped devices, pellets: slabs, or capsules (see, Goodell et al., Am. J. Hosp. Phann. 43:1454- 1461, 1986: Lanser et al., "Controlled release of macromolecules from polymers", in Biomedical polymers, Polymeric materials and phannaceuticals for biomedical use. Goldberg, Nakagim, A. (eds.) Academic Press, pp. 113-137, 1980: Rhine et al., J. Phann. Sci. 69:265-270, 1980; Brown et al., J. Pharm. Sci.

72:1181-1185. 1983: and Bawa et al., J. Controlled Release 1:259-267, 1985).

Preferably. anti-angiogenic compositions of the present invention (which comprise one or more anti-angiogenic factors, and a polymeric carrier) are fashioned in a manner appropriate to the intended use. Within preferred aspects of the present invention, the anti-angiogenic composition should be biocompatible, and release one or more anti-angiogenic factc-s over a period of several weeks to months. In addition, anti-angiogenic compositions of the present invention should preferably be stable for several months and capable of being produced and maintained under sterile conditions.

Within certain aspects of the present invention, anti-angiogenic compositions may be fashioned in any size ranging from nanospheres to microspheres from 0.1 um to 500 am), depending upon the particular use.

For example, when used for the purpose of tumor embolization (as discussed below), it is generally preferable to fashion the anti-angiogenic composition in microspheres of between 15 and 500 gm. preferably between 15 and 200 pm, and most preferably, between 25 and 150 Such nanoparticles may also be readily applied as a "spray", which solidifies into a film or coating.

Nanoparticles (also termed "nanospheres") may be prepared in a wide array of sizes, including for example, from 0.1 pm to 3 pm, from 10 pm to 30 jm, and from 30 pm to 100 pm (see Example 8).

Anti-angiogenic compositions may also be prepared, given the disclosure provided herein, for a variety of other applications. For example, for the -inistration of anti-angiogenic compositions to the cornea, the compo,..,ons of the present invention may be incorporated into polymers as nanoparticles (see generally, Kreuter J. Controlled Release 16:169-176, 1991; Couvreur and Vauthier, J. Controlled Release 17:187-198, 1991). Such nanoparticle may also be readily applied as a "spray", which solidifies into a film or coating. Nanoparticles (also termed "nanospheres") may be prepared in a wide array of sizes, including for example, from 0.1 pm to 3 pm, from. 10 pm to pm, and from 30 pm to 100 gm (see Example 8).

Anti-angiogenic compositions of the present invention may also be prepared in a varietx of "paste" or gel forms. For example, within one embodiment of the invention, anti-angiogenic compositions are provided which are liquid at one temperature temperature greater than 37°C, such as 50°C, 55°C or 60°C), and solid or semi-solid at another temperature ambient body temperature, or any temperature lower than 37 0 Such "thermopastes" may be readily made given the disclosure provided herein (see, Examples 10 and 14).

Within vet other aspects of the invention, the anti-angiogenic compositions of the present invention may he formed as a film. Preferably, such films are generally less than 5, 4, 3, 2, or 1. mm thick, more preferably less than 0.75 mm or 0.5 mm thick, and most preferably less than 500 gm to 100 gm thick.

Such films are preferably flexible with a good tensile strength greater than preferably greater than 100, and more preferably greater than 150 or 200 N/cm 2 good adhesive properties readily adheres to moist or wet surfaces), and has controlled permeability. Representative examples of such films are set forth below in the Examples (see Example 13).

Representative examples of the incorporation of anti-angiogenic factors such as into a polymeric carriers are described in more detail below in Examples 3, 4 and 8-15.

ArTT-RIA.. EMni 17l7in\n In addition to the compositions described above, the present invention also provides a variety of methods which utilize the above-described anti-angiogenic compositions. In particular, within one aspect of the present invention methods are provided for embolizing a blood vessel, comprising the step of delivering into the vessel a therapeutically effective amount of an antiangiogenic composition (as described above), such that the blood vessel .is effectively occluded. Therapeutically effective amounts suitable for occluding blood vessels may be readily determined given the disclosure provided below, and as described in Example 6. Within a particularly preferred embodiment, the anti-angiogenic composition is delivered to a blood vessel which nourishes a tumor (see Figure 13).

Briefly, there are a number of clinical situations bleeding, tumor development) where it is desirable to reduce or abolish the blood supply to an organ or region. As described in greater detail below, this may be accomplished by injecting anti-angiogenic compositions of the present invention into a desired blood vessel through a selectively positioned catheter (see Figure 13). The composition travels via the blood stream until it becomes wedged hi the vasculature, thereby physically (or chemically) occluding the blood vessel. The reduced or abolished blood flow to the selected area results in infarction (cell death due to an inadequate supply of oxygen and nutrients) or reduced blood loss from a damaged vessel.

For use in embolization therapy, anti-angiogenic compositions of the present invention are preferably non-toxic. thrombogenic. easy to inject down vascular catheters, radio-opaque. rapid and permanent in effect. sterile, and readily available in different shapes or sizes at the time of the procedure.

In addition, the compositions preferably result in the slow (ideally, over a period of several weeks to months) release of an anti-angiogenic factor. Particularly preferred anti-angiogenic compositions should have a predictable size of 200 arm after being injected into the vascular system. Preferably, they should not clump into larger particles either in solution or once injected. In addition, preferable compositions should not change shape or physical properties during storage prior to use.

Embolization therapy may be utilized in at least three principal ways to assist in the management of neoplasms: definitive treatment of tumors (usually benign); for preoperative embolization; and for palliative embolization. Briefly, benign tumors may sometimes be successfully treated by embolization therapy alone.. Examples of such tumors include simple tumrners of vascular origin haemangiomas), endocrine tumors such as parathyroid adenomas, and benign bone tumors.

For other tumors, renal adenocarcinoma), preoperative embolization may be employed hours or days before surgical resection in order to reduce operative blood loss, shorten the duration of the operation, and reduce the risk of dissemination of viable malignant cells by surgical manipulation of the tumor. Many tumors may be successfully embolized preoperatively, including for example nasopharyngeal tumors, glomus jugular tumors, meningiomas. chemodectomas, and vagal neuromas.

Embolization may also be utilized as a primary mode of treatment for inoperable malignancies, in order to extend the survival time of patients with advanced disease. Embolization may produce a marked improvement in the quality of life of patients with malignant tumors by alleviating unpleasant symptoms such as bleeding, venous obstruction and tracheal compression. The greatest benefit from palliative tumor embolization, hc-'ever, may be seen in patients suffering from the humoral effects of malignant endocrine tumors, wherein metastases from carcinoid tumors and other endocrine neoplasms such as insulinomas and glucagonomas may be slow growing, and yet cause great distress by virtue of the endocrine syndromes which they produce.

In general, embolization therapy utilizing anti-angiogenic compositions of the present invention is typically performed in a similar manner, regardless of the site. Briefly, angiography (a road map of the blood vessels) of the area to be embolized is first performed by injecting radiopaque contrast through a catheter inserted into an artery or vein (depending on the site to be embolized) as an X-ray is taken. The catheter may he inserted either percutaneously or by surgery. The blood vessel is then embolized by refluxing anti-angiogenic compositions of the present invention through the catheter, until flow is observed to cease. Occlusion may be confirmed by repeating the angiogram.

Embolization therapy generally results in the distribution of compositions containing anti-angiogenic factors throughout the interstices of the tumor or vascular mass to be treated. The physical bulk of the embolic particles clogging the arterial lumen results in the occlusion of the blood supply. In addition to this effect, the presence of an anti-angiogenic factor(s) prevents the formation of new blood vessels to supply the tumor or vascular mass, enhancing the devitalizing effect of cutting off the blood supply.

Therefore, it should be evident that a wide variety of tumors may be embolized utilizing the compositions of the present invention. Briefly, tumors are typically divided into two classes: benign and malignant. In a benign tumor the cells retain their differentiated features and do not divide in a completely uncontrolled manner. In addition, the tumor is localized and nonmetastatic. In a malignant tumor, the cells become undifferentiated, do not respond to the body's growth and hormonal signals, and multiply in a.n uncontrolled manner; the tumor is invasive and capable of spreading to distant sites (metastasizing).

Within one aspect of the present invention, metastases (secondary tumors) of the liver may be treated utilizing emholization therapy. Briefly, a catheter is inserted via the femoral or brachial artery and advanced into the hepatic artery by steering it through the arterial system under fluoroscopic guidance. The catheter is advanced into the hepatic arterial tree as far as necessary to allow complete blockage of the blood vessels supplying the tumor(s), while sparing as many of the arterial branches supplying normal structures as possible. Ideally this will be a segmental branch of the hepatic artery, but it could be that the entire hepatic artery distal to the origin of the gastroduodenal artery, or even multiple separate arteries, will need to be blocked depending on the extent of tumor and its individual blood supply. Once the desired catheter position is achieved, the artery is embolized by injecting anti-angiogenic compositions (as described above) through the arterial catheter until flow in the artery to be blocked ceases, preferably even after observation for 5 minutes. Occlusion of the artery may be confirmed by injecting radiopaque contrast through the catheter and demonstrating by fluoroscopy or X-ray film that the vessel which previously filled with contrast no longer does so.

The same procedure may be repeated with each feeding artery to be occluded.

As note above, both benign and malignant tumors may be embolized utilizing compositions of the present invention. Representative examples of benign hepatic tumors include Hepatocellular Adenoma, Cavernous Haemangioma, and Focal Nodular Hyperplasia. Other benign tumors, which are more rare and often do not have clinical manifestations, may also be treated. These include Bile Duct Adenomas, Bile Duct Cystadenomas, Fibromas, Lipomas, Leiomyomas, Mesotheliomas, Teratomas, Myxomas, and Nodular Regenerative Hyperplasia.

Malignant Hepatic. Tumors are generally subdivided into two categories: primary and secondary. Primary tumors arise directly from the tissue in which they are found. Thus, a primary liver tumor is derived originally from the cells which make up the liver tissue (such as hepatocytes and biliary cells).

Representative examples of primary hepatic malignancies which may be treated by arterial embolization include Hepatocellularcarcinoma, Cholangiocarcinoma, Angiosarcoma, Cystadenocar-cinoma, Squamous Cell Carcinoma, and Hepatoblastoma.

A secondary tumor, or metastasis, is a tumor which originated elsewhere in the body but has now spread to a distant organ. The common routes for metastasis are direct growth into adjacent structures, spread through the vascular or lymphatic systems, and tracking along tissue planes and body spaces (peritoneal fluid, cerebrospinal fluid, etc.). Secondary hepatic tumors are one of the most common causes of death in cancer patients and are by far and away the most common form of liver tumor. Although virtually any malignancy can metastasize to the liver, tumors which are most likely to spread to the liver include: cancer of the stomach, colon, and pancreas; melanoma; tumors of the lung, oropharynx, and bladder; Hodgkin's and non-Hodgkin's lymphoma; tumors of the breast, ovary, and prostate. Each one of the abovenamed primary tumors has numerous different tumor types which may be treated by arterial embolization (for example, there are over 32 different types of ovarian cancer).

As noted above, embolization therapy utilizing anti-angiogenic compositions of the present invention may also be applied to a variety of other clinical situations where it is desired to occlude blood vessels. Within one aspect of the present invention, arteriovenous malformation may be treated by administration of one of the above-described compositions. Briefly, arteriovenous malformations (vascular malformations) refers to a group of diseases wherein at least one (and most typically. many) abnormal communications between arteries and veins occur, resulting in a local tumor-like mass composed predominantly of blood vessels. Such disease may be either congenital or acquired.

Within one embodiment of the invention, an arteriovenous malformation may be treated by inserting a catheter via the femoral or brachial artery, and advancing it into the feeding artery under fluoroscopic guidance.

The catheter is preferably advanced as far as necessary to allow complete blockage of the blood vessels supplying the vascular malformation, while sparing as many of the arterial branches supplying normal structures as possible (ideally this will be a single artery, but most often multiple separate arteries may need to be occluded, depending on the extent of the vascular malformation and its individual blood supply). Once the desired catheter position is achieved, each artery may be embolized utilizing anti-angiogenic compositions of the present invention.

Within another aspect of the invention, embolization may be accomplished in order to treat conditions of excessive bleeding. For example, menorrhagia (excessive bleeding with menstruation) may be readily treated by embolization of uterine arteries. .Briefly, the uterine arteries are branches of the internal iliac arteries bilaterally. Within one embodiment of the invention, a catheter may be inserted via the femoral or brachial artery, and.advanced into each uterine artery by steering it through the arterial system under fluoroscopic guidance. The catheter should be advanced as far as necessary to allow complete blockage of the blood vessels to the uterus, while sparing as many arterial branches that arise from the uterine artery and supply normal structures as possible. Ideally a single uterine artery on each side may be embolized, but occasionally multiple separate arteries may need to be blocked depending on the individual blood supply. Once the desired catheter position is achieved, each artery may be embolized by administration of the anti-anrigiogenic compositions as described above.

In a like manner, arterial embolization may be accomplished in a variety of other conditions, including for example for acute bleeding, vascular abnormalities, central nervous system disorders. and hypersplenism.

USE OF A m-.A\GonrcEF1C CononMrnN.s AS CnATINC, FnR STENTS As noted above, the present invention also provides stents, comprising a generally tubular structure (which includes for example, spiral shapes), the surface of which is coated with a composition as described above.

Briefly, a stent is a scaffolding, usually cylindrical in shape, that may be inserted into a body passageway bile ducts), which has been narrowed by a disease process ingrowth by a tumor) in order to prevent closure or reclosure of the passageway. Stents act by physically holding open the walls of the body passage into which they are inserted.

A variety of stents may be utilized within the context of the present invention, including for example, esophageal stents, vascular stents, biliary stents, pancreatic stems. ureteric and urethral stents, lacrimal stents, eustachian tube stents, fallopian tube stems, and tracheal/bronchial stents.

Stents may be readily obtained from :ommercial sources, or constructed in accordance with well known techniques. Representative examples of stents include those described in U.S. Patent No. 4,776,337, entitled "Expandable Intraluminal Graft, and Method and Apparatus for Implanting and Expandable Intraluminal Graft", U.S. Patent No. 5,176,626, entitled "Indwelling Stent", U.S. Patent No. 5,147,370 entitled "Nitinol Stent for Hollow Body Conduits", U.S. Patent No. 5,064,435 entitled "Self-Expanding Prosthesis Having Stable Axial Length", U.S. Patent No. 5,052,998 entitled "Indwelling Stent and Method of Use", and U.S. Patent No. 5,041,126 entitled "Endovascular Stent and Delivery System, all of which are hereby incorporated by reference in their entirety.

Stents may be coated with anti-angiogenic compositions or antiangiogenic factors of the present invention using a variety of methods, including for example: by directly affixing to the stent an anti-angiogenic composition by either spraying the stent with a polymer/drug film, or by dipping the stent into a polymer/drug solution), by coating the stent with a substance such as a hydrogel which will in turn absorb the anti-angiogenic composition (or anti-angiogenic factor above), by interweaving anti-angiogenic composition coated thread (or the polymer itself formed into a thread) into the stent structure, by inserting the stent into a sleeve or mesh which is comprised of or coated with an anti-angiogenic composition, or constructing the stent itself with an anti-angiogenic composition. Within preferred embodiments of the invention, the composition should firmly adhere to the stent during storage and at the time of insertion, and should not be dislodged from the stent when the diameter is expanded from its collapsed size to its full expansion size. The anti-angiogenic composition should also preferably not degrade during storage, prior to insertion, or when warmed to body temperature after expansion inside the body. In addition, it should preferably coat the stent smoothly and evenly, with a uniform distribution of angiogenesis inhibitor, while not changing the stent contour. Within preferred embodiments of the invention, the antiangiogenic composition should provide a uniform, predictable, prolonged release of the anti-angiogenic factor into the tissue surrounding the stent once it has been deployed. For vascular stents, in addition to the above properties, the composition should not render the stent thrombogenic (causing blood clots to form), or cause significant turbulence in blood flow (more than the stent itself would be expected to cause if it was uncoated).

Within another aspect of the present invention, methods are provided for expanding the lumen of a body passageway, comprising inserting a stent into the passageway, the stent having a generally tubular structure, the surface of the structure being coated with an anti-angiogenic composition (or, an anti-angiogenic factor alone), such that the passageway is expanded. A variety of embodiments are described below wherein the lumen of a body passageway is expanded in order to eliminate a biliary, esophogeal, tracheal/bronchial, urethral or vascular obstruction. In addition, a representative example is described in more detail below in Example 7.

Generally, stents are inserted in a similar fashion regardless of the site or the disease being treated. Briefly, a preinsertion examination, usually a diagnostic imaging procedure. endoscopy, or direct visualization at the time of surgery, is generally first performed in order to determine the appropriate positioning for stent insertion. A guidewire is then advanced through the lesion or proposed site of insertion, and over this is passed a delivery catheter which allows a stent in its collapsed form to be inserted. Typically, stents are capable of being compressed, so that they can be inserted through tiny cavities via small catheters, and then expanded to a larger diameter once they are at the desired location. Once expanded, the stent physically forces the walls of the passageway apart and holds it open. As such. they are capable of insertion via a small opening, and yet are still able to hold open a large diameter cavity or passageway. The stent may be self-expanding the Wallstent and Gianturco stents), balloon expandable the Palmaz stent and Strecker stent), or implanted by a change in temperature the Nitinol stent).

Stents are typically maneuvered into place under radiologic or direct visual control, taking particular care to place the stent precisely across the narrowing in the organ being treated. The delivery catheter is then removed, leaving the stent standing on its own as a scaffold. A post insertion examination, usually an x-ray, is often utilized to confirm appropriate positioning.

Within a preferred embodiment of the invention, methods are provided for eliminating biliary obstructions, comprising inserting a biliary stent into a biliary passageway, the stent having a generally tubular structure, the surface of the structure being coated with a composition as described above, such that the biliary obstruction is eliminated. Briefly, tumor overgrowth of the common bile duct results in progressive cholestatic jaundice which is incompatible with life. Generally, the biliary system which drains bile from the liver into the duodenum is most often obstructed by a tumor composed of bile duct cells (cholangiocarcinoma), a tumor which invades the bile duct pancreatic carcinoma), or a tumor which exerts extrinsic pressure and compresses the bile duct enlarged lymph nodes).

Both primary biliary tumors, as well as other tumors which cause compression of the biliary tree may be treated utilizing the stents described herein. One example of primary biliary tumors are adenocarcinomas (which are also called Klatskin tumors when found at the bifurcation of the common hepatic duct). These tumors are also referred to as biliary carcinomas, choledocholangiocarcinomas, or adenocarcinomas of the biliary system. .Benign rumors which affect the bile duct adenoma of the biliary system), and, in rare cases, squamous cell carcinomas of the bile duct and adenocarcinomas of the gallbladder, may also cause compression of the biliary tree, and therefore, result in biliary obstruction.

Compression of the biliary tree is most commonly due to tumors of the liver and pancreas which compress and therefore obstruct the ducts.

Most of the tumors from the pancreas arise from cells of the pancreatic ducts.

This is a highly fatal form of cancer of all cancer deaths; 26,000 new cases per year in the with an average of 6 months survival and a 1 year survival rate of only 10%. When these tumors are located in the head of the pancreas they frequently cause biliary obstruction. and this detracts significantly from the quality of life of the patient. While all types of pancreatic tumors are generally referred to as "carcinoma of the pancreas." there are histologic subtypes including: adenocarcinoma, adenosquamous carcinoma, cystadeno-carcinoma.

and acinar cell carcinoma. Hepatic tumors. as discussed above, may also cause 24 compression of the biliary tree, and therefore cause obstruction of the biliary ducts.

Within one embodiment of the invention, a biliary stent is first inserted into a biliary passageway in one of several ways: from the top end by inserting a needle through the abdominal wall and through the liver (a percutaneous transhepatic cholangiogram or from the bottom end by cannulating the bile duct through an endoscope inserted through the mouth, stomach, or duodenum (an endoscopic retrograde cholangiogram or "ERCP"); or by direct incision during a surgical procedure. A preinsertion examination, PTC, ERCP, or direct visualization at the time of surgery should generally be performed to determine the appropriate position for stent insertion. A guidewire is then advanced through the lesion, and over this a delivery catheter is passed to allow the stent to be inserted in its collapsed form. If the diagnostic exam was a PTC, the guidewire and delivery catheter will be inserted via the abdominal wall, while if the original exam was an ERCP the stent will be placed via the mouth. The stent is then positioned under radiologic, endoscopic, or direct visual control taking particular care to place it precisely across the narrowing in the bile duct. The delivery catheter will be removed leaving the stent standing as a scaffolding which holds the bile duct open. A further cholangiogram will be performed to document that the stent is appropriately positioned.

Within yet another embodiment of the invention, methods are provided for eliminating esophageal obstructions, comprising inserting an esophageal stent into an esophagus, the stent having a generally tubular structure, the surface of the structure being coated with an anti-angiogenic composition as described above, such that the esophageal obstruction is eliminated. Briefly, the esophagus is the hollow tube which transports food and liquids from the mouth to the stomach. Cancer of the esophagus or invasion by cancer arising in adjacent organs cancer of the stomach or lung) results in the inability to swallow food or saliva. Within this embodiment, a preinsertion examination, usually a barium swallow or endoscopy should generally be performed in order to determine the appropriate position for stent insertion. A catheter or endoscope may then be positioned through the mouth, and a guidewire is advanced through the blockage. A stent delivery catheter is passed over the guidewire under radiologic or endoscopic control, and a stent is placed precisely across the narrowing in the esophagus. A post insertion examination, usually a barium swallow x-ray, may be utilized to confirm appropriate positioning.

Within other embodiments of the invention, methods are provided for eliminating tracheal/bronchial obstructions, comprising inserting a tracheal/bronchial stent into the trachea or bronchi, the stent having a generally tubular structure, the surface of which is coated with an anti-angiogenic composition as described abLve, such that the tracheal/bronchial obstruction is eliminated. Briefly, the trachea and bronchi are tubes which carry air from the mouth and nose to the lungs. Blockage of the trachea by cancer, invasion by cancer arising in adjacent organs cancer of the lung), or collapse of the trachea or bronchi due to chondromalacia (weakening of the cartilage rings) results in inability to breathe. Within this embodiment of the invention, preinsertion examination, usually an endoscopy, should generally be performed in order to determine the appropriate position for stent insertion. A catheter or endoscope is then positioned through the mouth, and a guidewire advanced through the blockage. A delivery catheter is then passed over the guidewire in order to allow a collapsed stent to be inserted. The stent is placed under radiologic or endoscopic control in order to place it precisely across the narrowing. The delivery catheter may then be removed leaving the stent standing as a scaffold on its own. A post insertion examination, usually a bronchoscopy, may be utilized to confirm appropriate positioning.

Within another embodiment of the invention, methods are provided for eliminating urethral obstructions, comprising inserting.a urethral stent into a urethra, the stent having a generally tubular structure, the surface of the structure being coated with an anti-angiogenic composition as described above, such that the urethral obstruction is eliminated. Briefly, the urethra is the tube which drains the bladder through the penis. Extrinsic narrowing of the urethra as it passes through the prostate, due to hypertrophy of the prostate, occurs in virtually every man over the age of 60 and causes progressive difficulty with urination. Within this embodiment, a preinsertion examination, usually an endoscopy or urethrogram should generally first be performed in order to determine the appropriate position for stent insertion, which is above the external urinary sphincter at the lower end. and close to flush with the bladder neck at the upper end. An endoscope or catheter is then positioned through the penile opening and a guidewire advanced into the bladder. A delivery catheter is then passed over the guidewire in order to allow stent insertion. The delivery catheter is then removed, and the stent exoanded in.a.olace. A post insertion examination, usually endoscopy or retrograde urethrogram, may be utilized to confirm appropriate position.

Within another embodiment of the invention, methods are provided for eliminating vascular obstructions, comprising inserting a vascular stent into a blood vessel, the stent having a generally tubular structure, the surface of the structure being coated with an anti-angiogenic composition as described above, such that the vascular obstruction is eliminated. Briefly, stents may be placed in a wide array of blood vessels, both arteries and veins, to prevent recurrent stenosis at the site of failed angioplasties, to treat narrowings that would likely fail if treated with angioplasty, and to treat post surgical narrowings dialysis graft stenosis). Representative examples of suitable sites include the iliac, renal, and coronary arteries, the superior vena cava, and in dialysis grafts. Within one embodiment, angiography is first performed in order to localize the site for placement of the stent. This is typically accomplished by injecting radiopaque contrast through a catheter inserted into an artery or vein as an x-ray is taken. A catheter may then be inserted either percutaneously or by surgery into the femoral artery, brachial artery, femoral vein, or brachial vein, and advanced into the appropriate blood vessel by steering it through the vascular system under fluoroscopic guidance. A stent may then be positioned across the vascular stenosis. A post insertion angiogram may also be utilized in order to confirm appropriate positioning.

USE OF A.'n-A\CNOGENIC CoMPosMrnns I SLRTIC..\. PROCEDURES As noted above, anti-angiogenic compositions may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention an anti-angiogenic compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, anti-angiogenic compositions in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within vet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh ladened with an anti-angiogenic composition may be utilized during ahdominal cancer resection surgery subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are provided for treating -tumor excision sites, comprising administering an antiangiogenic composition as described above to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic composition(s) (or anti-angiogenic factor(s) alone) are administered directly to the tumor excision site applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic composition(s) or factor(s)). Alternatively, the antiangiogenic composition(s) or factor(s) may be incorporated into known surgiral pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compositions are applied after, hepatic resections for malignancy, and after neurosurgical operations.

Within one aspect of the present invention, anti-angiogenic compositions (as described above) may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention antiangiogenic compositions may be administered to 'he site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited. Briefly, the brain is highly functionally localized; each specific anatomical region is specialized to carry out a specific function.

Therefore it is the location of brain pathology that is often more important than the type. A relatively small lesion in a key area can be far more devastating than a much larger lesion in a less important area. Similarly, a lesion on the surface of the brain may be easy to resect surgically, while the same. tumor located deep in the brain may not (one would have to cut through too many vital structures to reach it). Also, even benign tumors can be dangerous for several reasons: th'ey may grow in a key area and cause significant damage; even though they would be cured by surgical resection this may not be possible; and finally, if left unchecked they can cause increased intracranial pressure. The skull is an enclosed space incapable of expansion. Therefore, if something is growing in one location, something else must be being compressed in another location the result is increased pressure in the skull or increased intracranial pressure. If such a condition is left untreated., vital structures can be compressed, resulting in death. The incidence of CNS (central nervous system) malignancies is 8-16 per 100,000. Tne prognosis of primary malignancy of the brain is dismal, with a median survival of less than one year, even following surgical resection. These tumors, especially gliomas, are predominantly a local disease which recur within 2 centimeters of the original focus of disease after surgical removal.

Representative examples of brain tumors which may be treated utilizing the compositions and methods described herein include Glial Tumors (such as Anaplastic Astrocytoma, Glioblastoma Multiform, Pilocytic Astrocvtoma, Oligodendroglioma, Ependymoma. Myxopapillary Ependymoma, Subependymoma. Choroid Plexus Papilloma); Neuron Tumors Neuroblastoma, Ganglioneuroblastoma, Ganglioneuroma, and Medulloblastoma): Pineal Gland Tumors Pineoblastoma and Pineocytoma): Menigeal Tumors Meningioma, Meningeal Hemangiopericytoma. Meningeal Sarcoma); Tumors of Nerve Sheath Cells Schwannoma (Neurolemmoma) and Neurofibroma); Lymphomas Hodgkin's and Non-Hodgkin's Lymphoma (including numerous subtypes, both primary and secondary); Malformative Tumors Craniopharyngioma, Epidermoid Cysts, Dermoid Cysts and Colloid Cysts); and Metastatic Tumors (which can be derived from virtually any tumor, the most common being from lung, breast, melanoma, kidney, and gastrointestinal tract tumors).

OTHER THFR.APFLTnC USF-S OF ANn-AN.c.i-FNic Co.rnmsnoNs In addition to tumors, numerous other non-tumorigenic angiogenesis-dependent diseases which are characterized by the abnormal growth of blood vessels may also be treated with the anti-angiogenic compositions, or anti-angiogenic factors of the present invention.

Representative examples of such non-tumorigenic angiogenesis-dependent diseases include corneal neovascularization, hypertrophic scars and keloids, proliferative diabetic retinopathy, rheumatoid arthritis, arteriovenous malformations (discussed above), atherosclerotic plaques, delayed wound healing, hemophilic joints, nonunion fractures, Osler-Weber syndrome, psoriasis, pyogenic granuloma, scleroderma, tracoma, menorrhagia (discussed above) and vascular adhesions.

In particular, within one aspect of the present invention methods are provided for treating corneal neovascularization (including corneal graft neovascularization). comprising the step of administering a therapeutically effective amount of an anti-angioyenic composition (as described above) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions, however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates.

Blood vessels can enter the cornea in a variety of patterns and depths, depending upon the process which incites the neovascularization. These patterns have been traditionally defined by ophthalmologists in the following types: pannus trachomatosus, pannus leprosus, pannus phylctenulosus, pannus degenerativus, and glaucomatous pannus. The corneal stroma may also be invaded by branches of the anterior ciliary artery (called interstitial vascularizarion) which causes several distinct clinical lesions: terminal loops, a "brush-like" pattern, an umbel form, a lattice form, interstitial arcades (from episcleral vessels), and aberrant irregular vessels.

A wide variety of disorders can result in corneal neovascularization, including for example corneal infections trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.

While the cause of corneal neovascularization may vary, the response of the cornea to the insult and the subsequent vascular ingrowth is similar regardless of the cause. Briefly, the location of the injury appears to be of importance as only those lesions situated within a critical distance of the limbus will incite an angiogenic response. This is likely due to the fact that the angiogenic factors responsible for eliciting the vascular invasion are created at the site of the lesion, and must diffuse to the site of the nearest blood vessels (the limbus) in order to exert their effect. Past a certain distance from the limbus, this would no longer be possible and the limbic endothelium would not be induced to grow into the cornea. Several angiogenic factors are likely involved in this process, many of which are products of the inflammatory response. Indeed, neovascularization of the cornea appears to only occur in association with an inflammatory cell infiltrate, and the degree of angiogenesis is proportional to the extent of the inflammatory reaction. Corneal edema further facilitates blood vessel inrowth by loosening the corneal stromal framework and providing a pathway of "least resistance" through which the capillaries can grow.

Following the initial inflammatory reaction, capillary growth into the cornea proceeds in the same manner as it occurs in other tissues. The normally quiescent endothelial cells of the limbic capillaries and venules are stimulated to divide and migrate. The endothelial cells project away from their vessels of origin, digest the surrounding basement membrane and the tissue through which they will travel, and migrate towards the source of the angiogenic stimulus. The blind ended sprouts acquire a lumen and then anastomose together to form capillary loops. The end result is the establishment of a vascular plexus within the corneal stroma.

Anti-angiogenic compositions of the present invention are useful by blocking the stimulatory effects of angiogenesis promoters, reducing endothelial cell division, decreasing endothelial cell migration, and impairing the activity of the proteolytic enzymes secreted by the endothelium.

Within particularly preferred embodiments of the invention, an anti-angiogenic factor may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The antiangiogenic factor solution may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described .above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy.

Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.

Within other embodiments, the anti-angiogenic compositions described above may be injected direccly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature interspersed between the blood vessels and the normal cornea).

In most cases this would involve perilimbic corneal injection to "protect" the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustainedrelease form, injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.

Within another aspect of the present invention, methods are provided for treating hypertrophic scars and keloids. comprising the step of administering one of the above-described anti-angiogenic compositions to a hypertrophic scar or keloid.

Briefly, healing of wounds and scar formation occurs in three phases: inflammation, proliferation, and maturation. The first phase, inflammation, occurs in response to an injury whic. is severe enough to break the n. During this phase, which lasts 3 to 4 days, blood and tissue fluid form an adhesive coagulum and fibrinous network which serves to bind the wound surfaces together. This is then followed by a proliferative phase in which there is ingrowth of capillaries and connective tissue .from the wound edges, and closure of the skin defect. Finally, once capillary and fibroblastic proliferation has ceased, the rr turation process begins wherein the scar contracts and becomes less cellular, less vascular, and appears flat and white. This final phase may take between 6 and 12 months.

If too much connective tissue is produced and the wound remains persistently cellular, the scar may become red and raised. If the scar remains within the boundaries of the original wound it is referred to as a hypertrophic scar, but if it extends beyond the original scar and into the surrounding tissue, the lesion is r.,erred to as a keloid. Hypertrophic scars and keloids are produced during the second and third phases of scar formation. Several wounds are particularly prone to excessive endothelial and fibroblastic proliferation, including burns, open wounds, and intrcted wounds. With hypertrophic scars, some degree of maturation occurs and gradual improvement occurs. In the case of keloids however, an actual tumor is produced which can become quite large.

Spontaneous improvement in such cases rarely occurs.

32 Therefore, within one embodiment of the present invention either anti-angiogenic factors alone, or anti-angiogenic compositions as described above, are directly injected into a hypertrophic scar or keloid in order to prevent the progression of these lesions. The frequency of injections will depend upon the release kinetics of the polymer used (if present), and the clinical response.

This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development.

Within another aspect of the present invention methods are provided for treating neovascular glaucoma, comprising the step of administering a therapeutically effective amount of an anti-angiogenic composition to the eye, such that the formation of blood vessels is inhibited.

Briefly, neovascular glaucoma is a pathological condition wherein new capillaries develop in the iris of the eye. The angiogenesis usually originates from vessels located at the pupillary margin, and progresses across the root of the iris and into the trabecular meshwork. Fibroblasts and other connective tissue elements are associated with the capillary growth and a fibrovascular membrane develops which spreads across the anterior surface of the iris. Eventually this tissue reaches the anterior chamber angle where it forms synechiae. These synechiae in turn coalesce, scar, and contract to ultimately close off the anterior chamber angle. The scar formation prevents adequate drainage of aqueous humor through the angle and into the trabecular meshwork, resulting in an increase in intraocular pressure that may result in blindness.

Neovascular glaucoma generally occurs as a complication of diseases in which retinal ischemia is predominant. In particular, about one third of the patients with this disorder have diabetic retinopathy and 28% have central retinal vein occlusion. Other causes include chronic retinal detachment, end-stage glaucoma, carotid artery obstructive disease, retrolental fibroplasia, sickle-cell anemia, intraocular tumors, and carotid cavernous fistulas. In its early stages, neovascular glaucoma may be diagnosed by high magnification slitlamp biomicroscopy, where it reveals small, dilated, disorganized capillaries (which leak fluorescein) on the surface of the iris. Later gonioscopy demonstrates progressive obliteration of the anterior chamber angle by fibrovascular bands. While the anterior chamber angle is still open, 3)3 conservative therapies may be of assistance. However, once the angle closes surgical intervention is required in order to alleviate the pressure.

Therefore, within one embodiment of the invention antiangiogenic factors (either alone or in an anti-angiogenic composition, as described above) may be administered topically to the eye in order to treat early forms of neovascular glaucoma.

Within other embodiments of the invention, anti-angiogenic compositions may be implanted by injection of the composition into the region of the anterior chamber angle. This provides a sustained localized increase of anti-angiogenic factor, and prevents blood vessel growth into the area.

Implanted or injected anti-angiogenic compositions which are placed between the advancing capillaries of the iris and the anterior chamber angle can "defend" the open angle from neovascularization. As capillaries will not grow within a significant radius of the anti-angiogenic composition, patency of the angle could be maintained. Within other embodiments, the anti-angiogenic composition may also be placed in any location such that the anti-angiogenic.factor is continuously released into the aqueous humor. This would increase the antiangiogenic factor concentration within the humor, which in turn bathes the surface of the iris and its abnormal capillaries, thereby providing another mechanism by which to deliver the medication. These therapeutic modalities may also be useful prophylactically and in combination with existing treatments.

Within another aspect of the present invention, methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering a therapeutically effective amount of an anti-angiogenic composition to the eyes, such that the formation of blood vessels is inhibited.

Briefly, the pathology of diabetic retinopathy is thought to be similar to that described above for neovascular glaucoma. In particular, background diabetic retinopathy is believed to convert to proliferative diabetic retinopathy under the influence of retinal hypoxia. Generally, neovascular tissue sprouts from the optic nerve (usually within 10 mm of the edge), and from the surface of the retina in regions where tissue perfusion is poor. Initially the capillaries grow between the inner limiting membrane of the retina and the posterior surface of the vitreous. Eventually, the vessels grow into the vitreous and through the inner limiting membrane. As the vitreous contracts, traction is applied to the vessels, often resulting in shearing of the vessels and blinding of the vitreous due to hemorrhage. Fibrous traction from scarring in the retina may also produce retinal de:achment.

The conventional therapy of choice is panretinal photocoagulation to decrease retinal tissue, and thereby decrease retinal oxygen demands.

Although initially effective, there is a high relapse rate with new lesions forming in other parts of the retina. Complications of this therapy include a decrease in peripheral vision of up to 50% of patients, mechanical abrasions of the cornea, laser-induced cataract formation, acute glaucoma, and stimulation of subretinal neovascular growth (which can result in loss of vision). As a result, this procedure is performed only when several risk factors are present, and the riskbenefit ratio is clearly in favor of intervention.

Therefore, within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection of an anti-angiogenic factor(s) (or anti-angiogenic composition) into the aqueous humor or the vitreous, in order to increase the local concentration of antiangiogenic factor in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation. Within other embodiments of the invention, arteries which feed the neovascular lesions may be embolized (utilizing anti-angiogenic compositions, as described above) Within another aspect of the present invention, methods are provided for treating retrolental fibroblasia, comprising the step of administering a therapeutically effective amount of an anti-angiogenic factor (or anti-angiogenic composition) to the eye, such that the formation of blood vessels is inhibited.

Briefly, retrolental fibroblasia is a condition occurring in premature infants who receive oxygen therapy. The peripheral retinal vasculature, particularly on the temporal side, does not become fully formed until the end of fetal life. Excessive oxygen (even levels which would be physiologic at term) and the formation of oxygen free radicals are thought to be important by causing damage to the blood vessels of the immature retina.

These vessels constrict, and then become structurally obliterated on exposure to oxygen. As a result, the peripheral retina fails to vascularize and retinal ischemia ensues. In response to the ischemia, neovascularization is induced at the junction of the normal and the ischemic retina.

In 75% of the cases these vessels regress spontaneously.

However, in the remaining 25% there is continued capillary growth, contraction of the fibrovascular component. and traction on both the vessels and the. re'tina.

This results in vitreous hemorrhage and/or retinal detachment which can lead to blindness. Neovascular angle-closure glaucoma is also a complication of this condition.

As it is often impossible to determine which cases will spontaneously. resolve and which will progress in severity, co ventional treatment surgery) is generally initiated only in patients with established disease and a well developed pathology. This "wait and see" approach precludes early intervention, and allows the progression of disease in the 25% who follow a complicated course. Therefore, within one embodiment of the invention, topical administration of anti-angiogenic factors (or anti-angiogenic compositions, as described above) may be accomplished in infants which are at high risk for developing this condition in an attempt to cut down on the incidence of progression of retrolental fibroplasia. Within other embodiments, intravitreous injections and/or intraocular implants of an anti-angiogenic composition may be utilized. Such methods are particularly preferred in cases of established disease, in order to reduce the need for surgery.

Within another aspect of the present invention, methods are provided for treating rheumatoid arthritis, comprising the step of administering a therapeutically effective amount of an anti-angiogenic composition to a joint, such that the formation of blood vessels is inhibited.

Briefly, in rheumatoid arthritis articular damage is due to a combination of inflammation (including white blood cells and white blood cell products) and pannus tissue development (a tissue composed of neovascular tissue, connective tissue, and .inflammatory cells). Generally, chronic inflammation in itself is insufficient to result in damage to the joint surface, but a permanent deficit is created once fibrovascular tissue digests the cartilage tissue.

Within a preferred embodiment of the invention, anti-angiogenic factors (including anti-angiogenic compositions, as described above) rn-ry be administered by intra-articular injection, as a surgical paste, or as an oral agent contdining the angi-angiogenic factor thalidomide), in order to inhibit the formation of blood vessels within the joint. One representative example of such a method is set forth in more detail below in Example 19.

As noted above, within yet another aspect of the present invention, vascular grafts are provided comprising a synthetic tube, the surface of which is coated with an anti-angiogenic composition as described above.

Briefly, vascular gr'afts are synthetic tuhes. usually made of Dacron or Gortex, inserted surgically to bypass arterial hlockages. most frequently from the aorta to the femoral, or the femoral to the popliteal artery. A major problem which particularly complicates femoral-popliteal bypass grafts is the formation of a subendothelial scar-like reaction in the blood vessel wall called neointimal hyperplasia, which narrows the lumen within and adjacent to either end of the graft, and which can be progressive. A graft coated with or containing antiangiogenic factors (or anti-angiogenic compositions, as described above) may be utilized to limit the formation of neointimal hyperplasia at either end of the graft. The graft may then be surgically placed by conventional bypass techniques.

Anti-angiogenic compositions of the present invention may also be utilized in a variety of other manners. For example, they may be incorporated into surgical sutures in order to prevent stitch granulomas, implanted in the uterus (in the same manner as an IUD) for the treatment of menorrhagia or as a form of female birth control, administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis, attached to a monoclonal antibody directed against activated endothelial cells as a form of systemic chemotherapy, or utilized in diagnostic imaging when attached to a radioactively labelled monoclonal antibody which recognizes activated endothelial cells.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

EXAMPLE 1 PREPARATION OF A-SI-INVASIVE FACrOR The shoulder girdle and skull from a dogfish is excised, then scraped with a scalpel in order to remove all muscle and associated connective tissue from the cartilage. The cartilage is then homogenized with a tissue grinder, and extracted by continuous stirring at room temperature for 2 to 5 days in a solution containing 2.0 M guanidium hydrochloride and 0.02 M MES at pH After 2 to 5 days, the cartilage extract is passed through gauze netting in order to remove the larger constituents. The filtrate is then passed through an Amicon ultrafiltration unit which utilizes spiral-wound cartridges, with a molecular weight cutoff of 100,000. The filtrate (containing proteins with a molecular weight of less than 100,000 daltons) is then dialyzed against 0.02 M MES buffer (pH 6) with an Amicon ultrafiltration unit which retains proteins with a molecular weight of greater than 3,000 daltons. Utilizing this method, low molecular weight proteins and constituents are removed, as well as excessive amounts of guanidium HC1. The dialysate is concentrated to a final concentration 9 mg/ml.

EXAMPLE 2 ANALYSIS OF VARJOUS AGENTS FOR ATI-ALNGIOGENIc ACTIvrry A. Chick Chorioallantoic Membrane Assays Fertilized, domestic chick embryos were incubated for 3 days prior to shell-less culturing. In this procedure, the egg contents were emptied by removing the shell located around the air space. The interior shell membrane was then severed and the opposite end of the shell was perforated to allow the contents of the egg to gently slide out from the blunted end. The egg contents were emptied into round-bottom sterilized glass bowls and covered with petri dish covers. These were then placed into an incubator at 90% relative humidity and 3% CO-7 and incubated for 3 days.

Taxol (Sigma, St. Louis. MI) was mixed at concentrations of 1, 30ug per 10,ul aliquot of 0.5% aqueous methylcellulose. Since taxol is insoluble in water, glass beads were used to produce fine particles. Ten microliter aliquots of this solution were dried on parafilm for 1 hour forming disks 2mm in diameter. The dried disks containing taxol were then carefully placed at the growing edge of each CAM at day 6 of incubation. Controls were obtained by placing taxol-free methylcellulose disks on the CAMs over the same time course. After a 2 day exposure (day 8 of incubation) the vasculature was examined with the aid of a stereomicroscope. Liposyn II, a white opaque solution, was injected into the CAM to increase the visibility of the vascular details. The vasculature of unstained, living embryos were imaged using a Zeiss stereomicroscope which was interfaced with a video camera (Dage-MTI Inc., Michigan City, IN). These video signals were then displayed at 160 times magnification and captured using an image analysis system (Vidas, Kontron; Etching, Germany). Image negatives were then made on a graphics recorder (Model 3000; Matrix Instruments, Orangeburg,

NY).

The membranes of the 8 day-old shell-less embryo were flooded with 2% glutaraldehyde in 0.IM Na cacodylate buffer; additional fixative was injected under the CAM. After 10 minutes in situ, the CAM was removed and placed into fresh fixative for 2 hours at room temperature. The tissue was then washed overnight in cacodylate buffer containing 6% sucrose. The areas of interest were postfixed in 1% osmium tetroxide for 1.5 hours at 4 0 C. The tissues were then dehydrated in a graded series of ethanols, solvent exchanged with propylene oxide, and embedded in Spurr resin. Thin sections were cut with a diamond knife, placed on copper grids, stained, and examined in a Joel 1200EX electron microscope. Similarly, 0.5 mm sections were cut and stained with toluene blue for light microscopy.

At day 11 of development, chick embryos were used for the corrosion casting technique. Mercox resin (Ted Pella, Inc., Redding. CA) was injected into the CAM vasculature using a 30-gauge hypodermic needle. The casting material consisted of 2.5 grams of Mercox CL-2B polymer and 0.05 grams of catalyst (55% benzoyl peroxide) having a 5 minute polymerization time. After injection, the plastic was allowed to sit in situ for an hour at room temperature and then overnight in an oven at 65 0 C. The CAM was then placed in 50% aqueous solution of sodium hydroxide to digest all organic components.

The plastic casts were washed extensively in distilled water, air-dried, coated with gold/palladium, and viewed with the Philips 501B scanning electron microscope.

Results of the above experiments are shown in Figures 1-4.

Briefly, the general features of the normal chick shell-less egg culture are shown in Figure IA. At day 6 of incubation, the embryo is centrally positioned to a radially expanding network of blood vessels; the CAM develops adjacent to the embryo. These growing vessels lie close to the surface and are readily visible making this system an idealized model for the study of angiogenesis. Living, unstained capillary networks of the CAM can be imaged noninvasively with.a stereomicroscope. Figure 1B illustrates such a vascular area in which the cellular blood elements within capillaries were recorded with the use of a video/computer interface. The 3-dimensional architecture of such CAM capillary networks is shown by the corrosion casting method and viewed in the scanning electron microscope (Figure IC). These castings revealed underlying vessels which project toward the CAM surface where they form a single layer of anastomotic capillaries.

Transverse sections through the CAM show an outer ectoderm consisting of a double cell layer, a broader mesodermal layer containing capillaries which lie subjacent to the ectoderm, adventitial cells, and an inner, single endodermal cell layer (Figure ID). At the electron microscopic level, the typical structural details of the CAM capillaries are demonstrated. Typically, these vessels lie in close association with the inner cell layer of ectoderm (Figure IE) After 48 hours exposure to taxol at concentrations of 1, 5, 10, or 30,ug each CAM was examined under living conditions with a stereomicroscope equipped with a video/computer interface in order to evaluate the effects on angiogenesis. This imaging setup was used at a magnification of 160 times which permitted the direct visualization of blood cells within the capillaries; thereby blood flow in areas of interest could be easily assessed and recorded. For this study, the inhibition of angiogenesis was defined as an area of the CAM devoid of a capillary network ranging from 2 6 mm in diame-ter. Areas of inhibition lacked vascular blood flow and thus were only observed under experimental conditions of methylcellulose containing taxol; under control conditions of disks lacking taxol there was no effect on the developing capillary system. The dose-dependent, experimental data of the effects of taxol at different concentrations are shown in Table II.

TABLE II Anaiogenic Inhibition bvTaxol Taxol Concentration ig Embroys Evaluated Inhibition (Positive/Total) 31/31 100 16/21 76 5 18/25 72 1 6/15 Control 0/30 0 Typical taxol-treated CAMs (Figures 2A and 2B) are shown with the transparent methylcellulose disk centrally positioned over the avascular zone measuring 6 mm in diameter. At a slightly higher magnification, the periphery of such avascular zones is clearly evident (Figure 2C); the surrounding functional vessels were often redirected away from the source of taxol (Figures 2C and 2D). Such angular redirecting of blood flow was never observed under normal conditions. Another feature of the effects of taxol was the formation of blood islands within the avascular zone representing the aggregation of blood cells..

The associated morphological alterations of the taxo.l-treated CAM are readily apparent at both the light and electron microscopic levels. For the convenience of presentation, three distinct phases of general transition from the normal to the avascular state are shown. Near the periphery of the avascular zone the CAM is hallmarked by an abundance of mitotic cells within all three germ layers (Figures 3A and 4A). This enhanced mitotic division was also a consistent observation for capillary endothelial cells. However, the endothelial cells remained junctionally intact with no extravasation of blood cells. Wfth further degradation, the CAM is characterized by the breakdown and dissolution of capillaries (Figures 3B and 4B). The presumptive endothelial cells, typically arrested in mitosis, still maintain a close spatial relationship with blood cells and lie subjacent to the ectoderm: however, these cells are not junctionally linked. The most central portion of the avascular zone was characterized by a thickened ectodermal and endodermal layer (Figures 3C and 4C). Although these layers were thickened. the cellular junctions remained intact and the lavers maintained their structural characteristics. Within the mesoderm, scattered mitotically arrested cells were abundant; these cells did not exhibit the endothelial cell polarization observed in the former phase. Also, throughout this avascular region, degenerating cells were common as noted by the electron dense vacuoles and cellular debris (Figure 4C).

In summary, this study demonstrated that 48 hours after taxol application to the CAM, angiogenesis was inhibited. The blood vessel inhibition formed an avascular zone which was represented by three transitional phases of taxol's effect. The central, most affected area of the avascular zone contained disrupted capillaries with extravasated red blood cells; this indicated that intercellular junctions between endothelial cells were absent. The cells of the endoderm and ectoderm maintained their intercellular junctions and therefore.

these germ layers remained intact; however, they were slightly thickened. As the normal vascular area was aoproached, the blood vessels retained their junctional complexes and therefL e also remained intact. At the periphery of the taxol-treated zone, further blood vessel growth was inhibited which was evident by the typical redirecting or "elbowing" effect of the blood vessels (Figure 2D).

Taxol-treated avascular zones also revealed an abundance of cells arrested in mitosis in all three germ layers of the CAM; this was unique to taxol since no previous study has illustrated such an event. By being arrested in mitosis, endothelial cells could not undergo their normal metabolic functions involved in angiogenesis. In comparison, the avascular zone formed by suramin and cortisone acetate do not produce mitotically arrested cells in the CAM; they only prevented further blood vessel growth into the treated area. Therefore, even though agents are anti-angiogenic, there are many points in which the angiogenesis process may be targetted.

We also observed the effects of taxol over the 48 hour duration and noticed that inhibition of angiogenesis occurs as early as 9 hours after application. Histological sections revealed a similar morphology as seen in the first transition phase of the avascular zone at 48 hours illustrated in figure 3a and 4a. Also, we observed the revascularization process into the avascular zone previously observed. It has been found that the avascular zone formed by heparin and angiostatic steroids became revascularized 60 hours after application. In our study. taxol-treated avascular zones did not revascularize for at least 7 days after application implying a more potent long-term effect.

42 EXAMPLE 3 ENCAPSULATION OF SURAMIN One milliliter of 5% ELVAX (poly(ethylene-vinyl acetate) crosslinked with 5% vinyl acetate) in dichloromethane is mixed with a fixed weight of sub-micron ground sodium suramin. This mixture is injected into 5 ml of 5% Polyvinyl Alcohol in water in a 30 ml flat bottomed test rube.

Tubes containing different weights of the drug are then suspended in a multisample water bath at 400 for 90 minutes with automated stirring.. The mixes are removed, and microsphere samples taken for size analysis. Tubes are centrifuged at 10OOg for 5 min. The PVA supernatant is removed and saved for analysis (nonencapsulated drug). The microspheres are then washed (vortexed) in 5 ml of water and recentrifuged. The 5 ml wash is saved for analysis (surface bound drug). Microspheres are then wetted in 50 ul of methanol, and vortexed in I ml of DCM to dissolve the ELVAX. The microspheres are then warmed to and 5 ml of 50°C water is slowly added with stirring. This procedure results in the immediate evaporation of DCM, thereby causing the release of sodium suramin into the 5 ml of water. All three 5 ml samples were then assayed for drug content.

Sodium suramin absorbs uv/vis with a lambda max of 312nm.

The absorption is linear in the 0 to 100 ug/ml range in both water and 5% PVA.

The drug fluoresces strongly with an excitation maximum at 312nm, and emission maximum at 400nm. This fluorescence is quantifiable in the 0 to ug/ml range.

Results are shown in Figures 5-10. Briefly, the size distribution of microspheres appears to be unaffected by inclusion of the drug in the DCM (see Figures 5 and Good yields of microspheres in the 20 to 60 Am range may be obtained.

The encapsulation of suramin is very low (see Figure 8).

However as the weight of drug is increased in the DCM the total amount of drug encapsulated increased although the encapsulation decreased. As is shown in Figure 7. 50ug of drug may be encapsulated in 50 mg of ELVAX.

Encapsulation of sodium suramin in 5% PVA containing 10% NaCl is shown in Figures 9-10.

EXAMPLE 4 ENCAPsLLATnoN OF TAXOL Five hundred micrograms of either taxol or baccatin (a taxol analog, available from Inflazyme Pharmaceuticals Inc., Vancouver, British Columbia, Canada) are dissolved in 1 ml of a 50:50 ELVAX:poly-l-lactic acid mixture ini DCM. Microspheres are then prepared in a dissolution machine (Sixspindle dissolutior, tester, VanderKanp, Van Kel Industries Inc., in triplicate at 200 rpm, 42 0 C, for 3 hours. Microspheres so prepared are washed twice in water and sized on the microscope.

Determination of taxol encapsulation is undertaken in a uv/vis assay (uv/vis lamda max. at 237 nm, fluorescence assay at excitation 237, emission at 325 nm; Fluorescence results are presented in square brackets Utilizing the procedures described above, 58 ug kg) [75 gg of taxol may be encapsulated from a total 500 gg of starting material. This represents 12% [15% of the original weight, or 1.2% 0.25%) by weight of the polymer. After 18 hours of tumbling in an oven at 37 0 C, 10.3% of the total taxol had been released from the microspheres.

For baccatin, 100 gg [83 +/-23gg] of baccatin c. )e encapsulated from a total of 500 gg starting material. This represents. a [17% of the original weight of baccatin, and 2% by weight of the polymer. After 18 hours of tumbling in an oven at 37 0 C, 55% [60% of the baccatin is released from the microspheres.

EXAMPLE ANALYSIS OF SuRGICAL PASTE CONTAINING A.\T-ANGIOGENIC COMPOSmONS Fisher rats weighing approximately 300 grams are anesthetized, and a 1 cm transverse upper ahdominal incision is made. Two-tenths of a milliliter of saline containing I x 106 live 9L gliosarcoma cells (eluted immediately prior to use from tissue culture) are injected into 2 of the 5 hepatic lobes by piercing a 27 gauge needle I cm through the liver capsule. The abdominal wound is closed with 6.0 resorprible suture and skin clips and the GA terminated.

After 2 weeks, the tumor deposits will measure approximately 1 cm. At this time, both hepatic tumors are resected and the bare margin of the liver is packed with a hemostatic agent. The rats are divided into two groups: half is administered polymeric carrier alone, and the other half receives an antiangiogenic composition.

Rats are sacrificed 2, 7, 14, 21 and 84 days post hepatic resection.

In particular, the rats are euthanized by injecting Euthanyl into the dorsal vein of the tail. The liver, spleen, and both lungs are removed, and histologic analysis is performed in order to study the tumors for evidence of antiangiogenic activity.

EXAMPLE 6 EMBOLIZAIO OF RAT ARTEIES Fisher rats weighing approximately 300 grams are anesthetized.

Utilizing aseptic procedures, a 1 cm transverse upper abdominal incision is made, and the liver identified. Two-tenths of a milliliter of saline containing I million live 9L gliosarcoma ceils (eluted immediately prior from tissue culture) is injected into each of the 5 hepatic lobes by piercing a 27 gauge needle 1 cm through the liver capsule. One-tenth of a milliliter of normal saline is injected into the needle as it is withdrawn to ensure that there is no spillage of cells into the peritoneal cavity. A pledget of gelfoam is placed on each of the puncture sites to ensure hemostasis. The abdominal 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 standard diet for 14 days, at which time each tumor deposit will measure 1 cm in diameter. The same procedure is repeated using Westar rats and a Colon Cancer cell line (Radiologic Oncology Lab, M.D.

Anderson, Houston, Texas). In this instance. 3 weeks are required postinjection for the tumor deposits to measure 1 cm in diameter each.

After 2 or 3 weeks, depending on the rat species, the same general anesthetic procedure is followed and a midline abdominal incision is performed.

The duodenum is flipped and the gastroduodenal artery is identified and mobilized. Ties are placed above and below a cutdown site on the midportion of the gastroduodenal artery (GDA), and 0.03S inch polyethylene tubing is introduced in a retrograde fashion into the artery using an operating microscope. The tie below the insertion point will ligate the artery, while the one above will fix the catheter in place. Angiography is performed by injecting ml of 60% radiopaque contrast material through the catheter as an x-ray is taken. The hepatic artery is then embolized by refluxing particles measuring 200 pm through the gastroduodenal artery catheter until flow, observed via the operating microscope, is seen to cease for at least 30 seconds. Occlusion of the hepatic artery is confirmed by repeating an angiogram through the GDA catheter. Utilizing this procedure, one-half of the rats receive 15-200 pm particles of polymer alone, and the other half receive 15-200 pm particles of the polymer-anti-angiogenic factor composition. The upper GDA ligature is tightened to occlude the GDA as the catheter is withdrawn to ensure hemostasis, and the hepatic artery (although embolized) is left intact. The abdomen is closed with 6.0 absorbable suture and surgical clips.

The rats are subsequently sacrificed at 2. 7, 14, 21 and 84 days post-embolization in order to determine efficacy of the anti-angiogenic factor.

Briefly, general anesthetic is given, and utilizing aseptic precautions, a midline incision performed. The GDA is mobilized again, and after'placing a ligature near the junction of the GDA and the hepatic artery well above the site of the previous cutdown), a 0.038-inch polyethylene tubing is inserted via cutdown of the vessel and angiography is performed. The rat is then euthanized by injecting Euthanyl into the dorsal vein of the tail. Once euthanasia is confirmed, the liver is removed en bloc along with the stomach, spleen and both'lungs.

Histologic analysis is performed on a prepared slide stained with hematoxylin and eosin and stain. Briefly, the lungs are sectioned at 1 cm intervals to assess passage of embolic material through the hepatic veins and into the right side of circulation. The stomach and spleen are also sectioned in order to assess inadvertent immobilization from reflux of particles into the celiac access of the collateral circulation.

EXAMPLE 7 TI<A,NsI'LA.Vr,\no.x BILIARY STENT.S IN RATS General anesthetic is administered to 300 gram Fisher rats. A 1 cm transverse incision is then made in the upper abdomen, and the liver identified. In the most superficial lobe. 0.2 ml of saline containing I million cells of 9L gliosarcoma cells (eluted from tissue culture immediately prior to use) is injected via a 27 gauge needle to a depth of 1 crr into the liver capsule.

Hemostasis is achieved, after removal of the needle by placing a pledget of gelfoam at the puncture sites. Saline is injected as the needle is removed to ensure no spillage of cells into the peritoneal cavity or along the needle track.

The general anesthetic is terminated, and the animal returned to the animal care center and placed on a normal diet.

Two weeks later, general anesthetic is administered, and utilizing aseptic precautions, the hepatic lobe containing the tumor is identified through a midline incision. A 16 gauge angiographic needle is then inserted through the hepatic capsule into the tumor, a 0.038-irich guidewire passed through the needle, and the needle withdrawn over the guidewire. A number 5 French dilator is passed over the guide into the tumor and withdrawn. A number French delivery catheter is then passed over the wire containing a selfexpanding stainless steel Wallstent (5 mm in diameter and 1 cm long). The stent is deployed into the tumor and the guidewire delivery catheter is removed.

One-third of the rats have a conventional stainless steel stent inserted into the tumor, one-third a stainless steel stent coated with polymer, and one third a stent coated with the polymer-anti-angiogenic factor compound. The general anesthetic is terminated and the rat returned to the animal care facility.

A plain abdominal X-ray is performed at 2 days in order to assess the degree of stent opening. Rats are sacrificed at 2, 7, 14, 28 and 56 days poststent insertion by injecting Euthaniyl, and their livers removed en bloc once euthanasia is confirmed. After fixation in formaldehyde for 48 hours, the liver is' sectioned at 0.5 mm intervals; including severing the stent transversely using a fresh blade for each slice. Histologic sections stained with H and E are then analyzed to assess the degree of tumor ingrowth into the stent lumen.

EXAMPLE 8 MANLF.-CTURE OF MICROSPHERES Equipment which is preferred for the manufacture of microspheres described below include: 200 ml water jacketed beaker (Kimax or Pyrex), Haake circulating water bath. overhead stirrer and controller with 2 inch diameter (4 hblde. propeller cype stainless steel stirrer Fisher brand), 500 ml glass beaker. hot plate/stirrer (Corning brand), 4 X 50 ml polypropylene centrifuge tubes (Nalgene), glass scintillation vials with plastic insert caps, table top centrifuge (GPR Beckman), high speed centrifuge- floor model (JS 21 Beckman), Mettler analytical balr-ce (AJ 100, 0.1 mg), Mettler digital top loading balance (AE 163, .0.01 rr,, automatic pipetter (Gilson). Reagents include Polycaprolactone ("PCL" mol wt 10,000 to 20,000; Polysciences, Warrington Pennsylvania, USA), "washed" Ethylene Vinyl Acetate ("EVA" washed so as to remove the anti-oxidant BHT), Poly(DL)Iactic acid ("PLA" mol wt 15,000 to 25,000; Polysciences), Polyvinyl Alcohol ("PVA" mol wt 124,000 to 186,000; 99% hydrolyzed; Aldrich Chemical Co., Milwaukee WI, USA), Dichloromethane ("DCM" or "methylene chloride"; HPLC grade Fisher scientific), and distilled water.

A. Preparation of 5c% Polymer Solutinns Depending on the polymer solution being prepared, 1.00 g of PCL or PLA, or 0.50 g each of PLA and washed EVA is weighed directly into a 20 ml glass scintillation vial. Twenty milliliters of DCM is then added, and the vial tightly capped. The vial is stored a: om temperature (25°C) for one hour (occasional shaking may be used), or until all the polymer has dissolved (the solution should be clear). The solution may be stored at room temperature for at least two weeks.

B. Preparation of 5% Stock Solution of PVA Twenty-five grams of PVA is weighed directly into a 600 ml glass beaker. Five hundred milliliters of distilled. water is added, along with a 3 inch Teflon coated stir bar. The beaker is covered with glass to decrease evaporation losses, and placed into a 2000 ml glass beaker containing 300 ml of water (which acts as a water bath). The PVA is stirred at 300 rpm at 85°C (Coming hot plate/stirrer) for 2 hours or until fully dissolved. Dissolution of the PVA may be determined by a visual cE the solution sh,.uld be clear. The solution is then transferred to a glass screw top storage container and stored at 4°C for a maximum of two months. The solution, however should be warmed to room temperature before use or dilution.

C. Procedure for Producing Microspheres Based on the size of microspheres being made (see Table 100 ml of the PVA solution (concentrations given in Table III) is placed into the 200 ml water jacketed beaker. Haake circulating water bath is connected to this beaker and the contents are allowed to equilibrate at 27.C 0 C) for minutes. Based on the size of microspheres being made (see Table III), the start speed of the overhead stirrer is set, and the blade of the overhead stirrer placed half way down in the PVA solution. The stirrer is then started, and 10 mi of polymer solution (polymer solution used based on type of microspheres being produced) is then dripped into the stirring PVA over a period of 2 minutes using a 5 ml automatic pipetter. After 3 minutes the stir speed is adjusted (see Table III), and the solution stirred for an additional 2.5 hours. The stirring blade is then removed from the microsphere preparation, and rinsed with 10 ml of distilled water so that the rinse solution drains into the microsphere preparation. The microsphere preparation is then poured into a 500 ml beaker, and the jacketed water bath washed with 70 ml of distilled water, which is also allowed to drain into the microsphere preparation. The 180 ml microsphere preparation is then stirred with a glass rod, and equal amounts are poured into four polypropylene 50 ml centrifuge tubes. The tubes are then capped, and centrifuged for 10 minutes (force given in Table A 5 ml automatic pipetter or vacuum suction is then utilized to draw 45 ml of the PVA solution off of each microsphere pellet.

TABLE III PVA concentrations, stir speeds, and centrifugal force requirements for each diameter range of microspheres.

PRODUCTION MICROSPHERE DIAMETER RANGES STAGE 30 um to 100 am 10 um to 30 am I 0.1 um to 3 um PVA 2.5% 5% 3.5% (Le., Concentration dilute 5% stock undiluted stock) dilute 5% stock with distilled wate with distilled watei Starting Stir 500 rpm 500 rpm 3000 rpm Speed rpm rpm 200 rpm Adjusted Stir 500 rpm 500 rpm 2500 rpm Speed rpm 50 rpm 200 rpm Centrifuge Force 1000 g 1000 g 10 000 g 100 g 100 g 1000 g (Table top model) (Table top model) (High speed model)

I

Five milliliters of distilled water is then added to each centrifuge tube, which is then vortexed to resuspend the microspheres. The four microsphere suspensions are then pooled into one centrifuge tube along with ml of distilled water, and centrifuged for another 10 minutes (force given in Table This process is repeated two additional times for a total of three washes. The microspheres are then centrifuged a final time, and resuspended in 10 ml of distilled water. After the final wash, the microsphere preparation is transferred into a preweighed glass scintillation vial. The vial is capped, and left overnight at room temperature (25 0 C) in order to allow the microspheres to sediment out under gravity. Microspheres which fall in the size range of 0.1 um to 3 um do not sediment out under gravity, so they are left in the 10 ml suspension.

D. Drving of 10 am to 30 mrn or 30 um to 100 arr Diameter Micrnspheres After the microspheres have sat at room temperature overnight, a 5 ml automatic pipetter or vacuum suction is used to draw the supernatant off of the sedimented microspheres. The microspheres are allowed to dry in the uncapped vial in a drawer for a period of one week or until they are fully dry (vial at constant weight). Faster drying may be accomplished by leaving the uncapped vial under a slow stream of nitrogen gas (flow approx. 10 ml/min.) in the fume hood. When fully dry (vial at constant weight), the vial is weighed and capped. The labelled, capped vial is stored at room temperature in a drawer.

Microspheres are normally stored no longer than 3 months.

E. Drving of 0. 1 urn t 3 urn Diameter Micrnspheres This size range of microspheres will not sediment out, so they are left in suspension at 4°C for a maximum of four weeks. To determine the concentration of microspheres in the 10 ml suspension, a 200 l sample of the suspension is pipetted into a 1.5 ml preweighed microfuge tube. The tube- is then centrifuged at 10,000 g (Eppendorf table top microfuge), the supernatant removed, and the tube allowed to dry at 50°C overnight. The tube is then reweighed in order to determine the weight of dried microspheres within the tube.

F. Manufacture nfTaxnl Loaded Micrsphere In order to prepare taxol containing microspheres, an appropriate amount of weighed taxol (based upon the percentage of taxol to be encapsulated) is placed directly into a 20 ml glass scintillation vial. Ten milliliters of an appropriate polymer solution is then added to the vial containing the taxol, which is then vortexed until the taxol has dissolved.

Microspheres containing taxol may then be produced essentially as described above in steps through 51 EXAMPLE 9 MANLFACTLRE OF STE.T COATING Reagents and equipment which are utilized within the following experim. "s include (medical grade stents obtained commercially from a variety of manutacturers; the "Strecker" stent) and holding apparatus, 20 ml glass scintillation vial with cap (plastic insert type), TLC atomizer, Nitrogen gas tank, glass test tubes (various sizes from 1 ml and up), glass beakers (various sizes), Pasteur pipette, tweezers, Polycaprolactone ("PCL" mol wt 10,000 to 20,000; Polysciences), Taxol (Sigma Chemical Co., St. Louis, Mo., 95% purity), Ethylene vinyl acetate ("EVA" wa- d see previous), Poly(DL)lactic acid ("PL4" mol wt 15,000 to 25,000; Polyciences). dichloromethane ("DCM" HPLC grade, Fisher Scientific).

A. Procedure for Soraved Stents The following describes a typical method using a 3 mm crimped diameter interleaving metal wire stent of approximately 3 cm length. For larger diameter stents, larger volumes of polymer/drug solution are used.

Weigh sufficient polymer directly into a 20 ml glass scintillation vial and add sufficient DCM to achieve a 2% w/v solution. Cap the vial and mix the solution to dissolve the polymer (hand shaking). Assemble the stent in a vertical orientation. This can be accomplished using a piece of nylon and tying the stent to a retort stand. Position this stent holding apparatus 6 to 12 inches above the fume hood floor on a suitable support inverted 2000 ml glass beaker) to enable horizontal spraying. Using an automatic pipette, transfer a suitable volume (minimum 5 ml) of the 2% polymer solution to a separate 20 ml glass scintillation vial. Add an appropriate amount of taxol to the solution and dissolve it by hand shaking the capped vial.

To prepare f spraying, remove the cap of this vial and dip the barrel (only) of an TLC atomizer into the polymer solution. r')te that the reservoir of the atomizer need not be used in this procedure: th.; 20 ml glass vial acts as a reservoir. Connect the nitrogen tank to the gas inlet of the atomizer. Gradually increase the pressure until atomization and spraying begins. Note the pressure and use this pressure throughout the procedure. To spray the stent use 5 second oscillating sprays with a 15 second dry time between sprays. After 5 sprays. rotate the stent 90° and spray that portion of the stent.

Repeat until all sides of the scent have been sprayed. During the drv time, finger crimp the gas line to avoid wastage of the spray. Spraying is continued until a suitable amount of polymer is deposited on the scents. The amount may be based on the specific stent application in vivo. To determine the amount, weigh the stent after spraying has been completed and the stent has dried.

Subtract the original weight of the stent from the finished weight and this produces the amount of polymer (plus taxol) applied to the stent. Store the coated stent in a sealed container.

B. Procedure for Dipped Stents The following describes a typical method using a 3 mm crimped diameter interleaving metal wire stent of approximately 3 cm length. For larger diameter stents, larger volumes of polymer/drug solution are used in larger sized test tubes.

Weigh 2 g of EVA into a 20 ml glass scintillation vial and add ml of DCM. Cap the vial and leave it for 2 hours to dissolve (hand shake the vial frequently to assist the dissolving process). Weigh a known weight of taxol directly into a 1 ml glass test tube and add 0.5 ml of the polymer solution. Using a glass Pasteur pipette, dissolve the taxol by gently pumping the polymer solution. Once the taxol is dissolved, hold the test tube in a near horizontal position (the sticky polymer solution will not flow out). Using tweezers, insert the stent into the tube all the way to the bottom. Allow the polymer solution to flow almost to the mouth of the test tube by angling the mouth below horizontal and then restoring the test tube to an angle slightly above the horizontal. While slowly rotating the stent in the tube, slowly remove the stent (approximately seconds).

Hold the stent in a vertical position to dry. Some of the sealed perforations may pop so that a hole exists in the continuous sheet of polymer.

This may be remedied by repeating the previous dipping procedure, however repetition of the procedure can also lead to further popping and a general uneven build up of polymer. Generally, it is better to dip the s:ent just once and to cut out a section of stent that has no popped perforations. Store the dipped stent in a sealed container.

EXAMPLE ,MANUFACTUR OF SURGICAL "PASTES" As noted above, the present invention provides a variety of polymeric-containing drug compositions that may be utilized within a variety of clinical situations. For example, compositions may be produced: as a "thermopaste" that is applied to a desired site as a fluid, and hardens to a solid of the desired shape at a specified temperature body temperature); as a spray "nanospray") which may delivered to a desired site either directly or through a specialized apparatus endoscopy), and which subsequently hardens to a solid which adheres to the tissue to which it is applied; as an adherent, pliable, resilient, angiogeneis inhibitor-polymer film applied to a desired site either directly or through a specialized apparatus, and which preferably adheres to the site to which it is applied: and as a fluid composed of a suspension of microspheres in an appropriate carrier medium, which is applied to a desired site either directly or via a specialized apparatus, and which leaves a layer of microspheres at the application site.. Representative examples of each of the above embodiments is set forth in more detail below.

A. Procedure for Producing Thermnpaste Reagents and equipment which are utilized within the following experiments include a sterile glass syringe (1 ml). Corning hot plate/stirrer, ml glass scintillation vial, moulds 50 kil DSC pan or 50 ml centrifuge tube cap inner portion), scalpel and tweezers, Polycaprolactone ("PCL" mol wt 10,000 to 20,000; Polysciences, Warrington, Pennsylvania USA), and Taxol (Sigma grade 95% purity minimum).

Weigh 5.00 g of polycaprolactone directly into a 20 ml glass scintillation vial. Place the vial in a 600 ml beaker containing 50 ml of water.

Gently heat the beaker to 65°C and hold it at that temperature for 20 minutes.

This allows the polymer to melt. Thoroughly mix a known weight of taxol, or other angiogenesis inhibitor into the melted polymer at 65°C. Pour the melted polymer into a prewarmed (60C oven) mould. Use a spatula to assist with the pouring process. Allow the mould to cool so the polymer solidifies. Cut or break the polymer into small pieces (approximately 2 mm by 2 mm in size).

These pieces must fit into a 1 ml glass syringe. Remove the plunger from the 1 ml glass syringe (do not remove the cap from the tip) and place it on a balance.

Zero the balance.

Weigh 0.5 g of the pieces directly into the open end of the syringe.

Place the glass syringe upright (capped rtp downwards) into a 500 ml glass beaker containing distilled water at 65°C (corning hot plate) so that no water enters the barrel. The polymer melts completely within 10 minutes in this apparatus. When the polymer pieces have melted, remove the barrel from the water bath, hold it horizontally and remove the cap. Insert the plunger into the barrel and compress the melted polymer into a sticky mass at the tip end of the barrel. Cap the syringe and allow it to cool to room temperature.

For application, the syringe may be reheated to 60°C and administered as a liquid which solidifies when cooled to body temperature.

B. Procedure fnr Prnducing Nanosprav Nanospray is a suspension of small microspheres in saline. If the microspheres are very small under 1 pm in diameter) they form a colloid so that the suspension will not sediment under gravity. As is described in more detail below, a suspension of 0.1 im to 1 gm microparticles may be created suitable for deposition onto tissue through a finger pumped aerosol. Equipment and materials which may be utilized to produce nanospray include 200 ml water jacketed beaker (Kimax or Pyrex), Haake circulating water bath, overhead stirrer and controller with 2 inch diameter (4 blade, propeller type stainless steel stirrer; Fisher brand), 500 ml glass beaker, hot plate/stirrer (Corning brand), 4 X 50 ml polypropylene centrifuge tubes (Nalgene), glass scintillation vials with plastic insert caps, table top centrifuge (Beckman), high speed centrifuge floor model (JS 21 Beckman), Mettler analytical balance (AJ 100, 0.1 mg), Mettler digital top loading balance (AE 163, 0.01 mg), automatic pipetter (Gilson), sterile pipette tips, pump action aerosol (Pfeiffer pharmaceuticals) 20 ml, laminar flow hood, Polycaprolactone ("PCL" mol wt 10,000 to 20,000; Polysciences, Warrington, Pennsylvania USA), "washed" (see previous) Ethylene Vinyl Acetate Poly(DL)Iactic acid ("PLA" mol wt 15,000 to 25,000; Polysciences), Polyvinyl Alcohol ("PVA" mol wt 124,000 to 186,000; 99% hydrolyzed; Aldrich Chemical Co., Milwaukee, W1 USA), Dichloromethane ("DCM" or "methylene chloride:" HPLC grade Fisher scientific). Distilled water.

sterile saline (Becton and Dickenson or equivalent) 1. Preparation of 5% Polymer Solutions Depending on the polymer solution being prepared, weigh 1.00 g of PCL c: PLA or 0.50 g each of PLA and washed EVA directly into a 20 ml glass scintillation vial. Using a measuring cylinder, add 20 ml of DCM and tightly cap the vial. Leave the vial at room temperature (25°C) for one hour or until all the polymer has dissolved (occasional hand shaking may be used).

Dissolving of the polymer can be determined by a visual check; the solution should be clear. Label the vial with the name of the solution and the date it was produced. Store the solutions at room temperature and use within two weeks.

2. Preparation of 3.5% Stock Solution of PVA The solution can be prepared by following the procedure given below, or by diluting the PVA stock solution prepared for production of microspheres (see Example Briefly. 17.5 g of PVA is weighed directly into a 600 ml glass beaker, and 500 ml of distilled water is added. Place a 3 inch teflon coated stir bar in the beaker. Cover the beaker with a cover glass to reduce evaporation losses. Place the beaker in a 2000 ml glass beaker containing 300 ml of water. This will act as a water bath. Stir the PVA a' 300 rpm at 85°C (Corning hot plate/stirrer) for 2 hours or until fully dissolved.

Dissolving of the PVA can be determined by a visual check; the solution should be clear. Use a pipette to transfer the solution to a glass screw top storage container and store at 4 0 C for a maximum of two months. This solution-should be warmed to room temperature before use or dilution.

3. Procedure for Producing Nanospray Place the stirring assembly in a fume hood. Place 100 ml of the PVA solution in the 200 ml water jacketed beaker. Connect the Haake water bath to this beaker and allow the contents to equilibrate at 27°C for 10 minutes. Set the start speed of the overhead stirrer at 3000 rpm 200 rpm). Place the blade of the overhead stirrer half. way down in the PVA solution and start the stirrer. Drip 10 ml of polymer solution (polymer solution used based on type of nanospray being produced) into the stirring PVA over a period of 2 minutes using a 5 ml automatic pipetter. Afte- 3 minutes, adjust the stir speed to 2500 rpm 200 rpm) and leave the assembly for 2.5 hours.

After 2.5 hours. remove the stirring blade from the nanospray preparation and rinse with 10 ml of distilled water. Allow the rinse solution to go into the nanospray preparation.

56 Pour the microsphere preparation into a 500 ml beaker. Wash the jacketed water bath with 70 ml of distilled water. Allow the 70 ml rinse solution to go into the microsphere preparation. Stir the 180 ml microsphere preparation with a glass rod and pour equal amounts of it into four polypropylene 50 ml centrifuge tubes. Cap the tubes. Centrifuge the capped tubes at 10 000 g 1000 g) for 10 minutes. Using a 5 ml automatic pipetter or vacuum suction, draw 45 ml of the PVA solution off of each microsphere pellet and discard it. Add 5 ml of distilled water to each centrifuge tube and use a vortex to resuspend the microspheres in each tube. Using 20 ml of distilled water, pool the four microsphere suspensions into one centrifuge tube. To wash the microspheres, centrifuge the nanospray preparation for 10 minutes at 10 000 g 1000 Draw the supernatant off of the microsphere pellet. Add 40 ml of distilled water and use a vortex to resuspend the microspheres. Repeat this process two more times for a total of three washes. Do a fourth wash but use only 10 ml (not 40 ml) of distilled water when resuspending the microspheres.

After the fourth wash, transfer the microsphere preparation into a preweighed glass scintillation vial.

Cap the vial and let it to sit for 1 hour at room temperature to allow the 2 gm and 3 gm diameter microspheres to sediment out under gravity. After 1 hour, draw off the top 9 ml of suspension using a 5 ml automatic pipetter. Place the 9 ml into a sterile capped 50 ml centrifuge tube. Centrifuge the suspension at 10 000 g 1000 g) for 10 minutes. Discard the supernatant and resuspend the pellet in 20 ml-of sterile saline. Centrifuge the.suspension at 000 g 1000 g) for 10 minutes. Discard the supernatant and resuspend the pellet in sterile saline. The quantity of saline used is dependent on the final required suspension concentration (usually 10% Thoroughly rinse the aerosol apparatus in sterile saline and add the nanospray suspension to the aerosol.

C. Manufacture of Taxol Loaded Nnosprav To manufacture nanospray containing taxnl, use Taxol (Sigma grade 95% purity). To prepare the polymer drug stock solution, weigh the appropriate amount of taxol directly into a 20 ml glass scintillation vial. The appropriate amount is determined based on the percentage of taxol to be in the nanospray. For example, if nanospray containing 5% taxol was required. then the amount of taxol weighed would be 25 mg since the amount of polymer added is 10 ml of a 5% polymer in DCM solution (see next step).

Add 10 ml of the appropriate 5% polymer solution to the vial containing the taxol. Cap the vial and vortex or.hand swirl it to dissolve the taxol (visual check to ensure taxol dissolved). Label the vial with the date it was produced. This is to be used the day it is produced.

Follow the procedures as described above, except that polymer/drug taxol) stock solution is substituted for the polymer solution.

D. Procedure for Producing Film The term film refers to a polymer formed into one of many geometric shapes. The film may he a thin, elastic sheet of polymer or a 2 mm thick disc of polymer. This film is designed to be placed on exposed tissue so that any encapsulated drug is released from the polymer over a long period of time at the tissue site. Films may be made by several processes, including for example, by casting, and by spraying.

In the casting technique, polymer is either melted and poured into a shape or dissolved in dichloromethane and poured into a shape. The polymer then either solidifies as it cools or solidifies as the solvent evaporates, respectively. In the spraying technique, the polymer is dissolved in solvent and sprayed onto glass, as the solvent evaporates the polymer solidifies on the glass.

Repeated spraying enables a build up of polymer into a film that can be peeled from the glass.

Reagents and equipment which were utilized within these experiments -clude a small beaker, Corning hot plate stirrer, casting moulds 50 ml .ntrifuge tube caps) and mould holding apparatus, 20 ml glass scintillation vial with cap (Plastic insert type), TLC atomizer, Nitrogen gas tank, Polycaprolactone ("PCL" mol wt 10,000 to 20.000; Polysciences), Taxol (Sigma 95% purity), Ethanol, "washed" (see previous) Ethylene vinyl acetate Poly(DL)lactic acid "V mol wt 15,000 to 25,000; Polysciences), Dichloromethane (HPLC grade F,.ier Scientific).

1. Procedure for Producing Films Melt Casting Weigh a known weight of PCL directly into a small glass beaker.

Place the beaker in a larger beaker containing water (to act as a water bath) and put it on the hot plate at 70()°C for 15 minutes or until the polymer has fully melted. Add a known weight of drug to the melted polymer and stir the mixture thoroughly. To aid dispersion of the drug in the melted PCL, the drug may be suspended/dissolved in a small volume 10% of the volume of the melted PCL) of 100% ethanol. This ethanol suspension is then mixed into the melted polymer. Pour the melted polymer into a mould and let it to cool. After cooling, store the film in a container.

2. Procedure for Producing Films Solvent Casting Weigh a known weight of PCL directly into a 20 ml glass scintillation vial and add sufficient DCM to achieve a 10% w/v solution. Cap the vial and mix the solution. Add sufficient taxol to the solution to achieve the desired final taxol concentration. Use hand shaking or vortexing to dissolve the taxol in the solution. Let the solution sit for one hour (to diminish the presence of air bubbles) and then pour it slowly into a mould. The mould used is based on the shape required. Place the mould in the fume hood overnight.

This will allow the DCM to evaporate. Either leave the film in the mould to store it or peel it out and store it in a sealed container.

3. Procedure for Producing Films Sprayed Weigh sufficient polymer directly into a 20 ml glass scintillation vial and add sufficient DCM to achieve a 2% w/v solution. Cap the vial and mix the solution to dissolve the polymer (hand shaking). Assemble the moulds in a vertical orientation in a suitable mould holding apparatus in the fume hood.

Position this mould holding apparatus 6 to 12 inches above the fume hood floor on a suitable support inverted 2000 ml glass beaker) to enable horizontal spraying. Using an automatic pipette, transfer a suitable volume (minimum ml) of the 2% polymer solution to a separate 20 ml glass scintillation-vial. Add sufficient taxol to the solution and dissolve it by hand shaking the capped vial.

To prepare for spraying, remove the cap of this vial and dip the barrel (only) of an TLC atomizer into the polymer solution. Note: the reservoir of the atomizer is not used in this procedure the 20 ml glass vial acts as a reservoir.

Connect the nitrogen tank to the gas inlet of the atomizer.

Gradually increase the pressure until atomization and spraying begins. Note the pressure and use this pressure throughout the procedure. To spray the moulds use 5 second oscillating sprays with a 15 second dry time between sprays.

During the dry time, finger crimp the gas line to avoid wastage of the spray.

Spraying is continued until a suitable thickness of polymer is deposited on the mould. The thickness is based on the request. Leave the sprayed films attached to the moulds and store in sealed containers.

E. Procedure for Producing Nanopaste Nanopaste is a suspension of microspheres suspended in a hydrophilic gel. Within one aspect of the invention, the gel or paste can be smeared over tissue as a method of locating drug loaded microspheres close to the target tissue. Being water, based, the paste will soon become diluted with bodily fluids causing a decrease in the stickiness of the paste and a tendency of the microspheres to be deposited on nearby tissue. A pool of microsphere encapsulated drug is thereby located close to the target tissue.

Reagents and equipment which were utilized within these experiments include glass beakers, Carbopol 925 (pharmaceutical grade, Goodyear Chemical distilled water, sodium hydroxide (1 M) in water solution, sodium hydroxide solution (5 M) in water solution, microspheres in the 0.1 Am to 3 Am size range suspended in water at 20% w/v (See previous).

1. Preparation of 5% w/v Carbopol Gel Add a sufficient amount of carbopol to 1 M sodium hydroxide to achieve a 5% w/v solution. To dissolve the carbopol in the 1 M sodium hydroxide, allow the mixture to sit for approximately one hour. During this time period, stir the mixture using a glass rod. After one hour, take the pH of the mixture. A low pH indicates that the carbopol is not fully dissolved. The pH you want to achieve is 7.4. Use 5 M sodium hydroxide to adjust the pH. This is accomplished by slowly adding drops of 5 M sodium hydroxide to the mixture, stirring the mixture and taking the pH of the mixture. It usually takes approximately one hour to adjust the pH to 7.4. Once a pH of 7.4 is achieved, cover the gel and let it sit for 2 to 3 hours. After this time period, check the pH to ensure -it is still at 7.4. If it has changed, adjust back to pH 7.4 using 5 M sodium hydroxide. Allow the gel to sit for a few hours to ensure the pH is stable at 7.4. Repeat the process until the desired pH is achieved and is stable. Label the container with the name of the gel and the date. The gel is to be used to make nanopaste within the next week.

2. Procedure for Producing Nanopaste Add sufficient 0.1 am to 3 /m microspheres to water to produce a suspension of the microspheres. Put 8 ml of the 5% w/v carbopol gel in a glass beaker. Add 2 ml of the 20% microsphere suspension to the beaker.

Using a glass rod or a mixing spatula, stir the mixture to thoroughly disperse the microspheres throughout the gel. This usually takes 30 minutes. Once the microspheres are dispersed in the gel, place the mixture in a storage jar. Store the jar at 4"C. It must be used within a one month period.

EXAMPLE 11 CONTROLLED DELIVERY OF TAXOL FROM MICROSPHERES COMPOSED OF A BLEND OF ETHYLENE-VINYL-ACETATE COPOLYMER AND POLY (D,L LACTIC ACID). IN Vivo TESTING OF THE MICROSPHERES ON THE CAM ASSAY This example describes the preparation of taxol-loaded microspheres composed of a blend of biodegradable poly (d,l-lactic acid) (PLA) polymer and nondegradable ethylene-vinyl acetate (EVA) copolymer. In addition, the in vitro release rate and anti-angiogenic activity of taxol released from microspheres placed on a CAM are demonstrated.

Reagents which were utilized in these experiments include taxol, which is purchased from Sigma Chemical Co. (St. Louis, MO); PLA (molecular weight 15,000-25,000) and EVA (60% vinyl acetate) (purchased from Polysciences (Warrington, PA); polyvinyl alcohol (PVA) (molecular weight 124,000-186,000, 99% hydrolysed, purchased from Aldrich Chemical Co.

(Milwaukee, WI)) and Dichloromethane (DCM) (HPLC grade, obtained from Fisher Scientific Co). Distilled water is used throughout.

A. Preparation of microspheres Microspheres are prepared essentially as described in Example 8 utilizing the solvent evaporation method. Briefly, 5% w/v polymer solutions in mL DCM are prepared using blends of EVA:PLA between 35:65 to 90:10.

To 5 mL of 2.5% w/v PVA in water in a 20 mL glass vial is added 1 mL of the polymer solution dropwise with stirring. SLx similar vials are assembled in a six position overhead stirrer, dissolution testing apparatus (Vanderkamp) and stirred at 200 rpm. The temperature of the vials is increased from room temperature to 40 0 C over 15 mmin and held at 40 0 C for 2 hours. Vials are centrifuged at 500xg and the microspheres washed three times in water. At some EVA:PLA polymer blends, the microsphere samples aggregated during the washing stage due to the removal of the dispersing or emulsifying agent, PVA. This aggregation effect could be analyzed semi-quantitatively since aggregated microspheres fused and the fused polymer mass floated on the surface of the wash water. This surface polymer layer is discarded during the wash treatments and the remaining, pelleted microspheres are weighed.

The aggregation is determined from aggregation 1- (weight of pelleted microsDheres) x 100 initial polymer weight Taxol loaded microspheres w/w taxol) are prepared by dissolving the taxol in the 5% w/v polymer solution in DCM. The polymer blend used is 50:50 EVA:PLA. A "large" size fraction and "small" size fraction of microspheres are produced by adding the taxol/polymer solution dropwise into 2.5% w/v PVA and 5% w/v PVA, respectively. The dispersions are stirred at 40"C at 200 rpm for 2 hours, centrifuged and washed 3 times in water as described previously. Microspheres are air dried and samples are sized using an optical microscope with a stage micrometer. Over 300 microspheres are counted per sample. Control microspheres (taxol absent) are prepared and sized as described previously.

B. Encapsulation efficiency Known weights of taxol-loaded microspheres are dissolved in 1 mL DCM, 20 mL of 40% acetonitrile in water at 50°C are added and vortexed until the DCM had been evaporated. The concentration of taxol in the acetonitrile is determined by HPLC using a mobile phase of water:methanol:acetonitrile (37:5:58) at a flow rate of 1 mL/min (Beckman isocratic pump), a C8 reverse phase column (Beckman) and UV detection at 232 nm. To determine the recovery efficiency of this extraction procedure, known weights of taxol from 100-1000 4g are dissolved in 1 mL of DCM and subjected to the same extraction procedure in triplicate as described previously.

Recoveries are always greater than 85% and the values of encapsulation efficiency are corrected appropriately.

C. Drug release studies In 15 mL glass. screw capped tubes are placed 10 mL of 10 mM phosphate buffered saline (PBS), pH 7.4 and 35 mg taxol-loaded microspheres.

The tubes are tumbled at 37C and at given time intervals, centrifuged at 1500xg for 5 min and the supernatant saved for analysis. Microsphere pellets are resuspended in fresh PBS (10mL) at 37C and reincubated. Taxol concentrations are determined by extraction into 1 mnL DCM followed by evaporation to dryness under a stream of nitrogen, reconstitution in 1 mrL of acetonitrile in water and analysis using HPLC as previously described.

D. Scanning Electron Microscopy (SEM) Microspheres are placed on sample holders, sputter coated with gold and micrographs obtained using a Philips 501B SEM operating at 15 kV.

E. CAM Studies Fertilized, domestic chick embryos are incubated for.4 days prior to shell-less culturing. The egg contents are incubated at 90% relative humidity and 3% CO 2 for 2 days. On day 6 of incubation, 1 mg aliquots of 0.6% taxol loaded or control (taxol free) microspheres are placed directly on the CAM surface. After a 2 day exposure the vasculature is examined using a stereomicroscope interfaced with a video camera; the video signals are then displayed on a computer and video printed.

F. Results Microspheres prepared from 100% EVA are freely suspended in solutions of PVA but aggregated and coalesced or fused extensively on subsequent washing in water to remove the PVA. Blending EVA with an increasing proportion of PLA produced microspheres showing a decreased tendency to aggregate and coalesce when washed in water, as described in Figure 15A. A 50:50 blend of EVA:PLA formed microspheres with good physical stability, that is the microspheres remained discrete and well suspended with negligible aggregation and coulescence.

The size range for the "small" size fraction rrmicrospheres is determined to be >95% of the microsphere sample (by weight) between 10-30 p/m and for the "large" size fraction, 95% of the sample (by weight) between 30-100 /am. Representative scanning electron micrographs of taxol loaded 50:50 EVA:PLA microspheres in the "small" and "large" size ranges are shown in Figures 15B and 15C, respectively. The microspheres are spherical with a smooth surface and with no evidence of solid drug on the surface of the microspheres. The efficiency of loading 50:50 EVA:PLA microspheres with taxol is between 95-100% at initial taxol concentrations of between 100-1000 pm taxol per 50 mg polymer. There is no significant difference (Student t-test, p <0.05) between the encapsulation efficiencies for either "small" or "large" microspheres.

The time course of taxol release from 0.6% w/v loaded 50:50 EVA:PLA microspheres is shown in Figure 15D for "small" size (open circles) and "large" size (closed circles) microspheres. The release rate studies are carried out in triplicate tubes in 3 separate experiments. The release profiles are biphasic with an initial rapid release of taxol or "burst" phase occurring over the first 4 days from both size range microspheres. This is followed by a phase of much slower release. There is no significant difference between the release rates from "small" or "large" microspheres. Between 10-13% of the total taxol content of the microspheres is released in 50 days.

The taxol loaded microspheres w/v loading) are tested using the CAM assay and the results are shown in Figure 15E. The taxol microspheres released sufficient drug to produce a zone of avascularity in the surrounding tissue (Figure 15F). Note that immediately adjacent to the microspheres in Figures 15E and 15F) is an area in which blood vessels are completely absent (Zone further from the microspheres is an area of disrupted, non-functioning capillaries (Zone it is only at a distance of approximately 6 mm from the microspheres that the capillaries return to normal. In CAMs treated with control microspheres (taxol absent) there is a normal capillary network architecture.

Discussion Arterial chemoembolization is a invasive surgical .technique.

Therefore. ideally, a chemoembolic formulation of an anti-angiogenic and anticancer drug such as taxol would release the drug at the tumor site at concentrations sufficient for activity for a prolonged period of time, of the order of several months. EVA is a tissue compatible nondegradable polymer which has been used extensively for the controlled delivery of macromolecules over long time periods 100 days).

EVA is initially selected as a polymeric biomaterial for preparing microspheres with taxol dispersed in the polymer matrix. However, microspheres prepared with 100% EVA aggregated and coalesced almost completely during the washing procedure.

Polymers and copolymers based on lactic acid and glycolic acid are physiologically inert and biocompatible and degrade by hydrolysis to toxicologically acceptable products. Copolymers of lactic acid and glycolic acids have faster degradation rates than PLA and drug loaded microspheres prepared using these copolymers are unsuitable for prolonged, controlled release over several months. Dollinger and Sawan blended PLA with EVA and showed that the degradation lifetime of PLA is increased as the proportion of EVA in the blend is increased. They suggested that blends of EVA and PLA should provide a polymer matrix with better mechanical stability and control of drug release rates than PLA.

Figure 15A shows that increasing the proportion of PLA in a EVA:PLA blend decreased the extent of aggregation of the microsphere suspensions. Blends of 50% or less EVA in the EVA:PLA matrix produced physically stable microsphere suspensions in water or PBS. A blend of 50:50 EVA:PLA is selected for all subsequent studies.

Different size range fractions of microspheres could be prepared by changing the concentration of the emulsifier, PVA, in the aqueous phase.

"Small" microspheres are produced at the higher PVA concentration of 5% w/v whereas "large" microspheres are produced at 2.5% w/v PVA. All other production variables are the same for both microsphere size fractions. The higher contentration of :emulsifier gave a more viscous aqueous dispersion medium and produced smaller droplets of polymer/taxol/DCM emulsified in the aqueous phase and thus smaller microspheres. The taxol loaded microspheres contained between 95-100% of the initial taxol added to the organic phase encapsulated within the solid microspheres. The low water solubility of taxol favoured partitioning into the organic phase containing the polymer.

Release rates of Eaxol from the 50:50 EVA:PLA microspheres are very slow with less than 15% of the loaded taxol being released in 50 days. The initial burst phase of drug release may be due to diffusion of drug from the superficial region of the microspheres (close to the microsphere surface).

The mechanism of drug release from nondegradable polymeric matrices such as EVA is thought to involve the diffusion of water through the dispersed drug phase within the polymer, dissolution of the drug and diffusion of solute through a series of interconnecting, fluid filled pores. Blends of EVA and PLA have been shown to be immiscible or bicontinuous over a range of 30 to EVA in PLA. In degradation studies in PBS buffer at 37°C, following an induction or lag period, PLA hydrolytically degraded and eroded from the EVA:PLA polymer blend matrix leaving an inactive sponge-like skeleton.

Although the induction period and rate of PLA degradation and erosion from the blended matrices depended on the proportion of PL, in the matrix and on process history, there is consistently little or no loss of PLA until after 40-50 days.

Although some erosion of PLA from the 50:50 EVA:PLA microspheres may have occurred within the 50 days of the in vitro release r..e study (Figure 15C), it is likely that the primary mechanism of drug release from the polymer blend is diffusion of solute through a pore network in the polymer matrix.

At the conclusion of the release rate study, the microspheres are analyzed from the amount of drug remaining. The values for the percent of taxol remaining in the 50 day incubation microsphere samples are 94% 9% and 89% 12% for "large" .and "small" size fraction microspheres, respectively.

Microspheres loaded with 6mg per mg of polymer provided extensive inhibition of angiogenesis when placed on the CAM of the embryonic chick (Figures 15E and EXAMPLE 12 TAXOL ENCAPSULATION IN POLY(E-CAPROLACTONE) MICROSPHERES. INHIBITION OF ANGIOGENESIS ON THE CAM ASSAY BY TAXOL-LOADED MICROSPHERES This example evaluates the in vitro release rate profile of taxol from biodegradable microspheres of poly(e-caprolactone) and demonstrates the anti-angiogenic activity of taxol released from these microspheres when placed on the CAM.

~eaeftSwhich were utilized in these ex mei inldero poly~..Ca R a ets(P L (molecular we"ght 35,000 45,000; purchasedfo i polyscie c ne (WarriLgt dihooethane CM) from Fisher p lScien c eC C anada polyvi yl alcohol (P V P (M l ular$ W it ,0 an 18,000, 99% hydrolysed) fSom Ldrich Cheruca CoUMlak~Ws) n taxol from Sigma Chemical Co. (s.upisM. inledS atherise tatedghall chemiucals and reagents are used assupidDstlewarissd rOgut A- Preparatonlof micosphgres r prepared essentially as described in Exaed8 tilizinlg the solvent evpoato et o f Brifl.~ WWtxllae micospheres are prepared by d~ 5 n f0 Ig oftco and st90 ig f 1CL 00n 0 c PP aueos sluisn ~tifupged at 1000 m l o f C M a d d in g to lo0 m l 0f of mPaq u o s rs o ri d ce t ri fl at y O r~~m a 2 0 C fo hours. The suspensiori of aicdophehe 1 5 g "f o r 1 0 m i n u t e s B e c k m a n G P R t 'h e sed n m c o p e saremo e an dr h mirspee wased three times with water Theo wahe jcrosphees a ir.

dried overnight andsoe atie r o en.etaicrre s Cont aicToshre ta absent) are prepared as descfl e b ve M cospheres ar iz d usn tial i cros ande 2%t taxol are also prepared. Microshrsaeszduignopclmcocpewt a stage micrornew.

B. EnaP~Iaiio f effiienv kn wn eig t o g-loaded micospheres (about 5 g) is of acetoflitrile arid 2 ml distilled wate isiadde od prcptte the polymrer. The mixtureis~ntfgda100gor0mnueadth amout o taol encapsulated is calculated from the absobance f h supenatant measured In a UV spectrophotomt Diode Array SpectrophOtOmeter) at 232 rim.

C. rjj~ rejleaSe stilies m of taxol..loaded microspheres are suspendte. in 0m f 1 m ph pa ebu ffered saine. 91 7.4 (PBS) in screw .ca e tube. 5 The

O

tue are umblosae bufered at* 37oC arid at veri time intervals t9. mlho e na a i iS be d ove afe nd lO in h m icroSPheres to settle at h bottom), filtered through a 0.45 mm membrane filter and retained for taxol analysis. An equal volume of PBS is replaced in each tube to maintain sink conditions throughout the study. The filtrates are extracted with 3 x 1 ml DCM, the DCM extracts evaporated to dryness under a stream of nitrogen, redissolved in 1 ml acetonitrile and analyzed by HPLC using a mobile phase of water:methanol:acetonitrile (37:5:58) at a flow rate of Iml min" 1 (Beckman Isocratic Pump), a C8 reverse phase column (Beckman), and UV detection (Shimadzu SPD A) at 232 nm.

D. CAM studies Fertilized. domestic chick embryos are incubated for 4 days prior to shell-less culturing. On day 6 of incubation, 1 mg aliquots of 5% taxol-loaded or control (taxol-free) microspheres are placed directly on the CAM surface.

After a 2-day exposure the vasculature is examined using a stereomicroscope interfaced with a video camera; the video signals are then displayed on a computer and video printed.

E. Scanning electron microscopy Microspheres are placed on sample holders, sputter-coated with gold and then placed in a Philips 501B Scanning Electron Microscope operating at 15 kV.

F. Results The size range for the microsphere samples is between 30 100 um although there is evidence in all taxol-loaded or control microsphere batches of some microspheres falling outside this range. The efficiency of loading PCL microspheres with taxol is always greater than 95% for all drug loadings studied. Scanning electron microscopy demonstrated that the microspheres are all spherical and many showed a rough or pitted surface morphology. There appeared to be no evidence of solid drug on the surface of the microspheres.

The time courses of taxol release from 2% and 5% loaded PCL microspheres are shown in Figure 16A. The release rate profiles are biphasic. There is an initial rapid release of taxol or "burst phase" at all drug loadings. The burst phase occurred over 1-2 days at 1% and 2% taxol loading and over 3-4 days for 5% loaded microspheres. The initial phase of rapid release is followed by a phase of significantly slower drug release. For microspheres containing 1% or 2% taxol there is no further drug release after 21 days. At 5% taxol loading, the microspheres had released about 20% of the total drug content after 21 days.

Figure 16B shows CAMs treated with control PCL microspheres, and Figure 16C shows treatment with 5% taxol loaded microspheres. The CAM with the control microspheres shows a normal capillary network architecture.

The CAM treated with taxol-PCL microspheres shows marked vascular regression and zones which are devoid of a capillary network.

G. Discussion The solvent evaporation method of manufacturing taxol-loaded microspheres produced very high taxol encapsulation efficiencies of between 100%. This is due to the poor water solubility of taxol and its hydrophobic nature favouring partitioning in the organic solvent phase containing the polymer.

The biphasic release profile for taxol is typical of the release pattern for many drugs from biodegradable polymer matrices. Poly(ecaprolactone) is an aliphatic polyester which can be degraded by hydrolysis under physiological conditions and it is non-toxic and tissue compatible. The degradation of PCL is significantly slower than that of the extensively investigated polymers and copolymers of lactic and glycolic acids and is therefore suitable for the design of long-term drug delivery systems. The initial rapid or burst phase of taxol release is thought to be due to diffusional release of the drug from the superficial region of the microspheres (close to the microsphere surface). Release of taxol in the second (slower) phase of the release profiles is not likely due to degradation or erosion of PCL because studies have shown that under in vitro conditions in water there is no significant weight loss or surface erosion of PCL over a 7.5-week period. The slower phase of taxol release is probably due to dissolution of the drug within fluid-filled pores in the polymer matrix and diffusion through the pores. The greater release rate at higher taxol loading is probably a result of a more extensive pore network within the polymer matrix.

Taxol microspheres with 5% loading have been shown to release sufficient drug to produce extensive inhibition of angiogenesis when placed on the CAM. The inhibition of blood vessel growth resulted in an avascular zone as shown in Figure 16C.

EXAMPLE 13 TAXOL-LOADED POLYMERIC FILMS COMPOSED OF ETHYLENE VINYL ACETATE AND A SurFAcrTAN.

Two types of films are prepared esentially as described in Example 10: pure EVA films loaded with taxol and EVA/surfactant blend f!lms Pluronic F127, Span 80 and Pluronic L101) loaded with taxol.

The surfactants being examined are two hydrophobic surfactants (Span 80 and Pluronic L101) and one hydrophilic surfactant (Pluronic F127).

The pluroinc surfactants are themselves polymers, which is an attractive property since they can be blended with EVA to optimize various drug delivery properties. Span 80 is a smaller molecule which is in some manner dispersed in the polymer matrix, and does not form a blend.

Surfactants will be useful in modulating the release rates of taxol from films and optimizing certain physical parameters of the films. One aspect of the surfactant blend films which indicates that drug release rates can be controlled is the ability to vary the rate and extent to which the comnpound will swell in water. Diffusion of water into a polymer-drug matrix is critical to the release of drug from the carrier. Figures 17C and 17D show the degree of swelling of the films as the level of surfactant in the blend is altered. Pure EVA films do not swell to any significant extent in over 2 months. However, by increasing the level of surfactant added to the EVA it is possible to increase the degree of swelling of the compound, and by increasing hydrophiliciry swelling can also be-increased.

Results of experiments with these films are shown below in Figures 17A-E. Briefly, Figure 17A shows taxol release (in mg) over time from pure EVA films. Figure 17B shows the percentage of drug remaining for the same films. As can be seen from these two figures, as taxol loading increases percentage of taxol by weight is increased), drug release rates increase, showing the expected concentration dependence. As taxol loading is increased, the percent taxol remaining in the film also increases, indicating that higher loading may be more attractive for long-term release formulations.

physical strength and elasticity of the flms is assessed in Figure 17E. Briefly, Figure 17E shows stress/strain curves for pure EVA and EVA- 17E. Briefly, Figure 17E sho w s s trs n ofstress demonstrates that the Surfactant blend films. This crude measurement of stress demonstr27, and that the elasticity of films is increased with the addition of Pluronic F127, and that the elastrength (f filmstress on breaking) is increased in a concentration dependant tensile strength (stress on b r e a k i n g 7 Elasticity and strength are manner with the addition of Pluronic F127. Elastwhicit cand strength are important considerations in designing a film which can emipulated for particular clinical applications without causing permanent deformation of the compound.The above data demonstrates the ability of certain surfactant additives t control drg release rates and to alter the physical characteristics of additives to control drug release rates the vehicle.

EXAMPLE 14 NCORPORATING MErIOXYPOLYETHYLENE GLYCOL 350 (MEPEG) ITro POLY(E- CApRoLAcroNE) To DEVELOP A Fu.AON- FOR rTHE CONTROLLED DELIVERY OF TAXOL FROM A PASTE Reagents and equipment which were utilized within these Reagents and equipment w "MP n experiments include methoxypoyet hylene glycol 350 ("MePEG" Union Carbide, Danbury, CT). MePEG is liquid at room temperature, and has a freezing point of 100 to -5 0

C.

A. Preaa f MePEGPCL containng aste MePEG/PCL paste is prepared by first dissolving a quantity of taxol into MePEG, and then incorporating this into melted PCL. One advantage with this method is that no DCM is required.

B. Analys eli The melting point of PCL/MePEG polymer blends may be Thdetermined by differential scann inga calorimetry from 30 C to 70 0 C at a heating rated of 2.5C per minute. Results of this experiment are shown in Figures 18A rate of 2.50C per minute. Results and 8. refly, as shown in Figure 18A the melting point of the polymer nd 18B. Briefly, as ho wn by thermal analsis) is decreased by MePEG in a conentrd (ason determined by manner. ne melting point of the polymer blends as a concentration dependentis shown in Figure 18A. This lower melting point also translates into an increased time for the polymer blends to solidify point also trans lates igure 18B. A 30:70 blend of MePEG:PCL takes more than twice as long toidify from the fuid melt than does PC alone.

C. Measurement of brittleness ncorporation of MePEG into PCL appears to produce a less brittle solid, as compared to PCL alone. As a "rough" way of quantitating this, a webrittle soneedle is dropped from an equal height into polymer blends weightedng from 0% to 30o MePEG in PCL and the distance that the needle penetrates into the solidis then measured. The resulting graph is shown as Figure 8. Points are given as the average of four measurements 1 S.D.

ForFigure 18C. P ointses of comparison, a sample of paraffin wax is also tested and the needle penetrated into this a distance of 7.25 mm 0.3 m.

D .em ent of taxol el *no 0c 5% 10 o r 20 Pellets of polymer (PCL contain (0%BS, pH 7.4) t 3C, and MePEG) are incubated in phosphate buffered saline (PBS, pH 7.4)at 37 0 C, and change in polymer weight is measured over time. As can be seen in Figure change in polymer weight is meas cocentration of MePEG 18D, the amount of weight lost increases with the concentration of MePEG originally present in the blend. It is likely that this weight loss is due to the release of MePEG from the polymer matrix into the incubating fluid. This release of MePEG from the po .e d a MePEG/PCL blend would indicate that taxol will readily be released from a MePEG/PCL blend since taol is fndicate thadissolved in MePEG before incorporation into PCL.

since taxol is first dissolved in E. Effecn m Thermopastes are made up containing between 0.8% and MePEG in PCL. These are loaded with 1% taxol. The release of taxol over MePEG n PCL. Tese r e bduis monitore d u si ng H PL C As is shown in Figure 1E. the amount of N ePEG in the formulation does not affect the amount of taxol that is released.

72 F. Effect of varving quantities of taxol on the total amount of taxnl released from a 20% MePEG/PCL blend Thermopastes are made up containing 20% MePEG in PCL and loaded with between 0.2% and 10% taxol. The release of taxol over time is measured as described above. As shown in Figure 18F, the amount of taxol released over time increases with increased taxol loading. When plotted as the percent total taxol released, however, the order is reversed (Figure 18G). This gives information about the residual taxol remaining in the paste and, if assumptions are made about the validity of extrapolating this data, allows for a projection of the period of time over which taxol will be released from the MePEG Thermopaste.

G. Strength analysis of various MePEG/PCL blends A CT-40 mechanical strength tester is used to measure the strength of solid polymer "tablets" of diameter 0.88 cm and an average thickness of 0.560 cm. The polymer tablets are blends of MePEG at concentrations of 10% or 20% in PCL.

Results of this test are shown in Figure 18H, where both the tensile strength and the time to failure are plotted as a function of %MePEG in the blend. Single variable A.NOVA indicated that the tablet thicknesses within each group are not different. As can be seen from Figure 18H, the addition of MePEG into PCL decreased the hardness of the resulting solid.

EXAMPLE EFFECT OF TAXUL-LOADED THER.MOPASTE ON ANGIOGENESIS IN VIvo Fertilized, domestic chick embryos were incubated for 4 days prior to shell-less culturing as described in Example 2. The egg contents are removed from the shell and emptied into round-bottom sterilized glass bowls and covered with petri dish covers.

Taxol is incorporated into thermopaste at concentrations of and 20% essentially as described above (see Example 10), and used in the following experiments. Dried cut therropaste is then heatedllowing it to 60oolC andSix pressed between two sheets of parafilm, flatening it and allowing it to cool. Sixved embryos received 20% taxol-loaded thermoPaste and 6 embryos received embryos receivopaste prepared in this manner. One embryo died in each unloaded thermopaste preparedin control and treated grou; group leaving 5 embryos in each of the cotrol and treateding roax Unloaded thermopaste and thermopte onaing 20% taxoh C was also heated to 60"C and placed directly on the growing edge of each

CAM

at day 6 of incubation; two embryos each were treated in this manner.

There was no observable difference in the results obtained using the different methods of administration, indicating that the temperature of the paste at the time of application was not a factor in the outcome.

e heopaste with 10% taxol was applied to 11 CAMs and Thermopaste was applied to an additional 11 CAMs, while 5% taxoluloaded theropaste was applied to 10 CAMs and unloaded thermopaste was loaded thermopaste was applied to a d o 8 o e a applied to 10 other control CAMs. After a 2 day.exposure (day 8 of incubation) the vasculature was examined with the aid of a stereomicroscope Liposyn i11, a white opaque solution, was injected into the CAM to increase the visibility of th e n the embryos treated with 5% taxol-loaded paste, only 2 animals demonstrated maximum inhibition of angiogenesis, while the remaining 8 were only marginally affected. Of the animals treated with 10% taxol-loaded theronly marginally 2 showed maximal inhibition while the other 9 were only themarginally opasffecte d max marginally affected taxol-loaded thermopaste showed extensive areas of The 20% taxol-oaAs receiving this treatment. The avascularity (see Figure 19B) in all 5 of the CAsa receiving this treatment. The highest degree of inhibitionl was defined as a region of avascularity covering 6 highest degree of °inhibition was d e s treated with 20% taxol-loaded mm by 6 mm in size. All of the CAM treated wition thermopaste displayed this degree of angiogenesis inhibition.

h By comparison, the control (unloaded) thermopaste did not inhibit angiogemnesis on the CAM (see Figure 19A); this higher magnification view (note that the edge of the paste is seen at the top of the image) view (note that the edge of the pasto the paste are unaffected by the demonstrates that the vessels adjacen t observed is due to the sustained thermopaste. This uggestsomer itself or due to a secondary release of taxol and is not due to the polymer itself or due to a secondary pressure effect of the paste on the de g vascuature.

This study dem trtes that theropaste releases sufficient Tis nhi btor (In this case taxol) to inhibit the normal quantities of an g oenesinvasculature.

development of the

CAM

E XAMPLE 6 EFFECr OF t.x-LoA HE MOPASE ON TUMOR E C ROFHAND TUMOR ANGIOGF NESS IN Vrvo 0 Fertilized domestic chick embryos are incubated for 3 days prinor Fertilized contents are emptied by removing the o having their shells removed. ing the interior shell membrane, shell loed arou n d of he she, ae n alle egg contents to gently slide out from the blunted end. The contents are emptied into round-bottom sterilized glass boln covered with petri dish covers and incubated at relative humidity and 3h carbon dioxide (see Exampc 2).

MDAY-D

2 cells (a murine lymphoid tumor) are injected itu mice and allowed to grow into tumors weighing 0.5-.0 g. The mice are sacrificed, the tumor sites wiped with alcohol, excised, placed in sterile tissue culture media, and diced into 1 mm pieces under a laminar flow hood. Prior tO placing the dissected tumors onto the 9-day old chick embryos, CAM surfaces are gently scraped with a 30 gauge needle to insure tumor implantation. The tumors are then placed on the CAMs after 8 days of incubation (4 days after deshelling), and allowed to grow on the CAM for four days to establish a vascular supplY.

Four embyos are prepared utilizing this method, each embryo receiving 3 tumors. For these embOrS' one tumor receives 2076 taxol-loaded thermopaste, the second tumor unloaded thermopaste, and the third tumor no treatment.

The treatments are continued for two days befre the results were recorded.

Te t htens ar e MDAYD tumors secrete angiogenic factors which induce the ingrowth of capillaries (derived from the

CAM

mass and allow it to continue to grow in size. Since all the vessels of the tumor are derived from the CAM. while all the tumor cells are derived from the explant, it is Possible to assess the effect of therapeutic interventions on these Sndependentl. This assay has been used to determine the effectiveness of taxol-ioaded thermopaste on: inhibiting the vascularization of the tumor and hitig the roth of the tumor cells themselves.

Direct in vivo stereomicroscopic evaluation and histological examination of fixed tissues from this study demonstrated the following. In the tumors treated with 20% taxol-oaded thermopaste, there was a reduction in the number of the blood vessels which supplied the tumor (see Figures 20C and a reduction i the number of blood vessels within the tumor, and a reduction in the number of blood vessels in the periphery of the tumor (the area which is ty cally the most highly vascularized in a solid tumor) when compared to control t.,mors. The tumors began to decrease in size and mass during the two days the study was conducted. Additionally, numerous endothelial cells were seen to be arrested in cell division indicating that endothelial cell proliferation had been affected. mor cells were also frequently seen arrested in mitosis. All 4 embryos sho -d a consistent pattern with the 20% taxolioaded thermopaste suppressing tumor vascularity while the unloaded thermopaste had no effect.

By comparison, in CAMs treated with unloaded thermopaste, the tumors were well vascularized with an increase in the number and density of vessels when compared to that of the normal surrounding tissue, and vessedramatically more vessels than were observed in the tumors treated with taxolloaded paste. The newly formed vessels entered the tumor from all angles appearing like spokes attached to the center of a wheel (see Figures 20A and The control tumors continued to increase in size and mass during the course of the study. Histologically, numerous dilated thin-walled capillaries were seen in the periphery of the tumor and few endothelial were seen to be in cell division. The tumor tissue was we vascularized and viable throughout.

eAs an example, in two similarly-sized (initially, at the time of explantation) tumors placed on the same CAM the following data was obtained.

For the tumor treated with 20% taxol-loaded thermopaste the tumor measured 330 mmx 597 mm; the immediate periphery of the tumor has 14 blood vessels, while the tumor mass has only 3-4 small capillaries. For the tumor treated with unloaded termopaste the tumor size was 623 mm x 678 mm; the immediate periphery of tumor has 54 blood vessels, while the tumor mass has 12-14 small blood vessels. In addition, the surrounding CAM itself contained many more blood vessels as compared to the area surrounding the taxol-treated This study demonstrates that thermopaste releases sufficient quantities of angiogenesis inhibitor (in this case taxol) to inhibit the pathological angiogenesis which accompanies tumor growth and development. Under these conditions angiogenesis is maximally stimulated by the tumor cells which produce angiogenic factors capable of inducing the ingrowth of capillaries from the surrounding tissue into the tumor mass. The 20% taxol-loaded thermopaste is capable of blocking this process and limiting the ability of the tumor tissue to maintain an adequate blood supply. This results in a decrease in the tumor mass both through a cytotoxic effect of the drug on the tumor cells themselves and by depriving the tissue of the nutrients required for growth and expansion.

EXAMPLE 17 EFFECT OF ANGIOGENESIS INHIBrrOR-LOADED THER.MOPASTE ON TUMOR GROWTH In Kivo IN A ML'uuNE TUMOR MODEL The murine MDAY-D2 tumor model may be used to examine the effect of local slow release of the chemotherapeutic and anti-angiogenic compounds such as taxol on tumor growth, tumor metastasis, and animal survival. The MDAY-D2 tumor cell line is grown in a cell suspension consisting of 5% Fetal Calf Serum in alpha mem media. The cells are incubated at 37C in a humidified atmosphere supplemented with 5% carbon dioxide, and are diluted by a factor of 15 every 3 days until a sufficient number of cells are obtained. Following the incubation period the cells are examined by light microscopy for viability and then are centrifuged at 1500 rpm for 5 minutes.

PBS is added to the cells to achieve a dilution of 1,000,000 cells per ml.

Ten week old DBA/2j female mice are acclimatized for 3-4 days after arrival. Each mouse is then injected subcutaneously in the posteriolateral flank with 100,000 MDAY-D2 cells in 100 ml of PBS. Previous studies have shown that this procedure produces a visible tumor at the injection site in 3-4 days, reach a size of 1.0-1.7g by 14 days, and produces visible metastases in the liver 19-25 days post-injection. Depending upon the objective of the study a therapeutic intervention can be instituted at any point in the progression of the disease.

Using the above animal model, 20 mice are injected with 140,000 MDAY-D2 cells s.c. and the tumors allowed to grow. On day 5 the mice are divided into groups of 5. The tumor site was surgically opened under anesthesia, the local region treated with the drug-loaded thermopaste or control thermopaste without disturbing the existing tumor tissue, and the wound was closed. The groups of 5 received either no treatment (wound merely closed), 77 polymer (PCL) alone, 10% taxol-loaded thermopaste, or 20% taxol-loaded thermopaste (only 4 animals injected) implanted adjacent to the tumor site. On day 16, the mice were sacrificed, the tumors were dissected and examined (grossly and histologically) for tumor growth, tumor metastasis, local and systemic toxicity resulting from the treatment, effect on wound healing, effect on tumor vascularity, and condition of the paste remaining at the incision site.

The weights of the tumors for each animal is shown in the table below: Table IV Tumor Weights (gm) Animal No. Control Control 10% Taxol 20% Taxol (empty) (PCL) Thermopaste Thermopaste 1 1.387 1.137 0.487 0.114 2 0.589 0.763 0.589 0.192 3 0.461 0.525 0.447 0.071 4 0.606 0.282 0.274 0.042 5 0.353 0.277 0.362 Mean 0.6808 0.6040 0.4318 0.1048 Std. Deviation 0.4078 0.3761 0.1202 0.0653 P Value 0.7647 0.358 0.036 Thermopaste loaded with 20% taxol reduced tumor growth by over (average weight 0.105) as compared to control animals (average weight 0.681).

Animals treated with thermopaste alone or thermopaste containing 10% taxol had only modest effects on tumor growth; tumor weights were reduced by only and 35% respectively (Figure 21A). Therefore, thermopaste containing taxol was more effective in reducing tumor growth than thermopaste containing 10% taxol (see Figure 21C; see also Figure 21B).

Thermopaste was detected in some of the animals at the site of administration. Polymer varying in weight between 0.026 g to 0.078 g was detected in 8 of 15 mice. Every animal in the group containing 20% taxolloaded thermopaste contained some residual polymer suggesting that it was less susceptible to dissolution. Histologically. the tumors treated with taxol-loaded thermopaste contained lower cellularity and more tissue necrosis than control tumors. The vasculature was reduced and endothelial ces were frequently seen to be arrested in cell division. The taxol-loaded thermopaste did not appear to to be arrested in cell dvoe e i surrounding the rumor.

affect the integrity or cellularit of the skin or tissues ounding the tumor.

Grossly, wound healing was unaffected.

G IEXAM E-i PREVEN OF.

THE USE OF A,~NGIOGEN~ESIS-IHI TOR LOADED SURGICAL FILMS IN HE pREETION

OF

IATROGENIC METASTATC SEEDING OF TLUMOR CELLS DuING CANCER

RESECTION

0

SURGERY

le, pliable, stretchable drug-polymer compound would As a sterle, plable, Often it is desirable to isolate the be useful during cancer resection proceures. during res on operations to normal surrounding tissues from malignant tissujacent organs through inadv opertent prevent iatrogenic spread of the disease to aacet orgas through inadvertent contamination by cancer cells. A drug-loaded parafilm could be stretched contamina l tion by cancrior to manipulation of the tumor. This would be most aross normalaced around the liver and other abdominal contents during bowel usefl icancer rlaced around tho prevent intraeritoneal spread of the disease to the li0 caer. A ioegradable film could be left in situ to provide continued protection.

livencisr. A biodegradable n sites are also a common location of post-operative Incision sites aris thought to be due to contamination of the wocurrence ofwith tumor cells during the surgical procedure. To address these wound site with tumor cels dur he ability of angiogenesis issues, experiments are being conducted to determine the ability of angiogenesis inhibitor-loaded films to prevent this phenomenon- A. M erial and Methods m repared as described in Preparation of Surgical Fm. Sur cael cm x cm are prepared Example 10. Thin films meas PCL loaded with 5 taxeprepared containing either polymer alone (PCL)o epatic Tumor Mode. nitial study Wistar rats weighing a Hepatic Tumor Model.ent genera anesthesia and a 3-5 cm abdominal omael 300 S underwert gener a cm inision ,s apprincision is made alon the midline. I the largest hepatic lobe, a cm incision is incimade in the hepatic prenCh and part of the liver edge is resected.

A

concentration of 1 million live 9L Glioma tumor cells (eluted from tissue culture concendiation of the procedure) suspended in 100 ml of phosphate buffered saediately prior sito the procedut liver edge with a 30 gauge needle. The saline are deposited onto the cut I e g the tumor cells and surgical is then placed over the cut liver edge containing the tumor cells and affixed in place with Gelfoam. Two animals received PCL films containing taxol and two animals received films containing PCL alone. The abdominal wall is closed with 3.0 Dexon and skin clips. The general anesthetic is terminated and the animal is allowed to recover. Ten days later the animals are sacrificed and the livers examined histologically.

B. e-Sts Local tumour growth is seer in the 2 livers treated with polymer alone. Both livers tumour growith polymer plus taxol are completely free of tumour alone. Both livers treated I so of importance, the liver capsule had when examined histologicallY-. Also of importan the liver capsurface regenerated and grown completely over the polymeric film and the cut surface of the liver indicating that there is no significant effect on wound healing.

There is no evidence of local hepatic toxicity surrounding any (drugloaded or drug-free) of the surgical films.

C. Discussion This study indicates that surgical films placed around normal This study .ndcates u able of decreasing the tissues and incision sites during surgery ma be nto normale of surrounding incidence of accidental implantation of tumor cells into normal ur rounding tissue during resection of malignant tumors. This may help reduce the incidence of the significant problem of post-operative local recurrence of the disease.

EXAMPLE 19 IOGE-E.-IIBrrO-LOADED

BIODEGRADABLE

1N.TRA-ARTICLLAR INJECTION OF A. 'oGESl MIcROSPIRIERES IN THETR EATNIEr OF ARTIIRms Articular damage n arthritis is due to a combination of inflammation (including WBCs and WBC products) and pannus tissue development (a tissue composed on neovascular tissue, connective tissue, and development cels). Taxol has been chosen for the initial studies because it is a inflammatory cells). iaxo h potent inhibitor of neovascularization. In this manner, taxol in high local concentrations will prove to be a disease modifying agent in arthritis.

In order to determine whether microspheres have a deleterious effect on joints, the following experiments are conducted. Briefly, plain PCL and taxol-loaded microspheres are prepared as described previously in Example 8.

Three rabbits are injected intra-articularly with 0.5-5.0 10-30 gm, or 30-80 Am microspheres in a total volume of 0.2 mis (containing 0.5 mg of microspheres). The joints are assessed visually (clinically) on a daily basis.

After two weeks the animals are sacrificed and the joints examined histologically for evidence of inflammation and depletion of proteoglycans.

The rabbit inflammatory arthritis and osteoarthritis models are being used to evaluate the use of microspheres in reducing synovitis and cartilage degradation. Degenerative arthritis is induced by a partial tear of the cruciate ligament and meniscus of the knee. After 4 to 6 weeks, the rabbits develop erosions in the cartilage similar to that observed in human osteoarthritis. Inflammatory arthritis is induced by immunizing rabbits with bovine serum albumen (BSA) in Complete Freund's Adjuvent (CFA). After 3 weeks, rabbits containing a high titer of anti-BSA antibody receive an intraarticular injection of BSA (5 mg). Joint swelling and pronounced synovitis is apparent by seven days, a proteoglycan depletion is observed by 7 to 14 days, and cartilage erosions are observed by 4 to 6 weeks.

Inflammatory arthritis is induced as described above. After 4 days, the joints are injected with microspheres containing 5% taxol or vehicle.

One group of animals will be sacrificed on day 14 and another on day 28. The joints are examined histologically for inflammation and cartilage degradation.

The experiment is designed to determine if taxol microspheres can affect joint inflammation and cartilage matrix degradation.

Angiogenesis-inhibitor microspheres may be further examined in an osteoarthritis model. Briefly, degenerative arthritis is induced in rabbits as described above, and the joints receive an intra-articular injection of microspheres ta.xol or vehicle only) on day 4. The animals are sacrificed on day 21 and day 42 and the joints examined histologically for evidence of cartilage degradation.

Studies are conducted to assess angiogenesis inhibitors delivered via intra-articular microspheres as chondroprotective agents.

Results Unloaded PCL microspheres of differing sizes (0.5-5.0 urm, 10-30 Am, or 30-80 im) were injected intra-articularly into the rabbit knee joint.

Results of these experiments are shown in Figures 22A to D. Briefly, Figure 22A is a photograph of synovium from PBS injected joints. Figure 22B is a photograph of joints injected with microspheres. Figure 22C is a photograph of cartilage from joints injected with PBS, and Figure 22D is a photograph of cartilage from joints injected-with microspheres.

As can be seen from these photographs, histologically, there is no difference between joints receiving a microsphere injection and those whic. did not. Clinically, there was no evidence of joint inflammation during the 14 days the experiment was conducted. Grossly, there is no evidence of joint inflammation or cartilage damage in joints where microspheres are injected, as compared to untreated normal joints.

Conclusions Micrgpheres can be injected intra-articularly without causing any discernible changes toi0e joiqt surface. This indicates that this method may be an effective means of delivering a targeted, sustained-release of diseasemodifying agents to diseased joints, while minimizing the toxicity which could be associated with the systemic administration of such biologically active compounds.

As discussed above, microspheres can be formulated into specific sizes with defined drug release kinetics. It has also been demonstrated that taxol is a potent inhibitor of angiogenesis and that it is released from microspheres in quantities sufficient to block neovascularization on the CAM assay. Therefore, intra-articular administration of angiogenesis-inhibitor-loaded taxol-loaded) microspheres should be capable of blocking the neovascilarization that occurs in diseases such as rheumatoid arthritis and leads to cartilage destruction in the joint. In this manner the drug-loaded microspheres can act as a "chondroprotective" agent which protects the cartilage from irreversible destruction from invading neovascular pannus tissue.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of P:AOPERMKR\SPECI\71746.00-040 doc-18/02/04 82illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Claims (25)

1. A composition, comprising: an anti-angiogenic factor; and a polymeric carrier.

2. The composition of claim 1 wherein said anti-angiogenic factor is Anti-Invasive Factor.

3. The composition of claim 1 wherein said anti-angiogenic factor is retinoic acid and derivatives thereof.

4. The composition of claim 1 wherein said anti-angiogenic factor is selected from the group consisting of Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase- 2 Plasminogen Activator Inhibitor-1 and Plasminogen Activator Inhibitor- 2. A composition, comprising: taxol; and a polymeric carrier.

6. The composition according to any one of claims 1 to 5 wherein said composition is formed into microspheres having an average size of between 0.1 and 2100 pm.

7. The composition according to any one of claims 1 to 5 wherein said composition is formed into a film with a thickness of between 100 pm and 2 mm. p: oPER\JLR\IIjW-44.DIV -4/6193 86

8. The composition according to any one of claims 1 to 5 wherein said composition is liquid above 450C, and solid or semi-solid at 37 0 C.

9. The composition according to any one of claims 1 to 5 wherein said polymeric carrier is poly(ethylene-vinyl acetate) crosslinked with 40% vinyl acetate. The composition according to any one of claims 1 to 5 wherein said polymeric carrier is poly(lactic-co-glycolic acid).

11. The composition according to any one of claims 1 to 5 wherein said polymeric carrier is polycaprolactone.

12. The composition according to any one of claims 1 to 5 wherein said polymeric carrier is polylactic acid.

13. The composition according to any one of claims 1 to 5 wherein said polymeric carrier is a copolymer of poly(ethylene-vinyl acetate) crosslinked with 40% vinyl acetate, and polylactic acid.

14. The composition according to any one of claims 1 to 5 wherein said polymeric carrier is a copolymer of polylactic acid and polycaprolactone. A method for embolizing a blood vessel, comprising delivering into said vessel a therapeutically effective amount of composition according to any one of claims 1-14, such that said blood vessel is effectively occluded.

16. The method claim 13 wherein said blood vessel nourishes a tumor.

17. A stent, comprising a generally tubular structure, the surface of which is coated with a composition according to any one of claims 1-14. P:\OPER\JLR\71 IV 9 2.94.DIV 4/6196 -87

18. A method for expanding the lumen of a body passageway, comprising inserting a stent into the passageway, the stent having a generally tubular structure, the surface of said structure being coated with a composition according to any one of claims 1-14, such that said passageway is expanded.

19. A method for eliminating vascular obstructions, comprising inserting a vascular stent into a vascular passageway, the stent having a generally tubular structure, the surface of said structure being coated with a composition according to any one of claims 1-14, such that said vascular obstruction is eliminated. A method for eliminating biliary obstructions, comprising inserting a biliary stent into biliary passageway, the stent having a generally tubular structure, the surface of said structure being coated with a composition according to any one of claims 1-14, such that said biliary obstruction is eliminated.

21. A method for eliminating urethral obstructions, comprising inserting a urethral stent into a urethra, the stent having a generally tubular structure, the surface of said structure being coated with a composition according to any one of claims 1-14, such that said urethral obstruction is eliminated.

22. A method for eliminating esophageal obstructions, comprising inserting an esophageal stent into an esophagus, the stent having a generally tubular structure, the surface of said structure being coated with a composition according to any one of claims 1-14 such that said esophageal obstruction is eliminated.

23. A method for eliminating tracheal/bronchial obstructions, comprising inserting a tracheal/bronchial stent into the trachea or bronchi, the stent having a generally tubular structure, the surface of which is coated with a composition according to any one of claims 1-14, such that said tracheal/bronchial obstruction is eliminated. P:\OPER\J LR\71 192-94. DIV 4/6/98 -88

24. A method for treating a tumor excision site, comprising administering a composition according to any one of claims 1-14 to the resection margin of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at said site is inhibited. The method according to claim 24 wherein said anti-angiogenic composition is a thermopaste.

26. The method according to claim 24 wherein the step of administering comprising spraying a composition of nanospheres comprised of an anti-angiogenic composition into the resection margin of the tumor.

27. A method for treating corneal neovascularization, comprising administering a therapeutically effective amount of a composition according to any one of claims 1-14 to the cornea, such that the formation of blood vessels is inhibited.

28. The method of claim 23 wherein said composition further comprises a topical corticosteroid.

29. A method for inhibiting angiogenesis in patients with non-tumorigenic, angiogenesis- dependent diseases, comprising administering a therapeutically effective amount of a composition comprising taxol to a patient with a non-tumorigenic angiogenesis-dependent disease, such that the formation of new blood vessels is inhibited. A method for embolizing a blood vessel in a non-tumorigenic, angiogenesis-dependent diseases, comprising delivering to said vessel a therapeutically effective amount of a composition comprising taxol, such that said blood vessel is effectively occluded. P \OPER\JLR\71192-94.DIV 4/6/9

89- 31. A method for expanding the lumen of a body passageway, comprising inserting a stent into the passageway, the stent having a generaily tubular structure, the surface of said structure being coated with a composition comprising taxol, such that said passageway is expanded. 32. A method for eliminating vascular obstructions, comprising inserting a vascular stent into a vascular passageway, the stent having a generally tubular structure, the surface of said structure being coated with a composition comprising taxol, such that said vascular obstruction is eliminated. 33. A method for eliminating biliary obstructions, comprising inserting a biliary stent into a biliary passageway, the stent having a generally tubular structure, the surface of said structure being coated with a composition comprising taxol, such that said biliary obstruction is eliminated. 34. A method for eliminating urethral obstructions, comprising inserting a urethral stent into a urethra, the stent having a generally tubular structure, the surface of said structure being coated with a composition comprising taxol, such that said urethral obstruction is eliminated. A method for eliminating esophageal obstructions, comprising inserting an esophageal stent into an esophagus, the stent having a generally tubular structure, the surface of said structure being coated with a composition comprising taxol, such that said esophageal obstruction is eliminated. 36. A method for eliminating tracheal/bronchial obstructions, comprising inserting a tracheal/bronchial stent into the tracheal or bronchi, the stent having a generally tubular structure, the surface of said structure being coated with a composition comprising taxol, such that said tracheal/bronchial obstruction is eliminated. vPkoPrR\JLR\7II .DI .4,,698 90 Sa tumor excision site, comprising administering a composition comprising taxol to the resection margin of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at said site is inhibited. 38. A method for reatig cornea neovascularization, comprising administering a therapeutically effective amount of a composition comprising taxol to the cornea, such that the formation of new vessels is inhibited. 39. A pharmaceutical product, comprising: taxol, in a container; and a notice associated with said container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by said agency of said taxol, fotr human or veterinary administration to treat non- tumorigenic angiogenesis-dependent diseases. A stent for expanding the lumen of a body passageway, comprising a generally tubular structure coated with a composition comprising an anti-angiogenic factor and a polymeric carrier. 41. A stent according to claim 40 wherein said anti-angiogenic factor is a chemotherapeutic agent. 42. A stent according to claim 40 wherein said anti-angiogenic factor is selected from the group consisting of estramustine and methotrexate. 43. A stent according to claim 40 wherein said anti-angiogenic factor is taxol, or an analogue or derivative thereof. 44. A stent according to any one of claims 40 to 43 wherein said polymeric carrier comprises poly (carprolactone). P.\OPER\JLR\71 jqQ.94.DIV. 4 /619fl -91 A stent according to any one of claims 40 to 43 wherein said polymeric carrier comprises poly (lactic acid). 46. A stent according to any one of claims 40 to 43 wherein said polymeric carrier comprises poly (ethylene-vinyl acetate). 47. A stent according to any one of claims 40 to 43 wherein said polymeric carrier comprises a copolymer of poly caprolactone and poly lactic acid. 48. A stent according to any one of claims 40 to 47 wherein said stent is a vascular stent. 49. A stent according to. any one of claims 40 to 47 wherein said stent is a biliary stent. A stent according to any one of claims 40 to 47 wherein said stent is a urethral stent. 51. A stent according to any one of claims 40 to 47 wherein said stent is a esophageal stent. 52. A stent according to any one of claims 40 to 47 wherein said stent is a tracheal/bronchial stent. 53. A stent according to any one of claims 40 to 47 for treating narrowing of a body passageway. 54. A stent according to claim 53 for treating or preventing recurrent stenosis. Use of a composition comprising an anti-angiogenic factor for the manufacture of a medicament for treating arthritis. P.\OPER'JLR\711 92.9.DIV 416198 92 56. Use according to claim 55 wherein said anti-angiogenic factor is taxol, or an analogue or derivative thereof. 57. Use according to claims 55 or 56 wherein said anti-angiogenic factor further comprises a polymeric carrier. 58. Use according to claim 57 wherein said polymeric carrier comprises poly (caprolactone). 59. Use according to claim 57 wherein said polymeric carrier comprises poly (lactic acid). Use according to claim 57 wherein said polymeric carrier comprises poly (ethylene- vinyl acetate). 61. Use according to claim 57 wherein said polymeric carrier comprises poly (lactic-co- glycolic) acid. 62. Use according to any one of claims 55-61 wherein said arthritis is inflammatory arthritis. 63. Use according to any one of claims 55-62 wherein said arthritis is treated by intra- articular administration of said medicament. 64. Use of a composition comprising an anti-angiogenic factor and a polymeric carrier for coating a stent according to anyone of claims 40-54. Use of taxol, or an analogue or derivative thereof, for the manufacture of a medicament for anti-angiogenesis. 66. Use according to claim 65 wherein said medicament is for treating psoriasis. P \OPER\JLR\71192.94 DIV .4.6/98 93 67. Use according to claim 65 wherein said medicament is for treating vascular adhesions. 68. A stent according to any one of claims 40 to 43 wherein said polymeric carrier comprises gelatin. DATED this 18th day of February, 2004 Angiogenesis Technologies, Inc. AND The University of British Columbia By DAVIES COLLISON CAVE Patent Attorneys for the Applicant