WO2004070009A2 - Targeted multivalent macromolecules - Google Patents

Abstract

Targeted macromolecules comprising a dextran-coated liposome and more than one targeting entity are provided, as well as methods for their preparation and use.

Description

TARGETED MULTINALENT MACROMOLECULES

FIELD OF THE INVENTION The present invention concerns targeted agents suitable for a number of in vitro and in vivo applications, including therapeutics, imaging and diagnostics. More particularly, the present invention is concerned with macromolecules having more than one targeting and/or therapeutic entity.

BACKGROUND OF THE INVENTION Cancer remains one of the leading causes of death in the industrialized world. In the

United States, cancer is the second most common cause of death after heart disease, accounting for approximately one-quarter of the deaths in 1997. Clearly, new and effective treatments for cancer will provide significant health benefits. Among the wide variety of treatments proposed for cancer, targeted therapeutic agents hold considerable promise. In principle, a patient could tolerate much higher doses of a cytotoxic agent if the cytotoxic agent is targeted specifically to cancerous tissue, as healthy tissue should be unaffected or affected to a much smaller extent than the pathological tissue.

Integrins

The integrins are a class of proteins involved in the attachment of cells to matrix via RGD peptide sequences. Ruoslahti & Pierschbacher, Science (1987) 238:491-497. Their expression has been closely associated with many major disease processes involved in the formation of new blood vessels (angiogenesis) such as, osteoporosis, rheumatoid arthritis, macular degeneration and cancer. Folkman, Nature Medicine (1995) 1(1):27-31. The inhibition of the integrins is a new strategy to treat these diseases by either interfering directly with the function of these proteins (anti-angiogenesis) and/or the use of the integrins as an anchor for the delivery of pharmaceutical agents (vascular targeting). Schmitzer, New Eng. J. Med. (1998) 339(7):472-474; Eliceiri, & Cheresh, J. Clin. Invest. (1999) 103(9):1227-1230. Integrins are heterodimeric proteins having non-covalently linked α and β subunits that have multiple direct and indirect functions. Integrins regulate cell adhesion, they relay molecular cues in the cellular environment that influence cell survival and proliferation, and integrins activate phosphorylation of focal adhesion kinases (FAK) which in turn activate the Ras- extracellular signal-regulated kinase (ERK) signaling pathway. FAK, a cytoplasmic protein kinase, co-localizes with integrin clusters that interact with extracellular matrix molecules and FAK overexpression correlates with metastatic disease. Activation of FAK promotes cell survival and cell migration induced by integrins or growth factors.

Currently at least eleven different α subunits have been identified and at least six different β subunits have been identified. The various α subunits can combine with various β subunits to form distinct integrins. The integrin identified as α_vβ (also known as the vitronectin receptor) has been identified as an integrin that plays a role in various conditions or disease states including but not limited to tumor metastasis, solid tumor growth (neoplasia), osteoporosis, Paget's disease, humoral hypercalcemia of malignancy, angiogenesis, including tumor angiogenesis, antiangiogenesis, retinopathy, macular degeneration, arthritis, including rheumatoid arthritis, periodontal disease, psoriasis and smooth muscle cell migration (e.g., restenosis). Additionally, it has been found that such integrin inhibiting agents would be useful as antivirals, antifungals and antimicrobials. Thus, therapeutic agents that selectively inhibit or antagonize α_vβ would be beneficial for treating such conditions. It has been shown that the α_vβ₃ integrin binds to a number of Arg-Gly-Asp (RGD) containing matrix molecules, such as fibrinogen (Bennett et al, Proc. Natl. Acad. Sci. USA, Vol. 80 (1983) 2417), fibronectin (Ginsberg et al., J. Clin. Invest., Vol. 71 (1983) 619-624), and von Willebrand factor (Ruggeri et al, Proc. Natl. Acad. Sci. USA, Vol. 79 (1982) 6038). Compounds containing the RGD sequence mimic extracellular matrix ligands so as to bind to cell surface receptors. However, it is also known that RGD peptides in general are non-selective for RGD dependent integrins. For example, most RGD peptides that bind to α_vβ₃ also bind to α_vβ₅, α_vβι, and απ_bβni_a- Antagonism of platelet αn_bβπi_a (also known as the fibrinogen receptor) is known to block platelet aggregation in humans.

The α_vβ integrin is expressed at high levels in invasive cells, such as metastatic melanoma, and also in angiogenic cells. In comparison, expression levels of both molecules are low in pre-neoplastic cells or resting blood vessels. Thus, the α_vβ₃ integrin is considered a marker for tumor neovasculature.

In addition to their role in endothelial cell migration and adhesion, integrins, by binding to extracellular matrix molecules, can initiate pro-survival mechanisms to prevent apoptosis. It has been shown that the cytoplasmic domain of the unligated β₃ integrin subunit, which is highly conserved across mice, rats and humans, binds and activates caspase-8.

Activated caspases, in particular caspase-8, play an integral role in extrinsic and intrinsic apoptosis. Furthermore, it has been shown that FAK has a key role in cell survival, independent of matrix signaling . Thus, the activation of caspases and the interruption of the FAK signaling pathway can lead to apoptosis of invasive tumor cells.

Multivalency is a potentially powerful strategy for increasing the avidity of molecules for cell surface receptors. Mammen, et al., Angew. Chem. Int. Ed. (1998) 37:2754-2794. Polymers have been synthesized that contain multivalent arrays of RGD peptides and these materials have shown increased avidity to the integrin is in in vitro assays. Saiki, et al., Cancer Res. (1989) 49(14):3815-3822; Komazawa, et al., JBioact. Compat. Polym. (1993) 8:258-274; Oku, et al, Life Sci. (1996) 58(24):2263-2270; Kurohane, et al., Life Sci. (2000) 63(3):273-81; Maynard, et al., J. Am. Chem. Soc. (2001) 123:1275-1279. These materials also have been used to inhibit lung and liver metastasis in vivo in animal tumor models.

Targeted Therapeutics

Examples of the targeted therapeutic approach have been described in various patent publications and scientific articles. International Patent Application WO 93/17715 describes antibodies carrying diagnostic or therapeutic agents targeted to the vasculature of solid tumor masses through recognition of tumor vasculature-associated antigens. International Patent Application WO 96/01653 and U.S. Patent No. 5,877,289 describe methods and compositions for in vivo coagulation of tumor vasculature through the site-specific delivery of a coagulant using an antibody, while International Patent Application WO 98/31394 describes use of Tissue Factor compositions for coagulation and tumor treatment. International Patent

Application WO 93/18793 and U.S. Patent Nos. 5,762,918 and 5,474,765 describe steroids linked to polyanionic polymers which bind to vascular endothelial cells. International Patent Application WO 91/07941 and U.S. Patent No. 5,165,923 describe toxins, such as ricin A, bound to antibodies against tumor cells. U.S. Patent Nos. 5,660,827, 5,776,427, 5,855,866, and 5,863,538 also disclose methods of treating tumor vasculature. International Patent

Application WO 98/10795 and WO 99/13329 describe tumor homing molecules, which can be used to target drugs to tumors.

In Tabata, et al., Int. J. Cancer 1999 82:737-42, antibodies are used to deliver radioactive isotopes to proliferating blood vessels. Ruoslahti & Rajotte, Annu. Rev. Immunol. 2000 18:813-27; Ruoslahti, Adv. Cancer Res. 1999 76:1-20, review strategies for targeting therapeutic agents to angiogenic neovasculature, while Arap, et al., Science 1998 279:377-80 describe selection of peptides which target tumor blood vessels. It should be noted that the typical arrangement used in such systems is to link the targeting entity to the therapeutic entity via a single bond or a relatively short chemical linker. Examples of such linkers include SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane- 1-carboxylate) or the linkers disclosed in U.S. Patent No. 4,880,935, and oligopeptide spacers. Carbodiimides and N-hydroxysuccinimide reagents have been used to directly join therapeutic and targeting entities with the appropriate reactive chemical groups.

The use of cationic organic molecules to deliver heterologous genes in gene therapy procedures has been reported in the literature. Not all cationic compounds will complex with DNA and facilitate gene transfer. Currently, a primary strategy is routine screening of cationic molecules. The types of compounds which have been used in the past include cationic polymers such as polyethyleneamine, ethylene diamine cascade polymers, and polybrene. Proteins, such as polylysine with a net positive charge, have also been used. The largest group of compounds, cationic lipids; includes DOTMA, DOTAP, DMRIE, DC-chol, and DOSPA. All of these agents have proven effective but suffer from potential problems such as toxicity and expense in the production of the agents. Cationic liposomes are currently the most popular system for gene transfection studies. Cationic liposomes serve two functions: protect DNA from degradation and increase the amount of DNA entering the cell. While the mechanisms describing how cationic liposomes function have not been fully delineated, such liposomes have proven useful in both in vitro and in vivo studies. However, these liposomes suffer from several important limitations. Such limitations include low transfection efficiencies, expense in production of the lipids, poor colloidal stability when complexed to DNA, and toxicity.

Although conjugates of targeting entities with therapeutic entities via relatively small linkers have attracted much attention, far less attention has been focused on using large particles as linkers. Typically, the linker functions simply to connect the therapeutic and targeting entities, and consideration of linker properties generally focuses on avoiding interference with the entities linked, for example, avoiding a linkage point in the antigen binding site of an immunoglobulin.

Large particulate assemblies of biologically compatible materials, such as liposomes, have been used as carriers for administration of drugs and paramagnetic contrast agents. U.S.

Patent Numbers 5,077,057 and 5,277,914 teach preparation of liposome or lipidic particle suspensions having particles of a defined size, particularly lipids soluble in an aprotic solvent, for delivery of drugs having poor aqueous solubility. U.S. Patent No. 4,544,545 teaches phospholipid liposomes having an outer layer including a modified, cholesterol derivative to render the liposome more specific for a preselected organ. U.S. Patent No. 5,213,804 teaches liposome compositions containing an entrapped agent, such as a drug, which are composed of vesicle-forming lipids and 1 to 20 mole percent of a vesicle-forming lipid derivatized with hydrophilic biocompatible polymer and sized to control its biodistribution and recirculatory half life. U.S. Patent No. 5,246,707 teaches phospholipid-coated microcrystalline particles of bioactive material to control the rate of release of entrapped water-soluble biomolecules, such as proteins and polypeptides. U.S. Patent No. 5,158,760 teaches liposome encapsulated radioactive labeled proteins, such as hemoglobin.

Stabilization

The association of liposomes with polymeric compounds in order to avoid rapid clearance in the liver, or for other stabilizing effects, has been described. For example, Dadey, U.S. Patent No. 5,935,599 described polymer-associated liposomes containing a liposome, and a polymer having a plurality of anionic moieties in a salt form. The polymer may be synthetic or naturally-occurring. The polymer-associated liposomes remain in the vascular system for an extended period of time.

Polysaccharides are one class of polymeric stabilizer. Calvo Salve, et al., U.S. Patent 5,843,509 describe the stabilization of colloidal systems through the formation of lipid- polysaccharide complexes and development of a procedure for the preparation of colloidal systems involving a combination of two ingredients: a water soluble and positively charged polysaccharide and a negatively-charged phospholipid. Stabilization occurs through the formation, at the interface, of an ionic complex: aminopolysaccharide-phospholipid. The polysaccharides utilized by Calvo Salve, et al., include chitin and chitosan. Dextran is another polysaccharide whose stabilizing properties have been investigated.

Cansell, et al., J. Biomed. Mater. Res. 1999, 44:140-48, report that dextran or functionalized dextran was hydrophobized with cholesterol, which anchors in the lipid bilayer of liposomes during liposome formation, resulting in a liposome coated with dextran. These liposomes interacted specifically with human endothelial cells in culture. In Letourneur, et al., J. Controlled Release 2000, 65:83-91 , the antiproliferative functionalized dextran-coated liposomes were used as a targeting agent for vascular smooth muscle cells. Ullman, et al. Proc. Nat. Acad. Sci 91:5426-30 (1994) and Ullman, et al., Clin. Chem. 42:1518-26 (1996) describe the coating of polystyrene beads with dextran and the attachment of ligands, nucleic acids, and proteins to the dextran-polystyrene complexes.

Dextran has also been used to coat metal nanoparticles, and such nanoparticles have been used primarily as imaging agents. For example, Moore, et al., Radiology 2000, 214:568- 74, report that in a rodent model, long-circulating dextran-coated iron oxide nanoparticles were taken up preferentially by tumor cells, but also were taken up by tumor-associated macrophages and, to a much lesser extent, endothelial cells in the area of angiogenesis. Groman, et al., U.S. Patent No. 4,770,183, describe 10-5000 A superparamagnetic metal oxide particles for use as imaging agents. The particles may be coated with dextran or other suitable polymer to optimize both the uptake of the particles and the residence time in the target organ.

A dextran-coated iron oxide particle injected into a patient's bloodstream, for example, localizes in the liver. Groman, et al., also report that dextran-coated particles can be preferentially absorbed by healthy cells, with less uptake into cancerous cells.

Many of the liposomes disclosed in the prior art have undesirably poor stability. Thus, the prior art liposomes are more likely to rupture in vivo resulting, for example, in the untimely release of any therapeutic and/or diagnostic agent contained therein. Various studies have been conducted in an attempt to improve liposome stability. Such studies have included, for example, the preparation of liposomes in which the membranes or walls thereof comprise proteins, such as albumin, or materials which are apparently strengthened via crosslinking. See, e.g., Klaveness et al., WO 92/17212, in which there are disclosed liposomes which comprise proteins crosslinked with biodegradable crosslinking agents. A presentation was made by Moseley et al., at a 1991 Napa, California meeting of the Society for Magnetic Resonance in Medicine, which is summarized in an abstract entitled "Microbubbles: A Novel MR Susceptibility Contrast Agent." The microbubbles described by Moseley et al. comprise air coated with a shell of human albumin. Alternatively, membranes can comprise compounds which are not proteins but which are crosslinked with biocompatible compounds. See, e.g., Klaveness et al., WO 92/17436, WO 93/17718 and WO 92/21382.

Prior art techniques for stabilizing liposomes, including the use of proteins in the outer membrane, suffer from various drawbacks. The use in membranes of proteins, such as albumin, can impart rigidity to the walls of the bubbles. This results in bubbles having educed elasticity and, therefore, a decreased ability to deform and pass through capillaries. Thus, there is a greater likelihood of occlusion of vessels with prior art contrast agents that involve proteins. SUMMARY OF THE INVENTION

The present invention provides a targeted macromolecule comprising a liposome, said liposome comprising a stabilizing agent covalently attached to the surface of the liposome, and more than one targeting molecule, said targeting molecule being covalently attached to said stabilizing agent. The liposome may comprise l,2-dipalmitoyl-sn-glycero-3- phosphocholine; 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) and cholesterol. The targeting molecule is an integrin antagonist, including 3-{4-[2-(3,4,5,6- tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino}-2(S)-benzene-sulfonyl- aminopropionic acid. The integrin antagonist is attached to liposomes at loadings of about 5% (w/w), about 8% (w/w), about 10% (w/w), about 15% (w/w), about 20% (w/w), about 25%

(w/w), and about 30% (w/w).

The stabilizing agent is selected from the group consisting of dextran, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans, inulin, levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrose, dextrin, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch, natural homopolyner or heteropolymers containing one or more of the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, naturally occurring derivatives of the foregoing; proteins, albumin, polyalginates, and polylactide-glycolide copolymers, cellulose, cellulose (microcrystalline), methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, carboxymethylcellulose sodium 12, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, methoxycellulose, carboxydextran, aminodextran, dextran aldehyde, chitosan, carboxymethyl chitosan, poly(ethylene imine) and derivatives, polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes, Pluronics®, polyoxyethylene, and polyethylene terephthlate), polypropylenes, polypropylene glycol, polyurethanes, polyvinyl alcohol (PVA), polyvinyl chloride, polyvinylpyrrolidone, polyamides, nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers, polytetrafluoroethylene, acrylate, methacrylate, polymethylmethacrylate and derivatives thereof, polysorbate, carbomer 934P, magnesium aluminum silicate, aluminum monostearate, polyethylene oxide, polyvinylalcohol, povidone, polyethylene glycol, and propylene glycol, and in some embodiments is dextran or modified dextran.

A particular embodiment of the targeted macromolecule described herein is IA-DCL. The present invention also provides a method of preparing a targeted macromolecule, comprising providing a liposome; covalently associating the liposomes with a stabilizing agent to generate coated liposomes; attaching at least one linker to the coated liposomes to generate linker-attached liposomes via the stabilizing agent, wherein the linker facilitates attachment of a targeting molecule; and attaching at least one targeting molecule to the linker- attached liposome via the linker to prepare the targeted macromolecule.

In some embodiments of this method, the liposome comprises 1,2-dipalmitoyl-sn- glycero-3 -phosphocholine; 1 ,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine-N-(succinyl) and cholesterol.

In some embodiments of this method, the targeting molecule is an integrin antagonist, including 3-{4-[2-(3 ,4,5, 6-tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino}-2(S)- benzene-sulfonyl-aminopropionic acid, and which can be attached to liposomes at loadings of up to a member of the group consisting of about 5% (w/w), about 8% (w/w), about 10%

(w/w), about 15% (w/w), about 20% (w/w), about 25% (w/w), and about 30% (w/w).

In some embodiments, the liposome is prepared using a lipid solution in t-butanol. The stabilizing agent used in the method be selected from the group consisting of dextran, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans, inulin, levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrose, dextrin, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch, natural homopolyner or heteropolymers containing one or more of the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, naturally occurring derivatives of the foregoing; proteins, albumin, polyalginates, and polylactide-glycolide copolymers, cellulose, cellulose (microcrystalline), methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, carboxymethylcellulose sodium 12, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, methoxycellulose, carboxydextran, aminodextran, dextran aldehyde, chitosan, carboxymethyl chitosan, poly(ethylene imine) and derivatives, polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes, Pluronics®, polyoxyethylene, and polyethylene terephthlate), polypropylenes, polypropylene glycol, polyurethanes, polyvinyl alcohol (PVA), polyvinyl chloride, polyvinylpyrrolidone, polyamides, nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers, polytetrafluoroethylene, acrylate, methacrylate, polymethylmethacrylate and derivatives thereof, polysorbate, carbomer 934P, magnesium aluminum silicate, aluminum monostearate, polyethylene oxide, polyvinylalcohol, povidone, polyethylene glycol, and propylene glycol, especially, dextran or modified dextran.

The present invention also provides a targeted macromolecule prepared by these methods, including in one embodiment the targeted macromolecule designated as IA-DCL. The present inventions also provides methods for targeting an agent to a site of pathology comprising administering a targeted macromolecule of the invention to a patient in need thereof. Additionally, the invention provides method of treating a disease accompanied by a condition selected from the group consisting of neovascularization, aberrant vascular growth, and excessive or abnoπnal stimulation of endothelial cells, comprising administering a targeted macromolecule to a patient in need thereof. In some embodiments, the administration is intravenous.

The site of pathology or the disease includes solid tumors, blood-borne tumors, leukemias, tumor metastasis, malignant gliomas, anaplastic astrocytomas, anaplastic oligodendrogliomas, glioblastoma multiforme, benign tumors, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis, psoriasis; chronic inflammation; ocular angiogenic diseases, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neo vascular glaucoma, retrolental fibroplasia, rubeosis; arteriovenous malformations; ischemic limb angiogenesis; Osier-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma, wound granulation, intestinal adhesions, atherosclerosis, restenosis, scleroderma, and hypertrophic scars, and keloids.

Additionally, the invention provides a method for targeting a liposome containing a therapeutic agent comprising covalently associating the liposomes with a stabilizing agent to generate coated liposomes, attaching at least one linker to the coated liposomes to generate linker-attached liposomes via the stabilizing agent, wherein the linker facilitates attachment of a targeting molecule, attaching at least one targeting molecule to the linker-attached liposome via the linker, and administering the resulting composition to a patient in need thereof, in order to target the liposome.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1 A-I shows schematics of exemplary therapeutic constructs of the present invention. Lipid constructs that form micelles or vesicles are preferred carriers.

Figure 1 A shows a polymer-coated carrier with targeting agent 54 and an encapsulated therapeutic agent 56. The polymer coat 52 is external relative to vesicle 50. Figure IB shows a polymer-coated carrier with targeting agent 54 and a therapeutic agent 56 that is associated with the components of the vesicle. The polymer coat 52 is external relative to vesicle 50.

Figure 1C shows carrier 50 with targeting agent 54 and an encapsulated therapeutic agent 56. Figure ID shows carrier 50 with targeting agent 54 and a therapeutic agent 56 that is associated with the components of the vesicle.

Figure IE shows a polymer-coated carrier with targeting agent 54 and a therapeutic agent 56 that is associated with the surface of the vesicle. The polymer coat 52 is external relative to vesicle 50. Figure IF shows carrier 50 with targeting agent 54 and therapeutic agent 56, which is attached to the surface of the vesicle by covalent or non-covalent means.

Figure 1G shows a polymer-coated carrier with a therapeutic agent 56 that is associated with the surface of the vesicle by covalent or non-covalent means. The polymer coat 52 is external relative to vesicle 50. Figure 1H shows therapeutic agent 56 attached to the surface of carrier 50 by covalent or non-covalent means. Figure II shows a polymer-coated carrier with targeting agent 54 and therapeutic agent 56 that are associated with the polymer coat by covalent or non-covalent means. The polymer coat 52 is external relative to vesicle 50.

Figure 2. Coupling of a ligand Z-Y-L where Z is a chemically reactive moiety covalently attached to a spacer Y that is covalently attached to ligand L. This conjugation may require an activating agent such as a carbodiimide derivative or reducing agent.

Figure 3 shows the structure of N-succinyl-DPPE, sodium salt.

Figure 4 shows the structure of Ν-caproylamine-DPPE hydrochloride.

Figures 5-15 show exemplary lipids with a variety of functionalities for linking a lipid to a targeting entity or therapeutic entity, and showing various spacer groups.

Figure 16 shows the structure of a typical phosphatidylcholine lipid.

Figure 17A and 17B show the inhibition of tumor growth by IA-DCLs in the M21 melanoma model in nu/nu mice: A) Comparison of IA-DCLs, IA alone, DCLs alone, and saline. B) Additional experiment comparing control peptide cyclo(RGDfN) with IA-DCLs, DCLs, and saline. In both experiments, animals were dosed on days 0, 2, 4, and 6. Drug dose for IA, IA-DCLs, and cyclo(RGDfV) was 15 mg/kg fore each dose, and the DCL dose was equivalent to the DCL exposure in the IA-DCL groups. Volumes were calculated from measurements of subcutaneous, dorsal tumor cell implants.

Figure 18 shows normalized tumor volume eleven days post treatment sorted by treatment group. Grey lines indicate the mean tumor volume for the treatment group n=9.

Figure 19 is a scattergram of percentage human tumor cells in xenograft brain tissue as determined by FACS analysis with anti-human HLA class I. Samples were obtained from mice treated with IA-DCL and saline 20 days post tumor implantation. Each point represents one animal; lines indicate treatment mean. Figure 20 shows a time course of ^,4C-labeled IA-DCL and ¹⁴C -DCL in plasma

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed toward novel targeting molecules which bind specifically and with high avidity to biological targets and methods for their preparation. This invention relates to stabilized therapeutic and imaging agents, examples of which are shown schematically in Figure 1 A-1I which are comprised of a macromolecule, 50, a stabilizing agent, 52, a targeting entity 54, and/or a therapeutic or treatment entity, 56. As depicted in Figure 1 A and IB, the targeting and/or therapeutic entities may be associated with the lipid construct or the stabilizing entity. Figures IA, IB, IC, and ID show examples comprise both a therapeutic or targeting agent, but the agents of the invention may contain a therapeutic entity, a targeting entity, or both. Additionally, the therapeutic entity may be encapsulated within the lipid construct, or may be associated with the surface of the lipid construct or stabilizing agent. It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, a therapeutic entity refers to one or more therapeutic entities or at least one therapeutic entity. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising," "including," and "having" can be used interchangeably. The targeted agents of the present invention comprise more than one targeting entity.

In some embodiments, the targeted agents comprise three or more targeting entities. In other embodiments, the targeted agents comprise ten or more targeting entities. In other embodiments, the targeted agents comprise 100 or more targeting entities. In other embodiments, the targeted agents comprise 1000 or more targeting entities. Examples are provided herein describing the preparation of such multivalent targeting agents, including agents comprising 0.1-30 mol% of the targeting entity.

More particularly, this invention relates to therapeutic and imaging agents which are comprised of a lipid construct, more than one targeting entity, and a therapeutic or imaging entity. Liposomes

As used herein, lipid refers to an agent exhibiting amphipathic characteristics causing it to spontaneously adopt an organized structure in water wherein the hydrophobic portion of the molecule is sequestered away from the aqueous phase. A lipid in the sense of this invention is any substance with characteristics similar to those of fats or fatty materials. As a rule, molecules of this type possess an extended apolar region and, in the majority of cases, also a water-soluble, polar, hydrophilic group, the so-called head-group. Phospholipids are lipids which are the primary constituents of cell membranes. Typical phospholipid hydrophilic groups include phosphatidylcholine (Figure 16) and phosphatidylethanolamine moieties, while typical hydrophobic groups include a variety of saturated and unsaturated fatty acid moieties, including diacetylenes. Mixture of a phospholipid in water causes spontaneous organization of the phospholipid molecules into a variety of characteristic phases depending on the conditions used. These include bilayer structures in which the hydrophilic groups of the phospholipids interact at the exterior of the bilayer with water, while the hydrophobic groups interact with similar groups on adjacent molecules in the interior of the bilayer. Such bilayer structures can be quite stable and form the principal basis for cell membranes.

Bilayer structures can also be formed into closed spherical shell-like structures which are called vesicles or liposomes. The liposomes employed in the present invention can be prepared using any one of a variety of conventional liposome preparatory techniques. As will be readily apparent to those skilled in the art, such conventional techniques include sonication, chelate dialysis, homogenization, solvent infusion coupled with extrusion, freeze-thaw extrusion, microemulsification, as well as others. These techniques, as well as others, are discussed, for example, in U.S. Pat. No. 4,728,578, U.K. Patent Application G.B. 2193095 A, U.S. Pat. No. 4,728,575, U.S. Pat. No. 4,737,323, International Application PCT/US85/01161,

Mayer et al., Biochimica et Biophysica Acta, Vol. 858, pp. 161-168 (1986), Hope et al., Biochimica et Biophysica Acta, Vol. 812, pp. 55-65 (1985), U.S. Pat. No. 4,533,254, Mahew et al., Methods In Enzymology, Vol. 149, pp. 64-77 (1987), Mahew et al., Biochimica et Biophysica Acta, Vol. 75, pp. 169-174 (1984), and Cheng et al., Investigative Radiology, Vol. 22, pp. 47-55 (1987), and U.S. Ser. No. 428,339, filed Oct. 27, 1989. The disclosures of each of the foregoing patents, publications and patent applications are incorporated by reference herein, in their entirety. A solvent free system similar to that described in International Application PCT/US85/01161, or U.S. Ser. No. 428,339, filed Oct. 27, 1989, may be employed in preparing the liposome constructions. By following these procedures, one is able to prepare liposomes having encapsulated therein a gaseous precursor or a solid or liquid contrast enhancing agent.

The materials which may be utilized in preparing the liposomes of the present invention include any of the materials or combinations thereof known to those skilled in the art as suitable in liposome construction. The lipids used may be of either natural or synthetic origin. Such materials include, but are not limited to, lipids such as cholesterol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, lysolipids, fatty acids, sphingomyelin, glycosphingolipids, glucolipids, glycolipids, sulphatides, lipids with amide, ether, and ester- linked fatty acids, polymerizable lipids, and combinations thereof. As one skilled in the art will recognize, the liposomes may be synthesized in the absence or presence of incorporated glycolipid, complex carbohydrate, protein or synthetic polymer, using conventional procedures. The surface of a liposome may also be modified with a polymer, such as, for example, with polyethylene glycol (PEG), using procedures readily apparent to those skilled in the art. Lipids may contain functional surface groups for attachment to a metal, which provides for the chelation of radioactive isotopes or other materials that serve as the therapeutic entity. Any species of lipid may be used, with the sole proviso that the lipid or combination of lipids and associated materials incorporated within the lipid matrix should form a bilayer phase under physiologically relevant conditions. As one skilled in the art will recognize, the composition of the liposomes may be altered to modulate the biodistribution and clearance properties of the resulting liposomes.

Common adjuvants include cholesterol and alpha-tocopherol, among others. The lipid constructs may be used alone or in any combination which one skilled in the art would appreciate to provide the characteristics desired for a particular application. In addition, the technical aspects of lipid construct, vesicle, and liposome formation are well known in the art and any of the methods commonly practiced in the field may be used for the present invention. The therapeutic or treatment entity may be associated with the agent by covalent or non-covalent means. As used herein, associated means attached to by covalent or noncovalent interactions.

The membrane bilayers in these structures typically encapsulate an aqueous volume, and form a permeability baπier between the encapsulated volume and the exterior solution. Lipids dispersed in aqueous solution spontaneously form bilayers with the hydrocarbon tails directed inward and the polar headgroups outward to interact with water. Simple agitation of the mixture usually produces multilamellar vesicles (MLVs), structures with many bilayers in an onion-like form having diameters of 1-10 μm (1000- 10,000 nm). Sonication of these structures, or other methods known in the art, leads to formation of unilamellar vesicles (UVs) having an average diameter of about 30-300 nm. However, the range of 50 to 200 nm is considered to be optimal from the standpoint of, e.g., maximal circulation time in vivo. The actual equilibrium diameter is largely determined by the nature of the phospholipid used and the extent of incorporation of other lipids such as cholesterol. Standard methods for the formation of liposomes are known in the art, for example, methods for the commercial production of liposomes are described in U.S. Pat. No. 4,753,788 to Ronald C. Gamble and U.S. Pat. No. 4,935,171 to Kevin R. Bracken. Either as MLVs or UVs, liposomes have proven valuable as vehicles for drug delivery in animals and in humans. Active drugs, including small hydrophilic molecules and polypeptides, can be trapped in the aqueous core of the liposome, while hydrophobic substances can be dissolved in the liposome membrane. Other molecules, such as DNA or RNA, may be attached to the outside of the liposome for gene therapy or gene delivery applications. The liposome structure can be readily injected and form the basis for both sustained release and drug delivery to specific cell types, or parts of the body. MLVs, primarily because they are relatively large, are usually rapidly taken up by the reticuloendothelial system (the liver and spleen). The invention typically utilizes vesicles which remain in the circulatory system for hours and break down after internalization by the target cell. For these requirements the formulations preferably utilize UVs having a diameter of less than 200 nm, preferably less than 100 nm.

Therapeutic Entities The term "therapeutic entity" refers to any molecule, molecular assembly or macromolecule that has a therapeutic effect in a treated subject, where the treated subject is an animal, preferably a mammal, more preferably a human. The term "therapeutic effect" refers to an effect which reverses a disease state, arrests a disease state, slows the progression of a disease state, ameliorates a disease state, relieves symptoms of a disease state, or has other beneficial consequences for the treated subject. Therapeutic entities include, but are not limited to, drugs, including antibiotics, drugs such as doxorabicin, paclitaxel, and other chemotherapy agents including camptothecin and topotecan; small molecule therapeutic drugs, toxins such as ricin; radioactive isotopes; genes encoding proteins that exhibit cell toxicity, and prodrugs (drugs which are introduced into the body in inactive form and which are activated in situ).

Radioisotopes useful as therapeutic entities are described in Kairemo, et al., Acta Oncol. 35:343-55 (1996), and include Y-90, 1-123, 1-125, 1-131, Bi-213, At-211, Cu-67, Sc- 47, Ga-67, Rh-105, Pr-142, Nd-147, Pm-151, Sm-153, Ho-166, Gd-159, Tb-161, Eu-152, Er- 171, Re-186, and Re-188. Additional therapeutic agents include but are not limited to cytotoxic or cytostatic agents that target growth factors, cell cycle modulators, Bcl-2, TNF-α receptor, cyclin- dependent kinases, the Ras pathway, the EGFR pathway, and other relevant cellular pathways, proteins involved in multi-drug resistance including p-glycoprotein, tubulins, DNA, RNA, topoisomerases, telomerases, and kinases, and enzymes involved in DNA methylation. These therapeutic agents may be alkylating agents, cisplatinum and derivatives, pyrimidine and purine analogues, topoisomerase inhibitors, microtuble-targeting agents, estrogen derivatives, androgen derivatives, interferons, intercalating agents, and MDR inhibitors, for example. Specific agents include tubulin-binding molecules vincristine, vinblastine, vindesine, and vinorelbine.

In preferred embodiments of the present invention, the therapeutic entity is associated by covalent or non-covalent means with the macromolecule.

Stabilizing entities

The agents of the present invention preferably contain a stabilizing entity. As used herein, "stabilizing" refers to the ability to imparts additional advantages to the therapeutic or imaging agent, for example, physical stability, i.e., longer half-life, colloidal stability, and/or capacity for multivalency; that is, increased payload capacity due to numerous sites for attachment of targeting agents. As used herein, "stabilizing entity" refers to a macromolecule or polymer, which may optionally contain chemical functionality for the association of the stabilizing entity to the surface of the vesicle, and/or for subsequent association of therapeutic entities or targeting agents. The polymer should be biocompatible with aqueous solutions. Polymers useful to stabilize the liposomes of the present invention may be of natural, semi- synthetic (modified natural) or synthetic origin. A number of stabilizing entities which may be employed in the present invention are available, including xanthan gum, acacia, agar, agarose, alginic acid, alginate, sodium alginate, carrageenan, gelatin, guar gum, tragacanth, locust bean, bassorin, karaya, gum arabic, pectin, casein, bentonite, unpurified bentonite, purified bentonite, bentonite magma, and colloidal bentonite. Other natural polymers include naturally occurring polysaccharides, such as, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fϊicoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrose, dextrin, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch and various other natural homopolyner or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof. Other suitable polymers include proteins, such as albumin, polyalginates, and polylactide-glycolide copolymers, cellulose, cellulose (microcrystalline), methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, and calcium carboxymethylcellulose. Exemplary semi-synthetic polymers include carboxymethylcellulose, sodium carboxymethylcellulose, carboxymethylcellulose sodium 12, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose. Other semi-synthetic polymers suitable for use in the present invention include carboxydextran, aminodextran, dextran aldehyde, chitosan, and carboxymethyl chitosan. Exemplary synthetic polymers include poly(ethylene imine) and derivatives, polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes (such as, for example, polyethylene glycol, the class of compounds referred to as Pluronics®, commercially available from BASF, (Parsippany, N. J.), polyoxyethylene, and polyethylene terephthlate), polypropylenes (such as, for example, polypropylene glycol), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinyl chloride and polyvinylpyrrolidone), polyamides including nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers (such as, for example, polytetrafluoroethylene), acrylate, methacrylate, and polymethylmethacrylate, and derivatives thereof, polysorbate, carbomer 934P, magnesium aluminum silicate, aluminum monostearate, polyethylene oxide, polyvinylalcohol, povidone, polyethylene glycol, and propylene glycol.

Methods for the preparation of vesicles which employ polymers to stabilize vesicle compositions will be readily apparent to one skilled in the art, in view of the present disclosure, when coupled with information known in the art, such as that described and referred to in Unger, U.S. Pat. No. 5,205,290, the disclosure of which is hereby incorporated by reference herein in its entirety.

In a preferred embodiment, the stabilizing entity is dextran. In another preferred embodiment, the stabilizing entity is a modified dextran, such as amino dextran. In a further preferred embodiment, the stabilizing entity is poly(ethylene imine) (PEI). Without being bound by theory, it is believed that dextran may increase circulation times of liposomes in a manner similar to PEG. Additionally, each polymer chain (i.e. aminodextran or succinylated aminodextran) contains numerous sites for attachment of targeting agents, providing the ability to increase the payload of the entire lipid construct. This ability to increase the payload differentiates the stabilizing agents of the present invention from PEG. For PEG there is only one site of attacliment, thus the targeting agent loading capacity for PEG (with a single site for attachment per chain) is limited relative to a polymer system with multiple sites for attachment.

In other preferred embodiments, the following polymers and their derivatives are used. Poly(galacturonic acid), poly(L-glutamic acid), poly(L-glutamic acid-L-tyrosine), poly[R)-3- hydroxybutyric acid], poly(inosinic acid potassium salt), poly(L-lysine), poly(acrylic acid), poly(ethanolsulfonic acid sodium salt), poly(methylhydrosiloxane), poly( vinyl alcohol), poly(vinylpolypyrrolidone), poly(vinylpyrrolidone), poly(glycolide), poly(lactide), poly(lactide-co-glycolide), and hyaluronic acid. In other preferred embodiments, copolymers including a monomer having at least one reactive site, and preferably multiple reactive sites, for the attachment of the copolymer to the vesicle or other molecule.

In some embodiments, the polymer may act as a hetero- or homobifunctional linking agent for the attachment of targeting agents, therapeutic entities, proteins or chelators such as DTP A and its derivatives. In one embodiment, the stabilizing entity is associated with the vesicle by covalent means. In another embodiment, the stabilizing entity is associated with the vesicle by noncovalent means. Covalent means for attaching the targeting entity with the liposome are known in the art and described in the EXAMPLES section.

Noncovalent means for attaching the targeting entity with the liposome include but are not limited to attachment via ionic, hydrogen-bonding interactions, including those mediated by water molecules or other solvents, hyrdophobic interactions, or any combination of these. In a preferred embodiment, the stabilizing agent forms a coating on the liposome.

Targeting Entities

The term "targeting entity" refers to a molecule, macromolecule, or molecular assembly which binds specifically to a biological target. Examples of targeting entities include, but are not limited to, antibodies (including antibody fragments and other antibody- derived molecules which retain specific binding, such as Fab, F(ab')2, Fv, and scFv derived from antibodies); receptor-binding ligands, such as hormones or other molecules that bind specifically to a receptor; cytokines, which are polypeptides that affect cell function and modulate interactions between cells associated with immune, inflammatory or hematopoietic responses; molecules that bind to enzymes, such as enzyme inhibitors; nucleic acid ligands or aptamers, and one or more members of a specific binding interaction such as biotin or iminobiotin and avidin or streptavidin. Preferred targeting entities are molecules which specifically bind to receptors or antigens found on vascular cells. More preferred are molecules which specifically bind to receptors, antigens or markers found on cells of angiogenic neovasculature or receptors, antigens or markers associated with tumor vasculature. The receptors, antigens or markers associated with tumor vasculature can be expressed on cells of vessels which penetrate or are located within the tumor, or which are confined to the inner or outer periphery of the tumor. In one embodiment, the invention takes advantage of pre-existing or induced leakage from the tumor vascular bed; in this embodiment, tumor cell antigens can also be directly targeted with agents that pass from the circulation into the tumor interstitial volume.

Other targeting entities target endothelial receptors, tissue or other targets accessible through a body fluid or receptors or other targets upregulated in a tissue or cell adjacent to or in a bodily fluid. For example, targeting entities attached to carriers designed to deliver drugs to the eye can be injected into the vitreous, choroid, or sclera; or targeting agents attached to carriers designed to deliver drugs to the joint can be injected into the synovial fluid.

The targeting entity may have other effects, including therapeutic effects, in addition to specifically binding to a target. For example, the targeting entity may modulate the function of an enzyme target. By "modulate the function" it is meant altering when compared to not adding the targeting entity. In most cases, a preferred form of modulation of function is inhibition. Examples of targeting agents which may have other functions or effects are described herein. Other targeting entities that fall into this category include Combrestastatin A4 Prodrug (CA4P) (Oxigene/BMS) which may be used as a vascular targeting agent that also acts as an anti-angiogenesis agent, and Cidecin (Cubist Pharm/Emisphere) a cyclic lipopeptide used as a bactericidal and anti-inflammatory agent. Targeting entities attached to the macromolecules of the invention include, but are not limited to, small molecule ligands, such as carbohydrates, and compounds such as those disclosed in U.S. Patent No. 5,792,783 (small molecule ligands are defined herein as organic molecules with a molecular weight of about 5000 daltons or less); proteins, such as antibodies and growth factors; peptides, such as RGD-containing peptides (e.g. those described in U.S. Patent No. 5,866,540), bombesin or gastrin-releasing peptide, peptides selected by phage- display techniques such as those described in U.S. Patent No. 5,403,484, and peptides designed de novo to be complementary to tumor-expressed receptors; antigenic determinants; or other receptor targeting groups. These targeting entities can be used to control the biodistribution, non-specific adhesion, and blood pool half-life of the lipid constructs. For example, β-D-lactose targets the asialoglycoprotein (ASG) found in liver cells which are in contact with the circulating blood pool. Glycolipids can be derivatized for use as targeting entities by converting the commercially available lipid (DAGPE) or PEG-PDA amines into glycolipids.

In some embodiments, the targeting entity targets the liposomes to a cell surface. Delivery of the therapeutic or imaging agent can occur through endocytosis of the liposomes. Such deliveries are known in the art. See, for example, Mastrobattista, et al., Immunoliposomes for the Targeted Delivery of Antitumor Drugs, Adv. Drug Del. Rev. (1999) 40:103-27.

In one embodiment, the attachment is by covalent means. In another embodiment, the attachment is by non-covalent means. For example, antibody targeting entities may be attached by a biotin-avidin biotinylated antibody sandwich to allow a variety of commercially available biotinylated antibodies to be used on the coated liposome. In a preferred embodiment, the targeting entity is a small molecule ligand peptidomimetic which binds to chemokine receptors CCR4 and CCR5, VCAM, EGFR, FGFR, matrix metalloproteases (MMPs) including surface associated MMPs, PDGFR, P- and E-selectins, pleiotropin, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, Axl, α_vβ₃, α_vβ₅, ₅βι, α₄βι, αiβi, α₂β₂, or prostate specific membrane antigen (PSMA). Additional targets are described by E. Ruoslahti in Nature Reviews: Cancer, 2, 83-90 (2002). Further targets include the CD family of cell surface antigens including CDl through CD 178, and any target that is accessible to the targeting agent by administration to a patient including extracellular matrix components that are exposed in diseased tissue but less so in normal tissue. Examples of targeting entities which may be used in the targeted agents of the present invention include, but are not limited to Conivaptan (Yamanouchi Pharm.), a VI & V2 vasopressin receptor antagonist; GBC-590 (Abbott/GlycoGenesys), a lectin inhibitor useful in prevention of metastasis; Veletri (Actelion), an endothelin antagonist (tesosentan); VLA-4 Antagonist (Aventis) an agent with potential for treating rheumatoid arthritis, multiple sclerosis, cardiovascular disease and other conditions; Campath (Berlex/Millenium), a monoclonal antibody specific for CD52+ malignant lymphocytes; Tracleer (Actelion), an endothelin antagonist (bosentan) approved for the treatment of pulmonary arterial hypotension; and Natrecor (Scios), a natriuretic peptide that binds to vascular smooth muscle cells and endothelial cells.

In a preferred embodiment, the targeting entity is an integrin-specific molecule. The integrin specific molecule may be an RGD peptide or derivative thereof. Other integrin- specific molecules are described, for instance, in U.S. Pat. No. 5,561,148; U.S. Patent No.

6,204,280, International Publication No. WO 01/14338, and International Publication No. WO 01/14337. In a particularly preferred embodiment, the targeting entity is compound 10, 3-{4- [2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino}-2(S)-benzene-sulfonyl- aminopropionic acid, and the target is α_vβ₃. In another embodiment, the integrin-specific molecule is Cilengitide. In another particularly preferred embodiment, the targeting entity is a protease inhibitor such as N-acetyl-Leu-Val-Lys-aldehyde (Bachem Ν-1380) or Gly-Phe-Gly- aldehyde semicarbazone (Bachem C-3085) and the target is papain or cathespin B.

An antitumor agent can be a conventional antitumor therapy, such as cisplatin; antibodies directed against tumor markers, such as anti-Her2/neu antibodies (e.g., Herceptin); or tripartite agents, such as those described herein for vascular-targeted therapeutic agents, but targeted against the tumor cell rather than the vasculature. A summary of monoclonal antibodies directed against various tumor markers is given in Table I of U.S. Patent No. 6,093,399, hereby incorporated by reference herein in its entirety. In general, when the vascular-targeted therapy agent compromises vascular integrity in the area of the tumor, the effectiveness of any drug which operates directly on the tumor cells can be enhanced.

In one embodiment of the invention, a vascular-targeted therapeutic agent is combined with an agent targeted directly towards tumor cells. This embodiment takes advantage of the fact that the neo vasculature surrounding tumors is often highly permeable or "leaky," allowing direct passage of materials from the bloodstream into the interstitial space surrounding the tumor. Alternatively, the targeted therapeutic agent itself can induce permeability in the tumor vasculature. For example, when the agent carries a radioactive therapeutic entity, upon binding to the vascular tissue and irradiating that tissue, cell death of the vascular epithelium will follow and the integrity of the vasculature will be compromised.

Accordingly, in one embodiment, the vascular-targeted therapeutic agent has two targeting entities: a targeting entity directed towards a vascular marker, and a targeting entity directed towards a tumor cell marker. In another embodiment, an antitumor agent is administered with the vascular-targeted therapy agent. The antitumor agent can be administered simultaneously with the vascular-targeted therapy agent, or subsequent to administration of the vascular-targeted therapy agent. In particular, when the vascular- targeted therapy agent is relied upon to compromise vascular integrity in the area of the tumor, administration of the antitumor agent is preferably done at the point of maximum damage to the tumor vasculature. The size of the vesicles can be adjusted for the particular intended end use including, for example, diagnostic and/or therapeutic use. As the size of the macromolecule can be manipulated readily, the overall size of the vascular-targeted therapeutic agents can be adapted for optimum passage of the particles through the permeable ("leaky") vasculature at the site of pathology, as long as the agent retains sufficient size to maintain its desired properties (e.g., circulation lifetime, multi valency). Accordingly, the particles can be sized at

30, 50, 100, 150, 200, 250, 300 or 350 nm in size, as desired. In addition, the size of the particles can be chosen so as to permit a first administration of particles of a size that cannot pass through the permeable vasculature, followed by one or more additional administrations of particles of a size that can pass through the permeable vasculature. The size of the vesicles may preferably range from about 30 nanometers (nm) to about 400 nm in diameter, and all combinations and subcombinations of ranges therein. More preferably, the vesicles have diameters of from about 10 nm to about 500 nm, with diameters from about 40 nm to about 120 nm being even more preferred. In connection with particular uses, for example, intravascular use, including magnetic resonance imaging of the vasculature, it may be preferred that the vesicles be no larger than about 500 nm in diameter, with smaller vesicles being preferred, for example, vesicles of no larger than about 100 nm in diameter. It is contemplated that these smaller vesicles may perfuse small vascular channels, such as the microvasculature, while at the same time providing enough space or room within the vascular channel to permit red blood cells to slide past the vesicles. Further therapeutics contemplated for use in the invention include but are not limited to AGI-1067 (Atherogenics), for the treatment of restenosis, nystatin, an antifungal agent, and Gleevec, which blocks Bcr-Abl intracellular protein in white blood cells.

While one major focus of the invention is the use of vascular-targeted therapy agent against the vasculature of tumors in order to treat cancer, the agents of the invention can be used in any disease where neovascularization or other aberrant vascular growth accompanies or contributes to pathology. Diseases associated with neovascular growth include, but are not limited to, solid tumors; blood-borne tumors such as leukemias; tumor metastasis; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; chronic inflammation; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; arteriovenous malformations; ischemic limb angiogenesis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation. Diseases of excessive or abnormal stimulation of endothelial cells include, but are not limited to, intestinal adhesions, atherosclerosis, restenosis, scleroderma, and hypertrophic scars, i.e., keloids.

Differing administration vehicles, dosages, and routes of administration can be determined for optimal administration of the agents; for example, injection near the site of a tumor may be preferable for treating solid tumors. Therapy of these disease states can also take advantage of the permeability of the neo vasculature at the site of the pathology, as discussed above, in order to specifically deliver the vascular-targeted therapeutic agents to the interstitial space at the site of pathology.

Targeted Multivalent Agents

The liposome can be coupled to the targeting entity and the therapeutic entity by a variety of methods, depending on the specific chemistry involved. The coupling can be covalent or non-covalent. A variety of methods suitable for coupling of the targeting entity and the therapeutic entity to the macromolecule can be found in Hermanson, "Bioconjugate Techniques", Academic Press: New York, 1996; and in "Chemistry of Protein Conjugation and Cross-linking" by S.S. Wong, CRC Press, 1993. Specific coupling methods include, but are not limited to, the use of bifunctional linkers, carbodiimide condensation, disulfide bond formation, and use of a specific binding pair where one member of the pair is on the macromolecule and another member of the pair is on the therapeutic or targeting entity, e.g. a biotin-avidin interaction.

A schematic of the coupling of a ligand Z-Y-L where Z is a chemically reactive moiety covalently attached to a spacer Y that is covalently attached to ligand L is shown in Figure 2. This conjugation may require an activating agent.

Generally, prior to forming the linkage between the targeting entity and the lipid, macromolecule, and/or optionally, the spacer group, at least one of the chemical functionalities will be activated. One skilled in the art will appreciate that a variety of chemical functionalities, including hydroxy, amino, and carboxy groups, can be activated using a variety of standard methods and conditions. For example, a hydroxyl group of the ligand or lipid can be activated through treatment with phosgene to form the corresponding chloro formate. In addition, if the hydroxyl functionality is part of a sugar residue, then the hydroxyl group can be activated through reaction with di-(n-butyl)tin oxide to form a tin

5 complex.

Carboxy groups may be activated by conversion to the corresponding acyl halide. This reaction may be performed under a variety of conditions as illustrated in Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fourth Ed., at 388-89. In one embodiment, the acyl halide is prepared through the reaction of the carboxy containing

10 group with oxalyl chloride.

Typically, the lipid or macromolecule is linked covalently to a targeting entity using standard chemical techniques through their respective chemical functionalities. Optionally, the targeting entity can be coupled to the lipid or liking carrier through one or more spacer groups. The spacer groups can be equivalent or different when used in combination.

[ 5 The lipid-targeting agent complex is prepared by linking a lipid to a targeting entity

(or optionally to a spacer group which has been or will be attached to a targeting group) via their respective chemical functionalities. Preferably, the lipid (e.g., chemical functionality 1) is joined to the targeting entity, optionally via a spacer group, (e.g., chemical functionality 2) via the linkages shown in Table 1. Those of skill in the art will recognize that one can first

.0 attach the spacer either to the targeting agent or to the lipid. The chemical functionalities shown in Table 1 can be present on the targeting entity, spacer, or lipid, depending on the synthesis scheme employed. TABLE 1

One skilled in the art will readily appreciate that many of these linkages may be produced in a variety of ways and using a variety of conditions. For the preparation of esters, see, e.g., March, ibid., at 1157; for thioesters, see March, supra at 362-363, 491, 720-722, 829, 941, and 1172; for carbonates, see March, supra at 346-347; for carbamates, see March, supra at 1156-57; for amides, see March supra at 1152; for ureas and thioureas, see March supra at 1174; for acetals and ketals, see Greene et al. supra 178-210 and March supra at 1146; for acyloxyalkyl derivatives, see Prodrugs: Topical and Ocular Drug Delivery, K. B. Sloan, ed., Marcel Dekker, Inc., New York, 1992; for enol esters, see March supra at 1160; for N- sulfonylimidates, see Bundgaard et al., (1988) J. Med. Chem., 31:2066; for anhydrides, see March supra at 355-56, 636-37, 990-91, and 1154; for N-acylamides, see March supra at 379; for N-Mannich bases, see March supra at 800-02, and 828; for hydroxymethyl ketone esters, see Petracek et al. (1987) Annals NY Acad. Sci., 507:353-54; and for disulfides, see March supra at 1160.

A variety of ketal type linkages may be produced. Ketal type linkages that may be produced in the pharmaceutical agent-chemical modifier complexes of the present invention include, but are not limited to, imidazolidin-4-ones, see Prodrugs, supra; oxazolin-5-ones, see Greene et al. supra at 358; dioxolan-4-one, see Schwenker et al. (1991) Arch. Pharm. (Weinheim) 324:439; spirothiazolidines, see Bodor et al. (1982) Int. J. Pharm., 10:307 and

Greene et al. supra at 219 and 292; and oxazolidines, see March supra at 87 and Greene et al. supra at 217-218 and 266-267.

In a preferred embodiment, the targeting entity is attached to a carboxyl head group on the lipid. In another preferred embodiment, the targeting entity is attached to a maleimide or the alpha-methyl group of an acetamide.

Exemplary lipids with a variety of functionalities for linking a lipid to a targeting entity or therapeutic entity are shown in Figures 3-15. Additional linkages and functionalities, for example, for the attachment of nucleic acids, are described in Hale, et al., U.S. Patent No. 5,607,691. One or more spacer groups optionally may be introduced between the lipid and the targeting entity. Spacer groups typically contain two chemical functionalities and, typically do not carry a charge. Typically, one chemical functionality of the spacer group bonds to a chemical functionality of the lipid, while the other chemical functionality of the spacer group is used to bond to a chemical functionality of the targeting entity. Examples of chemical functionalities of spacer groups include hydroxy, mercapto, carbonyl, carboxy, amino, ketone, and mercapto groups. Spacer groups may also be used in combination. When a combination of spacer groups is used, the spacer groups may be different or equivalent. Preferred spacer groups include 6-aminohexanol, 6-mercaptohexanol, 10- hydroxydecanoic acid, glycine and other amino acids, 1,6-hexanediol, beta-alanine, 2- aminoethanol, cysteamine (2-aminoethanethiol), 5-aminopentanoic acid, 6-aminohexanoic acid, 3-maleimidobenzoic acid, phthalide, alpha-substituted phthalides, the carbonyl group, 5 aminal esters, and the like. Particularly preferred spacer groups are also depicted schematically in Figures 3-15, and include polyethylene glycol, and ethylene glycol derivatives with terminal amino groups.

The spacer can serve to introduce additional molecular mass and chemical functionality into the macromolecule-targeting entity complex. Generally, the additional mass 10 and functionality will affect the serum half-life and other properties of the pharmaceutical agent-chemical modifier complex. Thus, through careful selection of spacer groups, macromolecule-targeting entity complexes with a range of serum half-lives can be produced.

In addition, the nature of the linkage used to couple the spacer group to the chemical modifier or pharmaceutical agent may affect the serum half-life. L 5 Although discussion has thus far focused on the coupling of a single type of targeting entity to a macromolecule, in some embodiments, other entities can be coupled to the macromolecule or the macromolecule-targeting entity complex. Other entities which can be covalently bound to the macromolecule-targeting entity complex (optionally via a spacer group), will serve to affect or modify a chemical, physical, or biological property of the 10 complex, including providing a means for detection, for increasing the excretion half-life of the complex, for decreasing aggregation, for decreasing the inflammation and/or irritation accompanying the delivery of the pharmaceutical agent across membranes, and for facilitating receptor crosslinking.

An example of an additional entity which serves to provide a means for detection is a 15 radiolabeling site, including radiolabeled chelates for cancer imaging or radiotherapy and for assessing dose regiments in different tissues. Examples of complexes utilizing lipids containing sites for radiolabeling are described herein, and in copending U.S. Provisional Patent Application Serial No. 60/308,347.

A receptor crosslinking functionality modifier is essentially a targeting modifier. i0 Crosslinking of cell surface receptors is a useful ability for a pharmaceutical agent in that crosslinking is often a required step before receptor internalization. Thus, the crosslinking modifier can be used as a means to incorporate a pharmaceutical agent into a cell. In addition, the presence of two receptor binding sites (i.e., targeting modifiers) gives the pharmaceutical agent increased avidity.

A similar effect can also be obtained with an avidity modifier. In this case, each pharmaceutical agent will have a targeting modifier and an avidity modifier (i.e., a dimerization peptide). The dimerization of two peptides will effectively form one molecule with two targeting modifiers, thus allowing receptor crosslinking. With this bimolecular approach to crosslinking, the concentration dependence will be greater and increased targeting and crosslinking specificity can be obtained for tissues with high receptor density.

Alternatively, a functionality modifier may serve to prevent aggregation. Specifically, many peptide and protein pharmaceutical agents form dimers or larger aggregates which may limit their permeability or otherwise affect properties related to dosage form or bioavailability. For example, the hexameric form of insulin can be inhibited through the use of an appropriate functionality modifier and thus, result in greater diffusability of the monomeric form of insulin. Large numbers of therapeutic entities may be attached to one macromolecule that may also bear from several, about 4,000; about 6,000; 8,000; about 10,000; about 12,000; about 15,000, about 20,000, or up to about 24,000 targeting entities for in vivo adherence to targeted surfaces. The improved binding conveyed by multiple targeting entities can also be utilized therapeutically to block cell adhesion to endothelial receptors in vivo, for example. Blocking these receptors can be useful to control pathological processes, such as inflammation and control of metastatic cancer. For example, multi-valent sialyl Lewis X derivatized liposomes can be used to block neutrophil binding, and antibodies against NCAM-1 on liposomes can be used to block lymphocyte binding, e.g. T-cells.

Some lipids suitable for use in liposomes may have an active head group for attaching one or more therapeutic entities or targeting entities, a spacer portion for accessibility of the active head group; a hydrophobic tail for self-assembly into liposomes; and, optionally, a polymerizable group to stabilize the liposomes. Other suitable lipids include phosphatidylcholine and other lipids described herein.

Targeted liposomes which recirculate in the vasculature may include endothelial antigens which interact with the cell adhesion molecules or other cell surface receptors to retain a number of the targeted liposomes at the desired location. The high concentration of therapeutic entities in the liposomes render possible site-specific delivery of high concentrations of drugs or other therapeutic entities, while minimizing the burden on other tissues. The liposomes described herein are particularly well-suited since they maintain their integrity in vivo, recirculate in the blood pool, are rigid and do not easily fuse with cell membranes, and serve as a scaffold for attachment of both the antibodies/targeting entities and the therapeutic entities. The size distribution, particle rigidity and surface characteristics of the liposomes can be tailored to avoid rapid clearance by the reticuloendothelial system and the surface can be modified with ethylene glycol to further increase intravascular recirculation times. In one embodiment, the dextran-coated liposomes described herein were found to have blood pool half-lives of about 23 hours in mice.

Therapeutic Compositions The present invention is also directed toward therapeutic compositions comprising the therapeutic agents of the present invention. Compositions of the present invention can also include other components such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier. For example, compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, mannitol, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer, Tris buffer, histidine, citrate, and glycine, or mixtures thereof, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.

In one embodiment of the present invention, the composition can also include an immunopotentiator, such as an adjuvant or a carrier. Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, Freund's adjuvant; other bacterial cell wall components; aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins; viral coat proteins; other bacterial-derived preparations; gamma interferon; block copolymer adjuvants, such as Hunter's Titermax adjuvant (Naxcel™, Inc. Νorcross, Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark). Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, and glycols. One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).

Generally, the therapeutic agents used in the invention are administered to an animal in an effective amount. Generally, an effective amount is an amount effective to either (1) reduce the symptoms of the disease sought to be treated or (2) induce a pharmacological change relevant to treating the disease sought to be treated. For cancer, an effective amount includes an amount effective to: reduce the size of a tumor; slow the growth of a tumor; prevent or inhibit metastases; or increase the life expectancy of the affected animal.

Therapeutically effective amounts of the therapeutic agents can be any amount or doses sufficient to bring about the desired effect and depend, in part, on the condition, type and location of the cancer, the size and condition of the patient, as well as other factors readily known to those skilled in the art. The dosages can be given as a single dose, or as several doses, for example, divided over the course of several weeks.

The present invention is also directed toward methods of treatment utilizing the therapeutic compositions of the present invention. The method comprises administering the therapeutic agent to a subject in need of such administration. The therapeutic agents of the instant invention can be administered by any suitable means, including, for example, parenteral, topical, oral or local administration, such as intradermally, by injection, or by aerosol. In the preferred embodiment of the invention, the agent is administered by injection. Such injection can be locally administered to any affected area. A therapeutic composition can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration of an animal include powder, tablets, pills and capsules. Preferred delivery methods for a therapeutic composition of the present invention include intravenous administration and local administration by, for example, injection or topical administration.

For particular modes of delivery, a therapeutic composition of the present invention can be formulated in an excipient of the present invention. A therapeutic reagent of the present invention can be administered to any animal, preferably to mammals, and more preferably to humans. The particular mode of administration will depend on the condition to be treated. It is contemplated that administration of the agents of the present invention may be via any bodily fluid, or any target or any tissue accessible through a body fluid.

Preferred routes of administration of the cell-surface targeted therapeutic agents of the present invention are by intravenous, interperitoneal, or subcutaneous injection including administration to veins or the lymphatic system. While the primary focus of the invention is on vascular-targeted agents, in principle, a targeted agent can be designed to focus on markers present in other fluids, body tissues, and body cavities, e.g. synovial fluid, ocular fluid, or spinal fluid. Thus, for example, an agent can be administered to spinal fluid, where an antibody targets a site of pathology accessible from the spinal fluid. Intrathecal delivery, that is, administration into the cerebrospinal fluid bathing the spinal cord and brain, may be appropriate for example, in the case of a target residing in the choroid plexus endothelium of the cerebral spinal fluid (CSF)-blood barrier.

As an example of one treatment route of administration through a bodily fluid is one in which the disease to be treated is rheumatoid arthritis. In this embodiment of the invention, the invention provides therapeutic agents to treat inflamed synovia of people afflicted with rheumatoid arthritis. This type of therapeutic agent is a radiation synovectomy agent. Individuals with rheumatoid arthritis experience destruction of the diarthroidal or synovial joints, which causes substantial pain and physical disability. The disease will involve the hands (metacarpophalangeal joints), elbows, wrists, ankles and shoulders for most of these patients, and over half will have affected knee joints. Untreated, the joint linings become increasingly inflamed resulting in pain, loss of motion and destruction of articular cartilage. Chemicals, surgery, and radiation have been used to attack and destroy or remove the inflamed synovium, all with drawbacks. The concentration of the radiation synovectomy agent varies with the particular use, but a sufficient amount is present to provide satisfactory radiation synovectomy. For example, in radiation synovectomy of the hip, the concentration of the agent will generally be higher than when used for the radiation synovectomy of the wrist joints. The radiation synovectomy composition is administered so that preferably it remains substantially in the joint for 20 half-lifes of the isotope although shorter residence times are acceptable as long as the leakage of the radionuclide is small and the leaked radionuclide is rapidly cleared from the body.

The radiation synovectomy compositions may be used in the usual way for such procedures. For example, in the case of the treatment of a knee-joint, a sufficient amount of the radiation synovectomy composition to provide adequate radiation synovectomy is injected into the knee-joint. There are a number of different techniques which can be used and the appropriate technique varies on the joint being treated. An example for the knee joint can be found, for example, in Nuclear Medicine Therapy, J. C. Harbert, J. S. Robertson and K. D. Reid, 1987, Thieme Medical Publishers, pages 172-3.

The route of administration through the synovia may also be useful in the treatment of osteoarthritis. Osteoarthritis is a disease where cartilage degradation leads to severe pain and inability to use the affected joint. Although age is the single most powerful risk factor, major trauma and repetitive joint use are additional risk factors. Major features of the disease include thinning of the joint, softening of the cartilage, cartilage ulcers, and abraded bone.

Delivery of agents by injection of targeted carriers to synovial fluid to reduce inflammation, inhibit degradative enzymes, and decrease pain are envisioned in this embodiment of the invention.

Another route of administration is through ocular fluid. In the eye, the retina is a thin layer of light-sensitive tissue that lines the inside wall of the back of the eye. When light enters the eye, it is focused by the cornea and the lens onto the retina. The retina then transforms the light images into electrical impulses that are sent to the brain through the optic nerve.

The macula is a very small area of the retina responsible for central vision and color vision. The macula allows us to read, drive, and perform detailed work. Surrounding the macula is the peripheral retina which is responsible for side vision and night vision. Macular degeneration is damage or breakdown of the macula, underlying tissue, or adjacent tissue. Macular degeneration is the leading cause of decreased visual acuity and impairment of reading and fine "close-up" vision. Age-related macular degeneration (ARMD) is the most common cause of legal blindness in the elderly.

The most common form of macular degeneration is called "dry" or involutional macular degeneration and results from the thinning of vascular and other structural or nutritional tissues underlying the retina in the macular region. A more severe form is termed

"wet" or exudative macular degeneration. In this form, blood vessels in the choroidal layer (a layer underneath the retina and providing nourishment to the retina) break through a thin protective layer between the two tissues. These blood vessels may grow abnormally directly beneath the retina in a rapid uncontrolled fashion, resulting in oozing, bleeding, or eventually scar tissue formation in the macula which leads to severe loss of central vision. This process is termed choroidal neovascularization (CNN).

CΝN is a condition that has a poor prognosis; effective treatment using thermal laser photocoagulation relies upon lesion detection and resultant mapping of the borders. Angiography is used to detect leakage from the offending vessels but often CΝN is larger than indicated by conventional angiograms since the vessels are large, have an ill-defined bed, protrude below into the retina and can associate with pigmented epithelium.

Neovascularization results in visual loss in other eye diseases including neovascular glaucoma, ocular histoplasmosis syndrome, myopia, diabetes, pterygium, and infectious and inflammatory diseases. In histoplasmosis syndrome, a series of events occur in the choroidal layer of the inside lining of the back of the eye resulting in localized inflammation of the choroid and consequent scarring with loss of function of the involved retina and production of a blind spot (scotoma). In some cases, the choroid layer is provoked to produce new blood vessels that are much more fragile than normal blood vessels. They have a tendency to bleed with additional scarring, and loss of function of the overlying retina. Diabetic retinopathy involves retinal rather than choroidal blood vessels resulting in hemorrhages, vascular irregularities, and whitish exudates. Retinal neovascularization may occur in the most severe forms. When the vasculature of the eye is targeted, it should be appreciated that targets may be present on either side of the vasculature.

Delivery of the agents of the present invention to the tissues of the eye can be in many forms, including intravenous, ophthalmic, and topical. For ophthalmic topical administration, the agents of the present invention can be prepared in the form of aqueous eye drops such as aqueous suspended eye drops, viscous eye drops, gel, aqueous solution, emulsion, ointment, and the like. Additives suitable for the preparation of such formulations are known to those skilled in the art. In the case of a sustained-release delivery system for the eye, the sustained- release delivery system may be placed under the eyelid or injected into the conjunctiva, sclera, retina, optic nerve sheath, or in an intraocular or intraorbitol location. Intravitreal delivery of agents to the eye is also contemplated. Such intravitreal delivery methods are known to those of skill in the art. The delivery may include delivery via a device, such as that described in

U.S. Patent No. 6,251,090 to Avery.

Imaging

The present invention is directed to imaging agents displaying important properties in medical diagnosis. More particularly, the present invention is directed to magnetic resonance imaging contrast agents, such agents including gadolinium, ultrasound imaging agents, or nuclear imaging agents, such as Tc-99m, In-111, Ga-67, Rh-105, 1-123, Nd -147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, and Tl-201. Such imagaing agents may include chelator lipids described herein. An example of a commercially available chelator lipid comprising gadolinium includes diethylenetriaminepentaacetic acida,w-bis(8- stearoylamido-3,6-dioxaoctylamide) gadolinium salt.

This invention also provides a method of diagnosing abnormal pathology in vivo comprising, introducing a plurality of targeting image enhancing particles targeted to a molecule involved in the abnormal pathology into a bodily fluid contacting the abnormal pathology, the targeting image enhancing particles attaching to a molecule involved in the abnormal pathology, and imaging in vivo the targeting image enhancing particles attached to molecules involved in the abnormal pathology. Such methods are described in the EXAMPLES section, and also in copending U.S. Provisional Patent Application No. 60/308,347.

Exemplary lipid constructs and uses Dextran-coated liposomes (DCLs) are colloidally stable carriers for the covalent attachment of drugs or other targeting agents that target cell-surface receptors and other targets. DCLs as described herein comprise a liposome having dextran or modified dextran covalently attached to the surface of the liposome. Once a targeting agent has been attached to a DCL, it is referred to as a targeted DCL. In brief, targeted macromolecules comprising dextran-coated liposomes are prepared by providing a liposome, covalently attaching dextran or modified dextran to the liposomes to generate a dextran-coated liposome, attaching at least one linker to dextran-coated liposome to generate a linker-attached liposome, wherein the linker facilitates attachment of a targeting molecule and attaching at least one targeting molecule to the linker-attached liposome. Typically, a lipid solution is formed in a polar organic solvent, including lower alkyl alcohols, such as methanol, ethanol, propanols, butanols, and the like, with t-butanol being particularly preferred. The liposomes may be concentrated prior to coating with dextran or another stabilizing agent. Lipids which may be used to facilitate the covalent attachment of dextran to the liposomes includes N-succinyl-DPPE (DPPE-Suc)and Ν-caproylamine-DPPE (DPPE- Cap), described more fully in the EXAMPLES below. The α_vβ₃ integrin antagonist below

referred to herein as IA, compound 10, and 3-{4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)- ethyloxy]-benzoylamino}-2(S)-benzene-sulfonyl-aminopropionic acid is covalently linked to the surface of a DCL to produce a targeted DCL in an embodiment of the invention. The preparation of such liposomes is described in EXAMPLE 3. When the lipid is DPPE-Suc, this targeted DCL is referred to herein as IA-DCL or IA-DCL (DPPE-Suc). When the lipid is DPPE-Cap, the targeted DCL is referred to as IA-DCL (DPPE-Cap).

The IA was attached to DCLs at loadings of up to 5% (w/w), which represents about 4,000 1 A molecules per DCL, and the potency of the IA-DCL conjugate was similar to that observed for unmodified integrin antagonist. IA-DCLs inhibit the proliferation of HUNECs in vitro where the DCL carrier has no effect. It should be noted that the loading capacity of the DCL will vary with the stabilizing agent used. Stabilizing agents with additional reactive groups may generate higher loadings, including about 8% (w/w), about 10% (w/w), about 15% (w/w), and about 20% (w/w), about 25% (w/w), and about 30% (w/w), or up to 20,000

IA molecules per DCL.

The therapeutic advantages associated with IA-DCL stem from the unique properties of this active-carrier technology. IA-DCL binds to α_vβ₃ integrin, located on the surface of endothelial cells, blocking cell adhesion and migration and also causing apoptosis. Antagonism of the α_vβ₃ integrin represents a novel approach to treat cancer in two ways: directly by inhibiting angiogenic tumor growth and invasion, and indirectly by disruption of FAK and consequently, ERK signaling pathways resulting in apoptosis. Several IA molecules are attached to the surface of the coated liposome providing an increase in the potency of IA. In addition, the unique properties of IA-DCL result in sustained drug concentrations with minimal systemic exposure yielding less frequent administration and better safety, both of which are important therapeutic advances for patients. The in vitro and in vivo potency and selectivity of IA-DCL should translate into clinical efficacy. α_vβ integrin-targeted, dextran-coated liposomes are effective anti-tumor agents. These agents inhibit tumor growth in a human xenograft melanoma model and a human glioma model in vivo, and inhibit cell proliferation of endothelial cells in vitro. Interestingly, melanoma cells are known to be relatively resistant to radiotherapy.

IA-DCL demonstrated significantly better efficacy compared with buffer and DCL control groups as determined both by normalized tumor volume at day 11 and TNQT measurements, as shown in EXAMPLE 6. Of particular interest, significant reduction in tumor growth and increased survival was seen after treatment with IA-DCL as compared with the same dosage of free IA (15 mg/kg on days 0, 2, 4, 6). These data demonstrate directly the advantage of the covalent attachment of drugs to the dextran coated liposome platform. Different dosages of IA-DCL did not yield statistically significant results in tumor growth inhibition and were difficult to differentiate in the current study. Additional studies evaluating a longer duration of treatment and examining the pharmacokinetics of IA-DCL should help in dosage selection. There was no significant difference in tumor growth or survival following treatment with liquid vs. lyophilized formulations of IA-DCL. This implies that lyophilized material can be used for future studies. Histology (H & E, TUΝEL and anti-CD31 staining) results provide confirmation of the anti-angiogenic mechanism for IA-DCL and support the efficacy findings presented above. Pathology results indicate that there were no gross signs of toxicity following IA-DCL treatment.

Additionally, EXAMPLE 8 demonstrates that IA-DCL has a long half-life and a restricted distribution. There was a delay in reaching maximum radioactivity when using a labeled, compound, and this suggests a lack of instantaneous or rapid distribution of either IA- DCL or DCL. IA-DCL exhibited a similar time course as DCL. Some differences in the distribution phase, e.g., uptake in lymph, muscle, and kidney were greater and occurred more slowly for DCL than for IA-DCL. In addition, comparison of the AUC_LA_ST of liver and spleen to plasma AUC_LA_ST suggested considerable uptake of IA-DCL in the RES system, a known characteristic of liposomes. The tissue half-lives were similar to or longer than the half-life observed for plasma for both compounds.

Although only a few embodiments of the present invention have been described, it should be understood that the present invention may be embodied in many other specific forms without departing from the spirit or the scope of the present invention.

EXAMPLES

EXAMPLE 1. GENERAL METHODS All solvents and reagents used were of reagent grade. Solvent evaporations were performed under reduced pressure provided from house vacuum or a Welch direct drive vacuum pump at ≤ 40 °C 1H and ¹³C-NMR spectra were recorded on a JEOL FX90Q at 90MHz in CDC1₃, CD₃OD, D₂O or blends thereof as described for each case. (Note: although soluble in CDC1₃, the addition of CD₃OD to the lipids inhibits formation of inverted micelles and thus provided sharper spectra. Spectra were referenced to residual CHC1₃ (7.25 ppm) for

H experiments and the center line of CDCI₃ (77.00 ppm) for C experiments. MALDI-TOF mass spectrometry was performed on PerSeptive DE instrument (Mass Spectrometry, The Scripps Research Institute, La Jolla, CA). TLC was performed on glass backed Merck 60 F254 (0.2 mm; EM Separations, Wakefield, RI) and the developed plates routinely sprayed with eerie sulfate (1 %) and ammonium molybdate (2.5%) in 10% aqueous sulfuric acid and heated to ~ 150 °C Other developers include iodine (general use), 0.5% ninhydrin in acetone (for amines), and ultraviolet light (for chromophores).

Preparation of S-amino-2-hydroxypropyl dextran Dextran (40K, Amersham) ( 5.00 g) was added to 5 mL water with stirring. To this was added 7.5 mL 25% aqueous solution of Zn(BF₄)₂ (7 mmol) with stirring. Epichlorohydrin (25 mL, 319 mmol) was added in one portion and the reaction was heated to 80° for three hours. The reaction mixture was cooled to ambient and stirred overnight.

The reaction mixture was added dropwise to 350 mL acetone (addition required 45 minutes) with thorough stirring. The solid precipitate was collected by filtration, and redissolved in 60 mL water. The aqueous solution was added dropwise to 350 mL acetone

(addition required 45 minutes) with thorough stirring. Again the solid precipitate was collected by filtration, and redissolved in 60 mL water. 14N Aqueous ammonia (20 mL) was added and the reaction mixture stirred at ambient overnight.

The reaction mixture was added dropwise to 1000 mL methanol (addition required 45 minutes) with vigorous stirring. The product was collected by filtration, washed thoroughly with methanol and air-dried. The product was dissolved in 45 mL water and lyophilized to yield 4.86 g white solid. Determined amines to be 25.6/mole of dextran. 1H NMR (D O) - 5.2, s, 0.04; 5.05, s, 0.07; 4.8, s, 1; 2.7 bd d, 0.33.

Two modifications of this prep were run. Modification #1 was heated at 80°C six hours and worked up/ isolated as above. Modification #2 was heated 90°C three hours and worked up/ isolated as above. The NMR traces of the two modifications looked almost exactly like that of the original material, indicating that there was little or no effect caused by the modifications.

EXAMPLE 2. PREPARATION OF 4-[2-(3,4,5,6-TETRAHYDRO- PYRIMIDIN-2-YLAMINO)ETHYLOXY]BENZOYL-2-(S)AMINO

ETHYLSULFONYLAMINO-β-ALANINE

A. Preparation of N-Benzyloxycarbonyl-taurine sodium salt (2). Taurine, 1 (40g, 320 mmol) dissolved in 4N sodium hydroxide solution (80 mL) and water 1,200 mL). To this solution was added benzyloxycarbonyl chloride, (48 mL, 330 mmol) drop wise, with vigorous stirring during a period of 4 hours. The pH was maintained alkaline by the addition of 10% sodium bicarbonate solution (300 mL) and 4N sodium hydroxide solution (45 mL). The reaction mixture was then washed with ether (1000 mL) and the aqueous layer was spin evaporated to dryness, dried under high vacuum over phosphorous pentoxide overnight to yield 12.70 g (14.1%) of 2. 1H-NMR (D₂O): δ 7.50 (5H, s, Ar-H), 5.21 (2H, s, Ar-CH₂), 3.62 (2H, t, CH₂), 3.14 (2H, t, CH₂).

B. Preparation of 2-Benzyloxycarbonylaminoethanesulfonyl chloride (3). N-CBZ- Taurine sodium 2 (12.7 g, 32 mmol) was suspended in dry diethyl ether (30 mL) under a positive pressure of argon and treated with phosphorous pentachloride (7 g, 33.6 mmol) in 5 portions over 15minutes. The reaction was stirred for 4h, at ambient temperature. The solvent was removed by spin evaporation. Ice water (10 mL) was added and the residue was triturated after cooling the flask and the contents in an ice bath. More ice water (50 mL) was added and the product solidified. The solids were collected by filtration washed with ice water (20 mL) and dried over phosphorous pentoxide overnight to yield 6.95 g (78.0%) of 3. 1H-NMR (CDC1₃): δ 7.35 (5H, s, Ar-H), 5.12 (2H, s, Ar-CH₂), 3.89 (2H, t, CH₂) overlapping with 3.85 (2H, t, CH₂).

C. Prepration of Methyl 3-butyloxycarbonylamino-2-(S)benzyloxycarbonyl- aminoethylsulfonylaminopropionate (5). A mixture of the sulfonyl chloride 3 (21.6 g, 78.0 mmol) and methyl-3-N-butoxycarbonylamine-2-aminopropionate (4, 9.96g, 39.2 mmol) in anhydrous tetrahydrofuran (150 mL) under a positive pressure of argon was cooled in an ice bath. To this solution was added N-methylmorpholine (16 mL, 145 mmol) in anhydrous THF (275 mL) drop wise during a period of 30 min using a dropping funnel previously dried and under a positive pressure of argon. After lh stirring in the ice bath, by TLC it was observed that all the sulfonyl chloride (R_f = 0.65) had disappeared (eluent: ethyl acetate/hexane 1 :1); however, there was unreacted diaminopropionic acid (R_f = 0.1, ninhydrin spray) still present. More sulfonyl chloride (5.0 g, 18 mmol) was added during a period of 3h. The reaction was then filtered and spin evaporated to remove the solvent and dissolved in ethyl acetate (100 mL) and washed with cold dilute hydrochloric acid (20 mL), saturated sodium bicarbonate solution (20 mL) and saturated sodium chloride solution (20 mL) and dried over anhydrous sodium sulfate. The solvent removed by spin evaporation and dried under vacuum over night. The residue was recrystallized by first dissolving in ethyl acetate and then by adding equal volume of hexane to obtain 5 as a colorless solid 13.4 g (74.3 %). 1H-ΝMR (CDC1₃): δ 7.36 (5H, s, Ar-H), 5.83 (1H, d, NH), 5.55 (1H, t, NH), 5.12 (2H, s, Ar-CH₂), 5.06 (1H, t, NH),

4.26 (2H, m, CH), 3.79 (3H, s, CH₃), 3.70 (2H, dd, CH₂), 3.26 (2H, dd, CH₂), 1.43 (9H, s,

(CH₃)₃).

D. Preparation of 3-butyloxycarbonylamino-2-(S)-benzyloxycarbonylaminoethyl- sulfonylaminopropionic acid (6). A solution of the methyl ester 5 (13.3 g, 28.9 mmol) in tetrahydrofuran (160 mL) was cooled in an ice bath and to this solution was added a solution of lithium hydroxide (5.42 g, 128 mmol) in ice water (160 mL). The reaction mixture was slowly warmed to ambient temperature by removing the ice bath and the mixture was stirred at ambient temperature for lh. The organic solvent was then removed by spin evaporation. The residual aqueous portion was washed with diethyl ether (20 mL) and then acidified to pH 4 using diluted hydrochloric acid. This solution was cooled in an ice bath and then mixed with ethyl acetate (100 mL) and then further acidified to pH 1 using ice-cold diluted hydrochloric acid and immediately extracted with ethyl acetate (2 x 200mL). The ethyl acetate layer was washed with brine (50 mL) and dried over anhydrous sodium sulfate. The solvent was then removed by spin evaporation and dried under high vacuum overnight to obtain 13.3 g of a foamy solid, which was recrystallized from hexane/ethyl acetate (1 :1) to obtain 11.6 g (89.7 %) of 6. 1H-NMR (CDC1₃): δ 7.33 (5H, s, Ar-H), 6.12 (1 H, d, NH), 5.68 (IH, t, NH), 5.26 (IH, t, NH), 5.1 (2H, s, Ar-CH₂), 4.24 (2H, m, CH₂), 3.67 (IH, t, CH₂), 3.27 5 (2H, t, CH₂), 1.45 (9H, s, C(CH₃)₃).

E. Preparation of 3-amino-2-(S)-benzyloxycarbonylaminoethylsulfonylamino- propionic acid (7). N-BOC-β-amino acid 6 (11.5 g, 25.8 mmol) was treated with trifluoroacetic acid (68 mL) in methylene chloride (350 mL) for 1.5h and then spin evaporated to dryness. The residue was dissolved in water (200 mL) and lyophilized to obtain 7 as a solid 0 of 10.9 g (98.8 %) of the β-amino acid. 1H-NMR (CDC1₃): δ 7.30 (5H, s, Ar-H), 6.07 (IH, d,

NH), 5.61 (IH, t, NH), 5.20 (IH, t, NH), 5.17 (2H, s, Ar-CH₂), 4.11 (2H, m, CH₂), 3.53 (2H, t, CH₂), 3.32 (2H, t, CH₂). DCI-MS for C₁₃Hι₉N₃O₆S: m/z (ion) 346 (M+H) (calculated for Cι₃Hι₉N₃O₆S + H, m/z 346).

F. Preparation of 4-[2-(pyrimidin-2-ylamino ethyloxy]benzoyl-2-(S)- 5 benzyloxycarbonylaminoethylsulfonylamino-β-alanine (9). The benzoic acid derivative 8 (6.4 g, 24.7 mmol) and N-hydroxysuccinimide (3.6 g, 31 mmol) were dissolved in anhydrous dimethylsulfoxide (1 10 mL), under a positive pressure of argon and cooled in an ice bath. To this solution was added 1 -(3 (dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (4.9 g, 25.6 mmol). The solution was stirred at ice-cold temperature for lh and then allowed to

20 warm to ambient temperature and continued to stir at room temperature for another 24h. To this mixture was added a solution of the β-amino acid 7 (12.2 g, 25.8 mmol) followed by N- methymorpholine and stirred under argon for 3 days. The mixture was then poured into water 1 L) and acidified with diluted hydrochloric acid to pH 1.5 and extracted with ethyl acetate (5 x 500 mL). The organic phase was washed with saturated sodium chloride solution (50 mL)

>5 and then dried over anhydrous sodium sulfate. The solvent was removed by spin evaporation and the residue was triturated in ethyl acetate, filtered and dried under high vacuum to obtain 10.5 g (72.5 %) of 9. 1H-ΝMR (DMSO-d₆): δ 8.30 (2H, d Ar-H), 7.99 (2H, d, Ar-H), 7.34 (5H, s, Ar-H), 7.00 (2H, d, Ar-H), 6.60 (IH, dd, Ar-H), 5.01 (2H, s, CH₂), 4.15 (1 H, t, CH), 3.67 (2H, t, CH₂), 3.56 (2H, t, CH₂), 3.17 (2H, t, CH₂).

10 G. Preparation of 4-[^"2-(3 ,4.5.,6-Tetrahydropyrimidin-2-ylamino)ethyloxy]- benzoyl-2-(S)amino ethylsulfonylamino-β-alanine (10 = R'NH?). A solution of the pyrimidine derivative 10 (3.7 g, 6.4 mmol) was dissolved in acetic acid (190mL) and concentrated hydrochloric acid (17 mL). This solution was treated with 10 % palladium over carbon (1.62 g) and hydrogenated at 45 psi of hydrogen gas for 5h. The mixture was then filtered through celite and washed with water. The solvent was removed by spin evaporation and dried under high vacuum. The residue was dissolved in water ~ lOOmL) and pH adjusted to 7.0 with IN sodium hydroxide solution and then spin evapo ated to dryness. The residue was dissolved in methanol (20 mL) and filtered. The filtrate was spin evaporated and dissolved in water (275 mL) and lyophilized. The lyophilized product was then recrystallized from water to obtain 2.96 g (78.9 %) of pure product. 1H-NMR (D₂O): δ 7.80 (2H, d, Ar-H), 7.14 (2H, d, Ar-H), 4.49 (IH, s, CH_aH_b), 4.28 (IH, t, CH₂), 3.94 (IH, dd, CH_aH ), 3.61 (6Η, m, CH₂), 3.32 (4H, t, CH₂), 1.90 (2H, t, CH ). ES-MS for d₈H₂₈N₆O₆S: m/z (ion) 457 (M+H) (calculated for C₁₈H₂₈N₆O₆S + H, m/z 457).

H. Determination of chiral purity of 4-[2-(3 5.6-Tetr-^hydropyrimidin-2- ylamino ethylo y]benzoyl-2- S)-aminoethylsulfonylam To 1 mL of a solution of 10 (1.4 mg in 636 μL of water and 636 μL of acetone) was added 1 mL of a solution of Marfey's reagent (1.4 mg/mL). To the turbid solution was added 500 μL of acetone, 1.5 mL of water, and 400 μL of 1M NaHCO₃ solution and incubated at 40 °C for 24h. The solution was then neutralized with 200 μL of 2M hydrochloric acid solution and analyzed by HPLC. A control solution made without 10 was also treated similarly and analyzed by HPLC. A sample of 10 was epimerized by heating it to melt. The epimerized compound was treated similar to 10. The 10 sample showed only the SS diastereomer and the SR diastereoisomer was completely absent indicating the %ee was > 99 % (t_R = 12.2 min for SS diastereoisomer and 10.8 min for SR diastereoisomer)

EXAMPLE 3. Preparation of dextran coated liposomes containing DPPC, DPPE- succinate (DPPE-suc), and cholesterol The following lipids were used to formulate liposomes: l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (DPPE-Suc) and Cholesterol. Liposomes were prepared in the following proportions: 2000mg DPPC (55% molar ratio), 202mg DPPE-Suc (5% molar ratio) and 766.2mg Cholesterol (40% molar ratio). 60ml t-butanol was added to the dry lipids with rotation at approximately 65°C for 30 minutes. The resulting solution was faintly opalescent in appearance. The t-butanol solution was forcefully pipetted into rapidly 65°C stirring water. The resulting nearly clear solution was allowed to cool to 45°C and then filtered with a 0.2μ filter

The liposomes were then purified using tangential flow filtration (TFF). The TFF filter was washed with approximately 100ml 70% ethanol followed by 1600ml de-ionized water. The liposome suspension was continuously fed (initial flow rate ~21ml/min, final flow rate lOml/min) into the reservoir and then washed with 1 liter water (flow rate ~18ml/min). The liposome suspension was then concentrated to approximately 40ml and combined with two 7ml filter backwashes. The final 56ml liposome concentrate was filtered using 0.2μ syringe filters. Using dynamic light scattering (DLS) the liposome size was measured at 45.3nm with a polydispersity of 0.110 and a 5% to 95% size range of 26.6nm to 77.1nm. The liposome zeta potential was determined to be -68.86mN. The liposome concentration was determined to be 48.9mg/ml, the recovery for the liposome formulation and TFF filtration was determined to be 92.3%.

The liposomes were coated with dextran as follows. Amine modified 40,000 MW dextran (AmDex, 9.5 moles amine/mole dextran) was dissolved in 70ml 50mM HEPES pH7 at 50mg/ml. The pH of the solution was adjusted to approximately 7 with 6Ν hydrochloric acid. The suspension was diluted to 80ml and then filtered through a 0.2μ syringe filter. 55ml of the liposome suspension was added dropwise to 64.6ml of the AmDex solution with stirring. The mixture was visibly less clear than the initial liposome suspension. 2.4ml of a 1M l-(3-(dimethylamino)propyl)-3 -ethylcarbodiimide hydrochloride (EDC) solution in water was added dropwise to the coated liposome mixture with stirring. The reaction was allowed to proceed at room temperature overnight.

After reaction with EDC, 0.49 ml 2.5M ammonium sulfate was added to the coated liposomes in order to cap any remaining carboxyl groups on the liposome. After the addition of ammonium sulfate the pH was raised to 7.77 with IN sodium hydroxide. 1.23ml 1M EDC was then slowly added to the coated liposomes and allowed to react for four hours.

Excess AmDex and blocking reagents were removed by TFF. The filter was flushed with 1000 ml de-ionized water. In order to remove non-specifically bound material from the liposomes 5ml 5N sodium chloride was slowly added to the coated liposome mixture and allowed to incubate for 30 minutes with stirring just prior to filtration. The coated liposome mixture was filtered through a 0.45μ syringe filter and then a 0.2μ syringe filter and then placed in the TFF filter reservoir and concentrated to approximately 100 ml. The coated liposome mixture was continuously washed with 1 liter of lOmM HEPES, 200mM sodium chloride pH 7. The filtrate flow was approximately 4.7ml/min. The coated liposome mixture was then washed with 1 liter 50mM HEPES pH7 and then concentrated. The resulting coated liposome suspension volume, after combination with filter backwashes, was 82ml. Coated liposome size was measured with DLS to be 130.4nm with a polydispersity of 0.159 and a 5% to 95% size range of 69.3nm to 245.4nm. The liposome concentration was determined to be 43mg/ml, for a total coated liposome recovery of 3526mg.

After the removal of unattached AmDex free amine groups on the AmDex were converted to carboxyl groups by the addition of succinic anhydride to the coated liposome suspension. The pH of the coated liposome suspension was adjusted to 7.95 with ION sodium hydroxide. Then 2.14ml lOOmg/ml succinic anhydride in anhydrous dimethyl sulfoxide was quickly added to the coated liposome suspension with mixing. Reaction of a small aliquot of the succinylation mixture with 2,4,6-trinitrobenzene sulfonic acid indicated complete succinylation of amines.

The succinylation mixture was then filtered using TFF filtration. The TFF filter was flushed with 1 liter de-ionized water. The succinylation mixture was then placed in the TFF reservoir and washed with 500ml lOmM HEPES, 200mM sodium chloride, pH 7 and 1 liter lOmM HEPES, pH 7. The presence of free dextran in the mixture was monitored using size exclusion chromatography. The volume of the coated liposome suspension, after combination with filter backwashes was 66ml. Coated liposome size was measured with DLS to be 134.9nm with a polydispersity of 0.172 and a 5% to 95% size range of 70.1nm to 259.6nm. The liposome concentration was determined to be 44.6mg/ml, for a total coated liposome recovery of 2945mg.

Alternately, DPPE-Suc was replaced with N-capryolamine-DPPE (DPPE-cap) at the same molar ratio. The same procedure for preparing the liposomes was used, followed by the additional step of modifying DPPE-cap through reaction with succinic anhydride. In a smaller scale preparation, 500mg of DPPC, 51.3mg DPPE-Cap and 191.5mg of cholesterol were used to prepare liposomes in the molar ratio 55:5:40, respectively. A 25 fold molar excess of succinic anhydride was added dropwise to the liposome solution while the pH was maintained between 7.0 and 7.2 by the addition of IN NaOH. After the reaction was allowed to proceed for 1 hour, the succinylated liposomes were purified using TFF as described above and the remainder of the procedure described above followed. EXAMPLE 4. Preparation of small molecule integrin antagonist-attached, dextran coated liposomes containing DPPC, DPPE-succinate (DPPE-suc), and cholesterol

A small molecule integrin antagonist (I A, Compound 10) was coupled to the surface carboxyl groups on the dextran coated liposomes as follows. 2.6ml 50mg/ml IA in water was

5 added to 29ml (1300mg) dextran coated liposomes and diluted with 36ml lOmM HEPES, pH7. The pH of the resulting mixture was adjusted to 7 by adding IN sodium hydroxide. 1.35ml 1M EDC in water was added with mixing and the coupling reaction was allowed to proceed at room temperature overnight. The efficiency of the coupling reaction was monitored with size exclusion chromatography and estimated to be 47.5%. 0 The IA coupling reaction mixture was cleaned up with TFF filtration. The TFF filter was flushed with 200ml 70%ethanol followed by 1L de-ionized water. The IA coupling reaction mixture was transferred to the filter reservoir and washed with 500ml lOmM HEPES, 200mM NaCl, pH7 and then 500mL 150mM NaCl. Size exclusion chromatography was used to monitor the retentate and determine that free IA had been removed from the reaction

15 mixture. The suspension was concentrated and combined with two filter back washes. The resulting mixture was filtered through a 0.45μ syringe filter followed by a 0.2μ syringe filter and stored at 4°C. The recovered volume of the suspension was 21ml. Coated liposome size was measured with DLS to be 116.5nm with a polydispersity of 0.176 and a 5% to 95% size range of 60.1nm to 226. Onm. The liposome concentration was determined to be 53.4mg/ml,

>0 for a total recovery of 1122mg.

EXAMPLE 5. Efficacy Study to Evaluate Platform Composition and Compare Efficacy of IA-DCL and DM-Cilengitide in the M21 Human Melanoma Model.

In this study the pre clinical efficacy of the IA-DCL approach was compared to that of

-5 Desmethyl Cilengitide (DM-Cilengitide), a cyclic peptide IA similar to Cilengitide which is currently in phase II trials for solid tumors. Two doses of DM-Cilengitide were included, a high dose of 15 mg/kg as recommended in the investigator brochure, and a low dose of 4.5 mg/kg given in four doses. Formulations were injected intravenously via the tail vein, except that Cilengitide was injected intraperitoneally.

0 Five different IA targeted DCL platform candidates were tested for in vivo efficacy in this study. Four of the candidates were dextran-coated nanoparticles (DCLs); one was comprised of liposome only. Two platform candidates differed with respect to their linker lipid (IA-DCL (DPPE-Suc linker lipid)) vs. IA-DCL(DPPE-Cap) (DPPE-Cap linker lipid). An additional platform candidate contained a different filler lipid (DSPC instead of DPPC). Four of the platform candidates consisted of the IA targeting agent shown to be efficacious in previous studies. An untargeted liposome control was included in addition to vehicle only.

Efficacy was assessed in the human M21 melanoma model. Cells were implanted by subcutaneous injection in female NU/NU nude mice as previously described (Cheresh, D.A., Honsik, C.J., Staffileno, L.K., Jung, G., Reisfeld, R.A., Proc. Natl. Acad. Sci. USA, 82:5155-5159). Briefly, tumors were implanted by subcutaneous injection of 2.0 xlO⁶ M21 human melanoma cells. The M21 tumor cells were grown in tissue culture flasks in RPMI 1640 medium (Gibco catalog # 31800-089, lot # 1113507) with 10% fetal calf serum (Gemini catalog # 100-500, lot number A3070 IT), 2mM L-glutamine and Pen- Strep. The cell doubling time was approximately 24 hours. Cells were harvested using Trypsin-EDTA solution (containing 0.05% trypsin), resuspended in PBS at 20,000,000/ml, and kept on ice. The treatment regimen consisted of four doses (Injection volume: 6.67 ml/kg) every other day for one week. Normalized tumor volume and tumor volume quadrupling time (TVQT) were used to evaluate efficacy in this study. The candidates are referred to as follows:

Definitions and Measurement:

1) Lipid composition' mole percentage

2) Particle concentiatiorr dry weight

3) Size and size range: 0.2mg/ml of particle in lOmM HEPES ρH7.4 by DLS

4) IC50 of α_vβ₃ / FN-HRP or VN-Biotin challenging assay. IA or IA-particles Inhibiting 50% of maximum FN-HRP or VN- Biotin binding to α_vβ₃ PM value used as a control 5) Endotoxin (Eu/ml): LAL endotoxin testing.

*Note: Cilengitide is commercially available solution, diluted to target concentration.

SU050.1 Vehicle Control: 0.15M NaCl

SU050.2 Desmethyl-Cilengitide 2.25 mg/ml

SU050.3 Desmethyl-Cilengitide 0.675 mg/ml

SU050.4 IA-DCL

SU050.5 IA-DCL

10 SU050.6 IA-DCL (DPPE sue)

SU050.8 IA-DCL (DPPE sue)

SU050.9 IA-DCL (DSPC/DPPE sue)

15 days post implant mice with tumors between 47 and 225 mm³ were selected for

L5 treatment and randomly assigned to treatment groups as described in table 5-1.

Table 5-II summarizes the mouse weight data obtained at treatment and six days post treatment

Tumor volume quadrupling time (TVQT) is defined as the time required for a given tumor to show a four-fold increase in volume when compared with the tumor volume measured at the start of treatment. TVQT is calculated with the following formula:

TVQT = Day_finaι - r ~ .

-TVfina L

{ TNfina1-TV>nd to last ~] Dayfϊnai-Day_2nd to last J

^

Where:

Day_fmai = the day the animal was sacrificed N_fϊnai ⁼ the normalized tumor volume measured on the day the animal was sacrificed

Day_2ndto last ⁼ the day of the penultimate tumor measurement

TN2_nd to l_ast ⁼ the penultimate normalized tumor volume measurement

TVQT was defined as the primary endpoint for this study to determine the significance of treatment on the reduction in tumor growth rate.

1. Statistical Analyses

Treatment effects were compared using analysis of variance (AΝOVA) and Kruskal-Wallis statistical tests. These tests determine if the observed differences between treatment groups are due to chance alone. The AΝOVA tests the equality of the treatment means and is most reliable when there are no significant outliers in the data. The Kruskal Wallis test, on the other hand, considers the order, or rank of the tumors in a given group compared to other treatments and therefore minimizes the impact of outliers. The Kruskal-Wallis test looks for significant differences in the medians of the treatment populations and is more reliable when the data contains significant outliers. P-values calculated from these methods indicate the probability that a given result was obtained by chance.

Tukey's W procedure was used to make pairwise comparisons between treatment groups when an ANONA analysis indicated that a significant difference existed. An advantage of the Tukey procedure is that the overall error rate is controlled; the significance level calculated with this procedure takes into account all possible comparisons that can be made from the data. Results using Tukey's w procedure were confirmed with nonparametric tests. (Nonparametric tests were Mann- Whitney with Bonferroni correction and a multiple comparison procedure recommended by Conover (Practical Nonparametric Statistics, p290)).

2. Results and Discussion Normalized Tumor Volume

In order to compare treatment effects, tumor volumes were measured on the day of treatment and three times a week after treatment. The initial tumor volume (pre- treatment) was then used to normalize each tumor volume.

As specified in the protocol, animals were sacrificed once their tumors grew to four times the pretreatment volume. For this reason some animals in the buffer control group were sacrificed on Day 13. To allow comparison of the groups, the normalized tumor data from Day 13 was used for analysis

The normalized tumor volume data shown were compared using ANOVA and Kruskal-Wallis statistical tests. The p-values associated with these tests were 0.001 and 0.002 respectively. Both tests are significant at the 95% confidence limit and indicate that at least one treatment group shows significant anti-tumor efficacy. In order to determine exactly which treatments show efficacy a number of pairwise, or one to one, comparisons were made using Tukey's pairwise comparisons (Table 5-III). Table 5-III: Summary of P-values obtained using Tukey's W procedure with normalized

Table 5-III indicates that both IA-DCL(DPPEcap) and IA-DCL(DPPEsuc) treatments show significantly smaller tumor volumes when compared with buffer and untargeted nanoparticle controls. Nonparametric statistical tests confirm these results.

Tumor Volume Quadrupling Time

Tumor volume quadrupling time (TVQT) was used as a primary endpoint to evaluate tumor growth reduction for this study

Again, ANOVA and Kruskal-Wallis tests were used to compare TVQT values from different treatment groups. The p-value associated with each test was <0.001 and 0.001 respectively. Both tests are highly significant indicating that at least one treatment is significantly different from the others. Pairwise comparisons were made using Tukey's W procedure (Table 5-IV).

Table 5-IV: Summary of P-values obtained using Tukey's Pairwise Comparisons with TVQT data.

As with the normalized tumor volume data the TVQT data show statistically significant differences between IA-DCL(DPPEcap) and IA-DCL(DPPEsuc) treatments and treatment with buffer or untargeted DCL (20.7 and 23.0 days as compared to 11.7 and 11.9 days respectively). Again these results were confirmed by nonparametric statistical tests. In addition, table 5-IV shows that the increase in TVQT associated with IA-DCL(DPPEsuc) treatment was statistically significant when compared to low and high doses of DM- Cilengitide ( 23.0 days compared to 14.6 and 14.1 days respectively). In this case nonparametric tests do not agree with Tukey's method.

Conclusions

IA-targeted DCL therapy with both IA-dex NP (DPPEcap) and IA-DCL (DPPEsuc) demonstrated statistically significant efficacy compared to both untargeted NP and buffer control groups. Statistical significance was achieved as measured by both normalized tumor volume at day 13 and TVQT. In addition, both IA-dex NP (DPPEcap) and IA-DCL (DPPEsuc) demonstrated superior efficacy compared to DM-Cilengitide; the increase in

TVQT associated with IA-DCL(DPPEsuc) treatment was statistically significant when compared to low and high doses of DM-Cilengitide. This result is particularly encouraging given that DM-Cilengitide was included in this study to mimic the in vivo effects seen with Cilengitide, a cyclic peptide I A which is currently in phase II trials for solid tumors. The increased efficacy with the IA-dex NP formulations compared with DM-Cilengitide in the

M21 model seen here warrants additional efficacy studies in other tumor models.

The dose of 15 mg/kg chosen for the current study was based on information provided in the Cilengitide investigator brochure; since IA-DCL (DPPEsuc) was more efficacious than DM-Cilengitide it is possible that a lower dose may be preferable.

EXAMPLE 6. Efficacy of IA-DCL

This study compared IA-DCL with the same dose of free IA. The efficacy of different doses, as well as different foraiulations was investigated. Efficacy was assessed in the human M21 melanoma model. Cells were implanted by subcutaneous injection in female NU/NU nude mice as previously described (Cheresh, D.A., Honsik, C J., Staffileno, L.K., Jung, G., Reisfeld, R.A., Proc. Natl. Acad. Sci. USA, 82:5155-5159). Normalized tumor volume examined on day 11 (the latest time point at which all animals are still alive) and tumor volume quadrupling time (TVQT) were used to evaluate efficacy in this study. Test articles used were as follows:

SU051.1 Vehicle Control: 0.15M NaCl SU051.2 IA-DCL 44mg/ml SU051.3 IA-DCL 22 mg/ml SU051.4 IA-DCL llmg/ml SU051.5 IA-DCL 44mg/ml SU051.6 IA

SU051.7 DCL 44 mg/ml

SU050.8 IA-DCL 44 mg/ml lyophilized

Definitions and Measurement:

1) Lipid composition: mole percentage.

2) Particle concentration: dry weight.

₃) Size and size range: 0.2mg/ml of particle in lOmM HEPES pH7.₄ by DLS

4) ICsαof αvβ3 / FN-HRP or VN-Biotin challenging assay: IA or IA-particles Inhibiting 50% of maximum FN-HRP or VN-Biotin binding to αvβ3. T21 value used as a control.

5) Endotoxin (Eu/ml): LAL endotoxin testing.

6) Dextran quantity by anthrone assay.

7) T21: Targesome integrin antagonist Tumors were implanted as in EXAMPLE 5. Formulations were injected (6.67/ml/kg) intravenously via the tail vein. A total of four doses were given, once daily every other day (qod) for one week for groups 51.1, 2, 3, 4, 6, 7, 8, and a total of two doses were given, on days 0, 4 for group 51.5. 15 days post implant mice with tumors between 44 and 182 mm³ were selected for treatment and randomly assigned to treatment groups as described in table 6-1. Table 6-1: Description of treatment groups for efficacy study.

Table 6-II summarizes the mouse weight data obtained at treatment and six days post treatment Table 6-II: Summary of weight data by treatment group.

Tumor volume quadrupling time (TNQT) is defined as the time required for a given tumor to show a four-fold increase in volume when compared with the tumor volume measured at the start of treatment. TNQT is calculated as in EXAMPLE 5. Treatment effects for normalized tumor volume on day 11 were compared using analysis of variance (AΝONA) and Kruskal-Wallis statistical tests as in EXAMPLE 5.

The log rank test with Bonferroni correction was used to compare survival data as approximated by TNQT data. The log rank test is a nonparametric test that can accommodate incomplete (censored) data. The log rank test compares the actual survival rates for a given treatment group to the theoretical survival rate if no treatment effect were present.

In order to compare treatment effects, tumor volumes were measured on the day of treatment and three times a week after treatment. The initial tumor volume (pre-treatment) was then used to normalize each tumor volume (see appendix III for a complete summary of the tumor volume data). The eleventh day post treatment was the last tumor measurement before some animals were removed from the study due to large tumor burden. For this reason normalized tumor volume eleven days post treatment start was used to compare treatments in this study. One mouse in the group treated with four doses of 15 mg/kg IA- DCL showed a reduction in tumor volume from a maximum of 87mm³ on day 11 down to a tumor which was too small to measure (< 26mm³) on day 31. This mouse was sacrificed on day 60 and data from this mouse was censored. Figures 17A and 17B show the mean normalized tumor volume over time for selected comparisons between the treatment groups.

Data for normalized tumor volume were compared using AΝONA and Kruskal-Wallis statistical tests. The p-values associated with these tests were both highly significant (<0.0005). In order to determine exactly which treatments show efficacy a number of pairwise or one to one, comparisons were made using Tukey's pairwise comparisons (Table 6-III). Table 6-III: Summary of P-values obtained using Tukey's W procedure with normalized tumor volume measurements 11 days post treatment

Table 6-III indicates that tumors treated with any of the IA-DCL treatment regimes are significantly smaller than tumors treated with buffer or placebo (DCL). In addition, tumors in the higher dosage groups (15 mg/kg IA-DCL for four doses both lyophilized and non lyophilized material) were also significantly smaller than tumors treated with free IA. Non parametric statistical tests are consistent with these results.

Visual comparison of identical treatment using lyophilized and non-lyophilized material shown in Figure 18 indicates that there is no appreciable difference between these two treatments. The statistical analysis summarized in table 6-III above is consistent with this conclusion; however, it is important to note that the number of animals in this study was only sufficient to detect a two fold or greater difference in tumor volume between the treatment groups. Although it is reasonable to conclude that lyophilization does not adversely impact the efficacy of IA-DCL, statistically this study only confirms that any difference must have less than a two fold impact on efficacy.

Tumor volume quadrupling time (TNQT) was used as an estimate of survival. Average TVQTs were as follows: control, 8.7 ± 2.07; DCL, 9.3 ± 2.62; IA (free), 12.0 ± 3.72; IA-DCL- 3.75,4x, 17.0 ± 6.51; IA-DCL 7.5,4x, 16.1 ± 4.76; IA-DCL 15,2x, 17.8 ± 5.68; IA-DCL 15,4x, 26.3 ± 13.64; and IA-DCL lyo, 23.5 ± 7.46. The log rank test with the

Bonferroni Corrections was used to compare survival between treatment groups (The Bonferroni correction was applied by dividing α=0.05 by 28 (the total number of possible comparisons from this study). All IA-DCL treatment regimes show statistically significant longer survival than treatment with buffer or placebo (DCL). In addition, treatment of IA- DCL at high dosages (15mg/kg for four doses) is significantly better than treatment with free

IA alone. Histology

Twenty four hours following the final treatment dose two animals from each treatment group (median tumor volumes were selected) were sacrificed for histological staining. Slides were prepared with H&E, anti-CD31 and TUNEL stains from consecutive sections and were read by a pathologist who was blinded with respect to treatment groups . H & E staining demonstrates that in buffer control mice viable blood vessels are present within the tumor with many vessels localized around the tumor periphery. In contrast, in IA-DCL treated mice there is a large area of tumor necrosis. The TUNEL assay confirms the large area containing apoptotic cells in tumors from IA-DCL treated mice. In contrast, buffer control mice show fewer apoptotic foci. Anti-CD31 immunostaining enumerates blood vessels within the tumor; there are approximately 40% fewer vessels in IA-DCL treated mice compared with the control. As may be expected from the tumor volume results above, the number of blood vessels in IA treated mice is comparable to the control and the number of apoptotic cells is intermediate between the control and IA-DCL treated mice. DCL specimens look similar to the buffer control. These data confirm the anti-angiogenic mechanism of IA-DCL.

Pathology

Two to three animals from a subset of treatment groups were sent for necropsy twenty four hours following the last treatment dose. There were no gross signs of toxicity in any treatment group. The only significant lesions, possibly related to the drug, included the foamy cells primarily seen in the hepatic sinusoids and the inflammatory lesions in the pulmonary blood vessels of some mice. Large cells with foamy cytoplasm are commonly seen when liposome based delivery systems are used. In these mice, however, the numbers were extremely small as were the cells themselves. Nascular lesions were not seen in vessels in other organs, suggesting that the pulmonary vessels may be affected merely because the lung is so highly vascularized and pulmonary capillaries are the first small vessels that the drug encounters. In the lung, necrosis, PMΝs, or fibrinoid degeneration were NOT noted, indicating that the inflammatory lesions in the blood vessels were relatively mild to occasionally moderate, and not all the vessels were affected, nor did these vessel lesions result in thrombosis or perivascular hemorrhage. EXAMPLE 7. Efficacy Evaluation of IA-DCL in a Mouse Brain Tumor Model

Malignant gliomas include anaplastic astrocytomas, anaplastic oligodendrogliomas, and glioblastoma multiforme. These are the most common of the primary brain tumors and occur at a rate of approximately 6.08/100,000 individuals annually within the United States (Central Brain Tumor Registry of the U.S. 2000). Current treatment options include surgery, radiation therapy, and chemotherapy. Despite advances in surgical and radiation therapy, prognosis remains extremely poor and the median survival of 12 months from the time of diagnosis has not changed appreciably over the last 40 years. Novel therapeutic modalities are required to significantly alter the prognosis for patients with malignant gliomas. Among the new agents coming to clinical trial "are anti-angiogenic compounds, which offer particular promise for brain tumor patients.

Malignant gliomas are among the best- vascularized tumors in humans. Neuropathologist to histologically diagnose anaplastic astrocytomas and glioblastoma multiforme utilize the presence of endothelial proliferation or neovascularization. Tumor microvessel density has been shown to correlate with prognosis in breast cancer and metastasis in breast and prostate. Microvessel densities have also been preformed on astroglial tumors and found to correlate with prognosis and with elevated CSF levels of the angiogenic peptide bFGF in children with brain tumors. Angiogenesis can be divided into 3 phases: 1) initiation, 2) proliferation and migration, and 3) maturation . During the phase of proliferation and migration, endothelial cells (EC) undergo morphologic changes, secrete enzymes that allow degradation of the basement membrane and extracellular matrix (EM) and begin to migrate toward the tumor-directed stimulus. During this process of migration, integrins expressed by the EC must bind with EM ligands to ensure cell survival and progression of the angiogenic phenotype. Among the integrins involved with this process are αvβ3 and αvβ5. These integrins appear particularly important for angiogenesis within the central nervous system. The phenotypic description of the αv knockout noted the majority of the vasculature developed normally with the exception of the central nervous system which was characterized by dilated capillaries and hemorrhages. Although expressed by the EC during this activated state and involved in the process of angiogenesis, certain malignancies themselves will express these integrins to enhance invasive or migratory abilities. The malignant gliomas are among the tumors which have been identified as capable of expressing these integrins. Central nervous system malignancies appear to be an excellent system for the evaluation of anti-angiogenic therapies that seek to exploit the αβ integrins.

Intracerebral tumors were induced by allografting established glioma cell lines (U87) into Nude mice. Cells (1.0 xlO⁶) were injected through a 1-mm burr hole made 1.5mm to the right of the midline and 0.5-1.0mm anterior to the coronal suture. Tumor cells are loaded into a 250 μl Hamilton syringe fitted with a 30 gauge 0.5 inch needle and mounted to a sterotaxic holder. The needle is inserted through the burr hole to a depth of 2.5mm. Approximately 5 μl of cells are injected into the right caudate nucleus, needle is withdrawn, and the burr hole plugged with gel foam and animals to return to sterile microisolator cages. Previous studies have determined that these tumors are lethal with median survival times of 26-28 days. A tumor of ~30-50 μl volume can be found approximately 14 days from tumor implantation. Following tumor implantation, mice were randomized to 3 treatment groups: treatment with IA-DCL, DCL alone, or buffer.

The mice receiving DCL and buffer will be considered the control groups. Mice receiving IA-DCL will be the treatment group. Mice in the IA-DCL treatment group will receive IA-DCL intravenously beginning on day 7 post tumor implantation at a dose of 15mg IA/kg body weight. Mice in the DCL control group will receive DCL intravenously beginning on day 7 post tumor implantation at a DCL (drug carrier) dose matched to IA-DCL. The volume of all 3 treatments will be approximately 6.67 mL/kg or 200uL per 30 g mouse. The mice in all groups will undergo a total of six injections every other day, starting on day 7.

On day 20 all animals will be euthanized and the brains harvested immediately and processed for tumor volume analysis. See the following table for the Study Plan.

total 39

*Six doses to be given on day 7, 9, 11, 13, 15, 17 post tumor inoculation Based on injection volume of 6.67 mL/kg or 200 μl per 30g mouse

Tumor volume will be determined by thoroughly mincing the brain, passing through a 170 um pore mesh, resuspension in buffer, and the single cell suspension counted. Cells (500,000) will be incubated with FITC-conjugated mAb anti-human specific HLA-A, B, C; FITC-conjugated mouse IgG isotype control or anti-mouse specific Class I IgG washed and subjected to FACS analysis. The results will be presented as the percentage of human tumor cells for control and treated animals. Statistical Analysis: Based on a previous study using the same tumor/mouse model, we estimate that a sample of size 10 per group be used. From this data, the average tumor volume on day 20 of the control animals was 12.5 with S.D. = 5.9 and of the therapy group these were 5.3 and 2.3, respectively. A sample of size 10/group will allow us to detect with 80% power a real mean volume reduction from 12.5 to 5.3 in the vehicle versus drug treated animals.

Normalized tumor volume and tumor volume quadrupling time (TVQT) data will be used to evaluate efficacy in this study. Parametric and non-parametric statistical methods will be used to analyze normalized tumor volume and TNQT data and to calculate significance. Descriptive statistics, including mean, median, standard deviation and range, may be calculated.

Determine the ability of IA-DCL to inhibit the migration and invasion of endothelial cells. FAK phosphoiylation: Endothelial cell lines (IBE- immortalized mouse and human umbilical vein endothelial cells (HUNECs) will be plated on vitronectin-coated culture plates (lng NΝ/mL with 3 mL per P60). Cells will be exposed to varying concentrations of IA-

DCL, DCL or cyclo(RGDfN) for 6 hours. Detached as well as adherent cells will be collected and the cell pellets lysed using RIPA lysis buffer supplemented with protease inhibitors. Protein will be quantified in the cell lysate and separated on a 7.5% SDS-PAGE gel, transferred to nitrocellulose and western blotted for FAK phosphoiylation using a pY anti- FAK (Biosource International, Camarillo, CA). A chemiluminscent substrate will be utilized to develop the blot and band density will be quantified with an Alpha Innotech digital imaging system. Blots will be stripped and reprobed for total FAK using an anti-FAK antibody (Upstate Biotech, Lake Placid, ΝY). Ratios of phosphorylated to total FAK will be determined for each concentration of IA-DCL, DCL or cyclo(RGDfN). Adhesion assay: The ability of αvβ3 and αvβ5 expressing cells to adhere to ECM proteins is impaired when exposed to cyclic RGD containing peptides. A 96-well format will be utilized for this assay. Individual wells will be coated with vitronectin and plated with endothelial cells at a density of 35,000 per well. Cells will be exposed to varying concentrations of IA-DCL, DCL or cyclo(RGDfN) from 0 μM to lOOuM for a period of 2 hours. Plates will be washed twice with IX PBS and the adherent cell number quantified with

CyQuant (Molecular Probes, Eugene, OR). Cell number is represented as fluorescence and will be plotted in relation to IA-DCL, DCL or cyclo(RGDfN) concentration. The IC₅o will represent the inhibitory concentration at which adhered cells are 50% of control.

Transwell migration: We will use a modified two-well chamber (FluoroBlok, BD

Bioscience, Bedford, MA) with pore sizes of 0.4 μm filter separating the chambers. The invasion assays will utilize an inner chamber with a fluorescence block coating the bottom of the inner chamber. Endothelial cells will be loaded with a fluorescent tracing dye (NyBrant,

Molecular Probes, Eugene, OR). The inner chamber will be plated with human umbilical vein endothelial cells (HUNECs) and/or IBE cells and allowed to grow to 70-80% confluency in vitronectin or matrigel. The media in the inner chamber will be changed to media without serum and cells starved for 24 hours. The outer chamber media will be replaced with media +

10%FCS and the inner chamber will remain media without FCS however IA-DCL, DCL or cyclo(RGDfN) will be added at varying concentrations. At time intervals, the plate will be read from the bottom in order to only detect the cells that have successfully migrated through the vitronectin filter. Cell number is represented as fluorescence and will be plotted in relation to IA-DCL concentration. The IC₅o will represent the inhibitory concentration at which migrated cells are 50% of control.

Results

The ability of IA-DCL to inhibit adhesion of α_vβ₃ expressing tumor and endothelial cells to the extracellular matrix protein vitronectin was evaluated in vitro. Dose dependent inhibition was apparent with an IC₅o of 0.045μM and O.OOlμM for U251 glioma and HUNEC respectively (Table 7-1).

Table 7-1. IC₅o values for the inhibition of the in vitro binding of U251 glioma and HUNEC cells to vitronectin. IC₅₀ represents the inhibitory concentration at which adhered cells are 50% of control.

Anti-tumor efficacy of IA-DCL was evaluated in vivo in the U251 orthotopic glioma model in nude mice. Treatment with IA-DCL significantly reduced tumor growth when compared to freatment with saline alone (n = 10 per group; P = 0.02, Student's t test). The percentage of human tumor cells, as assessed by staining with an antibody to HLA class I, was reduced from 6.8% in control mice down to 3% following IA-DCL treatment (Figure 19), a reduction of approximately 50%.

EXAMPLE 8. Biodistribution and Pharmacokinetic Study of IA-DCL and DCL

The objective of this study was to assess the biodistribution and pharmacokinetics of ¹⁴C-labeled IA-DCL and ¹⁴C-labeled DCL in female nude mice, when administered intravenously. Seventy-two (72) female athymic (nude or nu/nu) mice (Mus museums) approximately 12 weeks of age (Simonson Laboratories, Gilroy, CA) were used in the study. Mice were selected for the study since mice are an accepted species frequently used in pre- clinical evaluation of drugs intended for human use. Nude mice in particular are frequently used for studies of anti-cancer agents. All animals were weighed on Day 0. Body weights ranged from 21.6 to 27.9 g. Each animal received a single intravenous (tail vein) bolus of

0.17 mL (approximately 1.1 μCi) of either test article on Day 0. Clinical observations, including morbidity, mortality, and overt signs of toxic or pharmacologic effect(s) were recorded for animals periodically throughout the in-life portion of the study. Three (3) mice per group were terminally bled by cardiocentesis at 0, 3, 15, 30 min and 1, 2, 4, 8, 12, 24, 36, and 48 hr post-dose. Blood for plasma (0.5 mL/sample) was collected and placed into labeled

Microtainers® with sodium heparin as anti-coagulant, centrifuged, and pipetted off into labeled Eppendorf® tubes (for at least 0.2 ml plasma) and frozen at -80°C. The plasma samples were separated into two aliquots, one to be held and one to be sent for scintillation counting. After bleeding, the mouse was infused with 5 mL of 0.9% NaCl and lung, liver, spleen, lymph nodes, kidney, heart, and muscle were collected. Pieces of the tissues from all three mice at each time point were pooled together into a single tared scintillation vial with combustion cones. The vials were then re-weighed to obtain organ weights. Scintillation counting was performed as follows: briefly, 50 μL of each of the three plasma samples for each timepoint were combined. The combined samples were diluted with 2 mL of water, 15 mL of scintillation cocktail was added, and the samples were counted in a Beckman LS6000IC scintillation counter. The tissue samples were combusted in a Harvey OX500 sample oxidizer and counted in either a Beckman LS6000IC or LS6500 scintillation counter together with appropriate controls. Pharmacokinetic analysis was performed using a non- compartmental model (Winnlin, Version 4.0). Peak (C_max) plasma radioactivity was attained at 30 min for IA-DCL and at 2 hr for DCL. The C_max for an IN drug normally occurs immediately after injection since there is no absorption phase with IN administration. Radioactivity was detected in plasma as early as 5 minutes after dosing for both ¹⁴C -IA-DCL 0 and ¹⁴C-DCL. Radioactivity was still detectable 48 hours after dosing in plasma and all tissues. The plasma half-lives were 23 and 18 hr for IA-DCL and DCL, respectively, and associated with clearances of 0.09 and 0.05 gm/hr. The volume of distribution at steady state (Nss) was <1.0 gm suggesting restriction to the vascular compartment. Except for the liver, tissue AUCs were much lower than the plasma AUCs for both test articles, suggesting limited 5 tissue distribution for both test articles. Figure 20 displays the time course of radioactivity over 48 hours for ¹⁴C -IA-DCL and ¹ C-DCL in plasma. The selected PK parameter estimates are displayed in Table 8-1.

Table 8-1 : IA-DCL and DCL PK Parameter Estimates in Plasma

>0

IA-DCL PK parameter estimates determined for the selected tissues are displayed in Table 8-2 below. Table 8-2

extrapolation of AUC to infinity >30%.

Comparison of AUC_LAS_T from various tissues to the AUC_LA_ST of plasma resulted in ratios <1 for the tissues. The AUC_LA_ST ratio was greatest for the liver (0.57) followed by spleen (0.32) > >lymρh (0.13)> kidney (0.08) > lung (0.08) > heart (0.04 ) = muscle (0.04). The PK parameter estimates for 14C-DCL are shown in Table 8-3.

Table 8-3: PK Parameter Estimates of 14C-DCL in Tissues

ND - Not determined; the r2 was too low for the regression line to determine λ, resulting in extrapolation of AUC to infinity >30%

As hypothesized, this preliminary study demonstrates a long half-life of C -IA-DCL and a restricted distribution. The delay in reaching maximum radioactivity in the plasma for DCL suggests a lack of instantaneous or rapid distribution of C -DCL, or sequestering and subsequent release back into the blood pool. ^l C-IA-DCL exhibited a similar time course as

¹⁴C -DCL although some differences in the distribution phase were observed, e.g. uptake in liver and spleen were lower and occurred more slowly for DCL than for IA-DCL. Lung, heart and muscle tissue AUC_LA_ST were approximately or less than 5% of the plasma AUC_LA_ST supporting the hypothesis of restricted distribution. In addition, comparison of the AUCL_AST of liver and spleen to plasma AUCLA_ST suggested considerable uptake of IA-DCL in the RES system, a known observation of liposomes. The tissue half-lives were similar to or longer than the half-life observed for plasma for both compounds. Doxil®, a marketed product of encapsulated doxorubicin in liposome, has an elimination half-life ranging from 22 to 28 hours in rats. In man, the half-life is reported to be 57 hours, allowing for an every 2 or 3 week administration. The elimination half-life of IA-DCL reported here in mice is similar to that of Doxil® in rats; thus, we hypothesize that IA-DCL will have a long half-life in man allowing for an infrequent (every 2 or 3 weeks) administration. In summary, the prolonged half-life and limited distribution observed in this study support the proposed advantages offered by this technology.

Claims

CLAIMS:What is claimed is:

1. A targeted macromolecule comprising a liposome, said liposome comprising a stabilizing agent covalently attached to the surface of the liposome, and more than one targeting molecule, said targeting molecule being covalently attached to said stabilizing agent.

2. The targeted macromolecule of Claim 1 , wherein the liposome comprises 1 ,2- dipalmitoyl-sn-glycero-3 -phosphocholine; 1 ,2-dipalmitoyl-sn-glycero-3 - phosphoethanolamine-N-(succinyl) and cholesterol.

3. The targeted macromolecule of Claim 1 , wherein the targeting molecule is an integrin antagonist.

4. The targeted macromolecule of Claim 3, wherein the targeting molecule is 3- {4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino}-2(S)-benzene- sulfonyl-aminopropionic acid.

5. The targeted macromolecule of claim 4, wherein the 3- {4-[2-(3 ,4,5,6- tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino}-2(S)-benzene-sulfonyl- aminopropionic acid is attached to liposomes at loadings of up to a member of the group consisting of about 5% (w/w), about 8% (w/w), about 10% (w/w), about 15% (w/w), about 20% (w/w), about 25% (w/w), and about 30% (w/w).

6. The targeted macromolecule of claim 5, wherein the 3- {4-[2-(3 ,4,5,6- tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino}-2(S)-benzene-sulfonyl- aminopropionic acid is attached to liposomes at loadings of up to about 5% (w/w).

7. The targeted macromolecule of claim 1 , wherein the stabilizing agent is selected from the group consisting of dextran, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans, inulin, levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, puUulan, glycogen, amylopectin, cellulose, dextran, dextrose, dextrin, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch, natural homopolyner or heteropolymers containing one or more of the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, naturally occurring derivatives of the foregoing; proteins, albumin, polyalginates, and polylactide-glycolide copolymers, cellulose, cellulose (microcrystalline), methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, carboxymethylcellulose sodium 12, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, methoxycellulose, carboxydextran, aminodextran, dextran aldehyde, chitosan, carboxymethyl chitosan, poly(ethylene imine) and derivatives, polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes, Pluronics®, polyoxyethylene, and polyethylene terephthlate), polypropylenes, polypropylene glycol, polyurethanes, polyvinyl alcohol (PNA), polyvinyl chloride, polyvinylpyrrolidone, poly amides, nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers, polytetrafluoroethylene, acrylate, methacrylate, polymethylmethacrylate and derivatives thereof, polysorbate, carbomer 934P, magnesium aluminum silicate, aluminum monostearate, polyethylene oxide, polyvinylalcohol, povidone, polyethylene glycol, and propylene glycol.

8. The targeted macromolecule of claim 7, wherein the stabilizing agent is dextran or modified dextran.

9. The targeted macromolecule of Claim 1 , wherein said targeted macromolecule is IA-DCL.

10. A method of preparing a targeted macromolecule, comprising: a) providing a liposome; b) covalently associating the liposomes with a stabilizing agent to generate coated liposomes; c) attaching at least one linker to the coated liposomes to generate linker- attached liposomes via the stabilizing agent, wherein the linker facilitates attachment of a targeting molecule; and d) attaching at least one targeting molecule to the linker-attached liposome via the linker, whereby a targeted macromolecule is prepared.

11. The method of Claim 10, wherein the liposome comprises 1 ,2-dipalmitoyl-sn- glycero-3 -phosphocholine; 1 ,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine-N-(succinyl) and cholesterol.

12. The method of Claim 10, wherein the targeting molecule is an integrin antagonist.

13. The method of Claim 3, wherein the targeting molecule is 3-{4-[2-(3,4,5,6- tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino}-2(S)-benzene-sulfonyl- aminopropionic acid.

14. The method of claim 13 , wherein the 3 - {4-[2-(3 ,4,5,6-tetrahydropyrimidin-2- ylamino)-ethyloxy]-benzoylamino}-2(S)-benzene-sulfonyl-aminopropionic acid is attached to liposomes at loadings of up to a member of the group consisting of about 5% (w/w), about 8% (w/w), about 10% (w/w), about 15% (w/w), about 20% (w/w), about 25% (w/w), and about 30% (w/w).

15. The method of claim 14, wherein the 3-{4-[2-(3,4,5,6-tetrahydropyrimidin-2- ylamino)-ethyloxy]-benzoylamino}-2(S)-benzene-sulfonyl-aminopropionic acid is attached to liposomes at loadings of up to about 5% (w/w).

16. The method of claim 10, wherein the liposome is prepared using a lipid solution in t-butanol.

17. The method of claim 10, wherein the stabilizing agent is selected from the group consisting of dextran, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans, inulin, levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, puUulan, glycogen, amylopectin, cellulose, dextran, dextrose, dextrin, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch, natural homopolyner or heteropolymers containing one or more of the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, naturally occurring derivatives of the foregoing; proteins, albumin, polyalginates, and polylactide-glycolide copolymers, cellulose, cellulose (microcrystalline), methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, carboxymethylcellulose sodium 12, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, methoxycellulose, carboxydextran, aminodextran, dextran aldehyde, chitosan, carboxymethyl chitosan, poly(ethylene imine) and derivatives, polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes, Pluronics®, polyoxyethylene, and polyethylene terephthlate), polypropylenes, polypropylene glycol, polyurethanes, polyvinyl alcohol (PNA), polyvinyl chloride, polyvinylpyrrolidone, polyamides, nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers, polytetrafluoroethylene, acrylate, methacrylate, polymethylmethacrylate and derivatives thereof, polysorbate, carbomer 934P, magnesium aluminum silicate, aluminum monostearate, polyethylene oxide, polyvinylalcohol, povidone, polyethylene glycol, and propylene glycol.

18. The method of claim 17, wherein the stabilizing agent is dextran or modified dextran.

19. A targeted macromolecule prepared by the method of claim 10.

20. The targeted macromolecule of claim 19, wherein the targeted macromolecule is IA-DCL.

21. A method for targeting an agent to a site of pathology comprising administering a compound of Claim 10 to a patient in need thereof.

22. The method of claim 21, wherein the site of pathology is selected from the group consisting of solid tumors, blood-borne tumors, leukemias, tumor metastasis, malignant gliomas, anaplastic astrocytomas, anaplastic oligodendrogliomas, glioblastoma multiforme, benign tumors, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis, psoriasis; chronic inflammation; ocular angiogenic diseases, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; arteriovenous malformations; ischemic limb angiogenesis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma, wound granulation, intestinal adhesions, atherosclerosis, restenosis, scleroderma, and hypertrophic scars,and keloids.

23. A method for targeting a liposome containing a therapeutic agent comprising: a) covalently associating the liposomes with a stabilizing agent to generate coated liposomes; b) attaching at least one linker to the coated liposomes to generate linker- attached liposomes via the stabilizing agent, wherein the linker facilitates attachment of a targeting molecule; c) attaching at least one targeting molecule to the linker-attached liposome via the linker; and d) administering the resulting composition to a patient in need thereof; whereby a liposome is targeted.

24. A method of treating a disease accompanied by a condition selected from the group consisting of neovascularization, aberrant vascular growth, and excessive or abnormal stimulation of endothelial cells, comprising administering a compound of Claim 1 to a patient in need thereof.

25. The method of claim 24, wherein the disease is selected from the group consisting of solid tumors, blood-borne tumors, leukemias, tumor metastasis, malignant gliomas, anaplastic astrocytomas, anaplastic oligodendrogliomas, glioblastoma multiforme, benign tumors, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis, psoriasis; chronic inflammation; ocular angiogenic diseases, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; arteriovenous malformations; ischemic limb angiogenesis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma, wound granulation, intestinal adhesions, atherosclerosis, restenosis, scleroderma, and hypertrophic scars,and keloids.

26. The method of claim 12, wherein the administration is intravenous.