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• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
  • A61K51/1217—Dispersions, suspensions, colloids, emulsions, e.g. perfluorinated emulsion, sols
  • A61K51/1227—Micelles, e.g. phospholipidic or polymeric micelles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
  • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
  • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
  • A61K51/1217—Dispersions, suspensions, colloids, emulsions, e.g. perfluorinated emulsion, sols
  • A61K51/1234—Liposomes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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• • A—HUMAN NECESSITIES
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  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

• 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 macromolcules having more than one targeting and/or therapeutic entity.
• cancer is the second most common cause of death after heart disease, accounting for approximately one-quarter of the deaths in 1997.
• new and effective treatments for cancer will provide significant health benefits.
• targeted therapeutic agents hold considerable promise.
• 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.
• doxorubicin anthracycline antibiotic doxorubicin
• doxorubicin anthracycline antibiotic
• Doxorubicin has wide activity against a number of human neoplasms and is used extensively both as a single agent and in combination regimens.
• Doxorubicin can be administered in its free form, however, this use of free doxorubicin is linked to toxicity in the form of both an acute and a chronic form of cardiomyopathy.
• doxorubicin There are two US Food and Drug Administration approved liposomal formulations of doxorubicin currently available, with several additional liposomal formulations being researched either in the laboratory or in clinical trials. These liposomal formulations reduce the toxicity of doxorubicin, as these systems tend to sequester the drug away from organs such as the heart, with greater accumulation in liver, spleen and tumors. Overall, the use of liposomal doxorubicin allows for a greater lifetime cumulative dose of doxorubicin to be administered.
• the taxanes are a group of drugs that includes paclitaxel (Taxol®) and docetaxel (Taxotere®), which are used in the treatment of cancer.
• Taxanes block cell division by the promotion and stabilization of microtubule assemblies. This induced stability dispruts the kinetics and equilibrium of microtubule-dependent cytoplasmic structures that are required for such functions as mitosis, maintenance of cellular morphology, shape changes, neurite formation, locomotion, and secretion, thereby damaging the cells.
• FDA U.S. Food and Drug Administration
• Paclitaxel was later approved as initial treatment for ovarian cancer in combination with cisplatin. Women with epithelial ovarian cancer are now generally treated with surgery followed by a taxane and a platinum (another type of anticancer drug).
• paclitaxel for the treatment of breast cancer that recurred within 6 months after adjuvant chemotherapy (chemotherapy that is given after the primary treatment to enhance the effectiveness of the primary treatment), or that spread (metastasized) to nearby lymph nodes or other parts of the body.
• chemotherapy that is given after the primary treatment to enhance the effectiveness of the primary treatment
• paclitaxel is also used for other cancers, including AIDS-related Kaposi's sarcoma and lung cancer.
• Docetaxel a compound that is structurally similar to paclitaxel, has been approved by the FDA to treat advanced breast, lung, and ovarian cancer. Both paclitaxel and docetaxel have unpleasant side effects, and neither is currently available in a liposomal formulation. Camptothecin and topotecan are other therapeutic agents which exhibit an in vivo antitumor effect, thought to be mediated through the inhibition of angiogenesis. Clements, et al., Cancer Chemother. Pharmacol. (1999) 44:411-16. This publication, and all other patents, patent applications, and publications referred to herein are incorporated by reference herein in their entirety. Integrins
• the integrins are a class of proteins involved in the attachment of cells to matrix via
• 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., J Bioact. Compat. Polym.
• the various ⁇ subunits can combine with various ⁇ subunits to form distinct integrins.
• the integrin identified as ⁇ v ⁇ 3 (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).
• integrin inhibiting agents would be useful as antivirals, antifungals and antimicrobials.
• therapeutic agents that selectively inhibit or antagonize ⁇ v ⁇ 3 would be beneficial for treating such conditions.
• the ⁇ v ⁇ 3 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.
• RGD peptides in general are non-selective for RGD dependent integrins.
• RGD peptides that bind to ⁇ v ⁇ 3 also bind to ⁇ v ⁇ s, ⁇ v ⁇ , and ⁇ n ⁇ ia- Antagonism of platelet ⁇ b ⁇ la (also known as the fibrinogen receptor) is known to block platelet aggregation in humans.
• Ginsberg et al., U.S. Pat. No. 5,306,620 disclose antibodies that react with integrin so that the binding affinity of integrin for ligands is increased. As such these monoclonal antibodies are said to be useful for preventing metastasis by immobilizing melanoma tumors. Brown, U.S. Pat. No. 5,057,604 discloses the use of monoclonal antibodies to ⁇ v ⁇ 3 integrins that inhibit RGD-mediated phagocytosis enhancement by binding to a receptor that recognizes RGD sequence containing proteins. Plow et al., U.S. Pat. No.
• 5,149,780 disclose a protein homologous to the RGD epitope of integrin ⁇ subunits and a monoclonal antibody that inhibits integrin-ligand binding by binding to the ⁇ 3 subunit. That action is said to be of use in therapies for adhesion-initiated human responses such as coagulation and some inflammatory responses.
• VEGF vascular endothelial growth factor
• VEGF is unique among angiogenic growth factors in its ability to induce a transient increase in blood vessel permeability to macromolecules (hence its original and alternative name, vascular permeability factor) (Dvorak et ⁇ .(1979) J. Immunol. 122:166-174; Senger et ⁇ /.(1983) Science 219:983-985; Senger et /.(1986) Cancer Res. 46:5629-5632).
• Increased vascular permeability and the resulting deposition of plasma proteins in the extravascular space assists the new vessel formation by providing a provisional matrix for the migration of endothelial cells (Dvorak et /.(1995) Am.
• VEGF and its receptors contribute to tumor growth were recently obtained by a demonstration that the growth of human tumor xenografts in nude mice could be inhibited by neutralizing antibodies to VEGF (Kim et al. (1993) Nature 362:841-844), by the expression of dominant-negative VEGFR2 (Millauer et al. (1996) Cancer Res.56: 1615-1620; Millauer et al. (1994) Nature 367:576-579), by low molecular weight inhibitors of VEGF receptor inhibitors (Strawn et al. (1966) Cancer Res. 56:3540-3545), or by the expression of antisense sequence to VEGF mRNA (Saleh et al. (1996) Cancer Res.
• VEGF antagonists Asano et al. (1995) Cancer Res. 55:5296-5301; Warren et al. (1995) J. Clin. Invest. 95:1789-1797; Claffey et al. (1996) Cancer Res. 56:172-181; Melnyk et al. (1996) Cancer Res. 56:921-924. Inhibitors of VEGF signaling may thus have broad clinical utility as anticancer agents.
• VEGF signaling In addition to cancer, as noted above, other proliferative diseases characterized by excessive neovascularization such as psoriasis, age-related macular degeneration, diabetic retinopathy and rheumatoid arthritis could be treated with antagonists of VEGF signaling.
• VEGF occurs in several forms (VEGF-121, VEGF-145, VEGF-165, VEGF-189, VEGF-206) as a result of alternative splicing of the VEGF gene that consists of eight exons (Houck et al. (1991) Mol. Endocrin. 5:1806-1814; Tischer et al. (1991) J. Biol. Chem. 266:11947-11954; Poltorak et al. (1997) J. Biol. Chem. 272:7151-7158).
• the three smaller forms are diffusable, while the larger two forms remain predominantly localized to the cell membrane as a consequence of their high affinity for heparin.
• VEGF-165 and VEGF-145 also bind to heparin (as a consequence of containing basic exon 7- and exon 6-encoded domains, respectively), albeit with somewhat lower affinity compared with VEGF-189 (that contains both exons 6 and 7).
• VEGF-165 appears to be the most abundant form in most tissues (Houck et al. (1991) Mol. Endocrinol. 5:1806-1814; Carmeliet et al. (1999) Nature Med. 5:495-502).
• VEGF-121 the only alternatively spliced form that does not bind to heparin, appears to have a somewhat lower affinity for the receptors (Gitay-Goren et al. (1996) J. Biol. Chem. 271:5519-5523) as well as lower mitogenic potency (Keyt et al. (1996) J. Biol. Chem. 271:7788-7795).
• VEGF vascular endothelial growth factor receptor 1
• Flt-1 Flt-1
• Flk-1/KDR Flk-1/KDR
• VEGFR2 is by far the more abundant receptor (Brown et al. (1997) in Regulation of Angiogenesis, supra). In vivo, however, in quiescent endothelial cells, both receptors are expressed at low levels (Kremer et al. (1997) Cancer Res.57:3852-3859; Barleon et al. (1997) Cancer Res.57:5421-5425).
• Both receptors are substantially upregulated when endothelial cells are activated by a variety of stimuli.
• Hypoxia for example, induces an increase in expression of both VEGFRl and VEGFR2 in endothelial cells (Tuder et al. (1995) J. Clin. Invest. 95:1798- 1807; Gerber et al. (1997) J. Biol. Chem. 272:23659-23667; Brogi et al. (1996) J. Clin. Invest. 97:469-476; Kremer et al. (1997) Cancer Res. 57:3852-3859).
• hypoxia leads to both direct activation via the flt-1 promoter that contains the hypoxia- inducible-f actor- 1 (HIF-1) consensus binding site (Gerber et al. (1997) J. Biol. Chem., supra) and indirect activation via hypoxia-induced VEGF (Barleon et al. (1997) Cancer Res., supra).
• VEGF-induced upregulation of VEGFRl is mediated by both VEGFRl and VEGFR2 (Barleon et al. (1997) Cancer Res., supra).
• VEGFR2 is upregulated by VEGF (through VEGFR2, but not VEGFRl) (Kremer et al. (1997) Cancer Res., supra; Wilting et al. (1996) Dev.
• VEGFR2 in endothelial cells is also upregulated by bFGF and this accounts in part for the synergistic activation of endothelial cells by VEGF and bFGF (Pepper et al. (1998) Exp. Cell Res. 241:414-425).
• VEGFRl and VEGFR2 expression may be sensitive to variations in blood flow (Tuder et al. (1995) J. Clin. Invest., supra).
• porcine aortic endothelial (PAE) cells transfected with the flt-1 or kdr receptor genes have suggested that VEGFR2 is the primary transducer in endothelial cells of VEGF-mediated signals related to changes in cell morphology and mitogenicity (Waltenberger et al. (1994) J. Biol. Chem. 269:26988-26995).
• stimulation of flt-1 -transfected PAE cells with VEGF did not appear to produce detectable changes.
• VEGFRl VEGF signaling through VEGFRl induces migration of monocytes and upregulation of tissue factor expression in both endothelial cells and monocytes.
• VEGF signaling through VEGFRl induces migration of monocytes and upregulation of tissue factor expression in both endothelial cells and monocytes.
• the extracellular domain of VEGFR2 is retained on a cation exchange resin only in the presence of VEGFRl and that the VEGFR2 retention is enhanced when both VEGFRl and VEGF were present
• Kendall et al. have concluded that the two receptors have some affinity for one another and that this interaction is stabilized by VEGF (Kendall et al.
• VEGFR1/R2 heterodimer constitutes at least a fraction of the binding- competent VEGF receptor.
• VEGFRl and VEGFR2 are expressed predominantly on endothelial cells, they have also been detected on some non-endothelial cells.
• VEGFRl is expressed on trophoblasts (Charnockjones et al. (1994) Biol. Reprod. 51:524-530), monocytes (Barleon et al. (1996) Blood, supra), hematopoietic stem cells and megakaryocytes/platelets (Katoh et al. Cancer Res. 55:5687-5692), renal mesangial cells (Takahashi et al. (1995) Biochem. Biophys. Res. Commun. 209:218-226) and pericytes (Yamagishi et al. (1999) Lab.
• VEGFRl In monocytes, VEGFRl is responsible for the VEGF-mediated induction of migration and tissue factor expression (Clauss et al. (1996) J. Biol. Chem., supra; Barleon et al. (1996) Blood, supra; Hiratsuka et al. (1998) Proc. Natl. Acad. Sci., supra). In pericytes, VEGFRl may mediate the recently described ability of VEGF to act as a mitogen and chemotactic factor (Yamagishi et al. (1999) Lab. Invest., supra). The role of VEGFRl in trophoblasts and mesangial cells remains to be elucidated.
• VEGFR2 has been detected on hematopoietic stem cells, megakaryocytes/platelets and retinal progenitor cells (Katoh et al. (1995) Cancer Res. 55:5687-5692; Yang et al. (1996) J. Neurosci. 16:6089-6099). VEGFRl and VEGFR2 expression has also been reported on malignant cells including leukemia cells (Katoh et al. (1995) Cancer Res., supra) and melanoma cells (Gitay-Goren et al. (1993) Biochem. Biophys. Res. Commun. 190:702-709).
• 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.
• 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.
• 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.
• linkers include SMCC (succinimidyl 4-[N- maleimidomethyl]cyclohexane-l-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.
• 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.
• 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.
• 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.
• 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. 5,246,707 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.
• Liposomes containing polymerized lipids for non-covalent immobilization of proteins and enzymes are described in Storrs et al., "Paramagnetic Polymerized Liposomes: Synthesis, Characterization, and Applications for Magnetic Resonance Imaging," J. Am. Chem. Soc. (1995) 117(28):7301- 7306; and Storrs et al., "Paramagnetic Polymerized Liposomes as New Recirculating MR Contrast Agents," JMRI (1995) 5(6):719-724.
• 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.
• 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.
• Magnetic resonance imaging is an imaging technique which, unlike X-rays, does not involve ionizing radiation.
• MRI may be used for producing cross-sectional images of the body in a variety of scanning planes such as, for example, axial, coronal, sagittal or orthogonal.
• MRI employs a magnetic field, radio-frequency energy and magnetic field gradients to make images of the body.
• the contrast or signal intensity differences between tissues mainly reflect the Tl (longitudinal) and T2 (transverse) relaxation values and the proton density in the tissues.
• Tl longitudinal
• T2 transverse relaxation values
• proton density proton density in the tissues.
• a contrast medium may be designed to change either the Tl, the T2 or the proton density.
• MRI requires the use of contrast agents. If MRI is performed without employing a contrast agent, differentiation of the tissue of interest from the surrounding tissues in the resulting image may be difficult.
• paramagnetic contrast agents involve materials which contain unpaired electrons. The unpaired electrons act as small magnets within the main magnetic field to increase the rate of longitudinal (Tl) and transverse (T2) relaxation.
• Paramagnetic contrast agents typically comprise metal ions, for example, transition metal ions, which provide a source of unpaired electrons.
• these metal ions are also generally highly toxic. For example, ferrites often cause symptoms of nausea after oral administration, as well as flatulence and a transient rise in serum iron.
• the gadolinium ion which is complexed in Gd-DTPA, is highly toxic in free form.
• the various environments of the gastrointestinal tract including increased acidity (lower pH) in the stomach and increased alkalinity (higher pH) in the intestines, may increase the likelihood of decoupling and separation of the free ion from the complex.
• the metal ions are typically chelated with ligands.
• Ultrasound is another valuable diagnostic imaging technique for studying various areas of the body, including, for example, the vasculature, such as tissue microvasculature.
• diagnostic techniques involving nuclear medicine and X-rays generally involve exposure of the patient to ionizing electron radiation. Such radiation can cause damage to subcellular material, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins. Ultrasound does not involve such potentially damaging radiation.
• ultrasound is inexpensive relative to other diagnostic techniques, including CT and MRI, which require elaborate and expensive equipment. Ultrasound involves the exposure of a patient to sound waves. Generally, the sound waves dissipate due to absorption by body tissue, penetrate through the tissue or reflect off of the tissue.
• the reflection of sound waves off of tissue forms the basis for developing an ultrasound image.
• sound waves reflect differentially from different body tissues. This differential reflection is due to various factors, including the constituents and the density of the particular tissue being observed.
• Ultrasound involves the detection of the differentially reflected waves, generally with a transducer that can detect sound waves having a frequency of one to ten megahertz (MHz). The detected waves can be integrated into an image which is quantitated and the quantitated waves converted into an image of the tissue being studied.
• ultrasound also generally involves the use of contrast agents.
• Exemplary contrast agents include, for example, suspensions of solid particles, emulsified liquid droplets, and gas-filled bubbles (see, e.g., Hilmann et al., U.S. Pat. No. 4,466,442, and published International Patent Applications WO 92/17212 and WO 92/21382).
• Widder et al., published application EP-A-0 324 938 disclose stabilized microbubble-type ultrasonic imaging agents produced from heat- denaturable biocompatible protein, for example, albumin, hemoglobin, and collagen.
• liposomes or vesicles are useful as contrast agents.
• the effectiveness of liposomes as contrast agents depends upon various factors, including, for example, the size and/or elasticity of the bubble.
• liposomes disclosed in the prior art have undesirably poor stability.
• 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.
• microbubbles A Novel MR Susceptibility Contrast Agent.
• the microbubbles described by Moseley et al. comprise air coated with a shell of human albumin.
• 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.
• the present invention provides a targeted macromolecule comprising a linking carrier and more than one targeting entity.
• the targeted macromolecule comprises three or more targeting entities, ten or more targeting entities, 100 or more targeting entities, and 1000 or more targeting entities, or is present at a concentration from 0.1 to 10 mole percent.
• the linking carrier may be a liposome, may comprise polymerizable lipids, or may be a polymerized vesicle.
• the targeting entity may be associated with the linking carrier by covalent or non- covalent means.
• the targeting entity may target the targeted macromolecule to a cell surface, or may have a vascular target, a tumor cell target.
• the targeting entity is an integrin-specific molecule, such as an
• RGD peptide or and RGD peptidomimetic, such as 3- ⁇ 4-[2-(3,4,5,6-tetrahydropyrimidin-2- ylamino)-ethyloxy]-benzoylamino ⁇ -2(S)-benzene-sulfonyl-aminopropionic acid.
• the present invention provides a macromolecule comprising more than one 3- ⁇ 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino ⁇ -2(S)- benzene-sulfonyl-aminopropionic acid moiety.
• the targeted macromolecule may include a compound of the formula: wherein the compound is associated with the linking carrier by non-covalent or covalent means. This compound is also provided by the present invention.
• the targeting entity is a tyrosine kinase specific molecule, such as the compounds AG1433 or SU1498.
• the targeting entity has a target selected from the group consisting of P-selectin, E-selectin, pleiotropin, G-protein coupled receptors, endosialin, endoglin, VEGF receptors, PDGF receptor, EGF receptor, FGF receptors, the matrix metalloproteases including MMP2 and MMP9, and prostate specific membrane antigen (PSMA).
• the targeting entity is an enzyme modulator.
• the targeted macromolecule of further comprises a therapeutic entity.
• the therapeutic entity may be associated with the linking carrier via a chelator lipid, such as N,N-bis[[[[(13',15'-pentacosadiynamido-3,6- doxaoctyl)carbamoyl]methyl](carboxymethyl)amino]ethyl]glycine.
• the therapeutic entity is Y-90, Bi-213, At-211, Cu-67, Sc-47, Ga-67, Rh-105, Pr-142, ⁇ d-147, Pm-151, Sm-153, Ho-166, Gd-159, Tb-161, Eu-152, Er- 171, Re-186, or Re-188.
• the therapeutic entity is 90 Y and the targeting entity is 3- ⁇ 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino ⁇ -2(S)-benzene- sulfonyl-aminopropionic acid.
• the targeted macromolecule further comprises a stabilizing entity, such as a natural polymer, a semi-synthetic polymer, and a synthetic polymer, such as dextran, modified dextran, and poly (ethylene imine).
• the stabilizing entity provides the capacity for multivalency.
• the invention provides a method of treating a patient comprising administering a therapeutic agent to a patient in need thereof in a sufficient amount, said therapeutic agent comprising a targeted macromolecule, said targeted macromolecule comprising a liposome or polymerized vesicle, more than one targeting entity, and a therapeutic entity.
• the invention provides a method of therapeutic treatment, comprising the step of introducing into a bodily fluid contacting an area of desired treatment a the targeted macromolecule.
• the targeted macromolecule further comprises a detectable entity, such as a metal ion, or a radioactive metal ion, including Tc-99m, In-Ill, Ga-67, Rh-105, Nd -147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, or Tl- 201.
• a detectable entity such as a metal ion, or a radioactive metal ion, including Tc-99m, In-Ill, Ga-67, Rh-105, Nd -147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, or Tl- 201.
• the invention further provides a method of imaging a patient comprising administering an imaging agent to a patient in need thereof, said imaging agent comprising a targeted macromolecule, said targeted macromolecule comprising more than one targeting entity and a detectable entity; and imaging the patient.
• the imaging may include magnetic resonance imaging or nuclear scintigraphy.
• the imaging of a patient may comprise imaging a tumor.
• the present invention provides a targeted therapeutic agent comprising a linking carrier, a therapeutic entity associated with the linking carrier, and at least one targeting entity.
• the agent has three or more targeting entities, ten or more targeting entities, 100 or more targeting entities, and 1000 or more targeting entities, or is present at a concentration from 0.1 to 10 mole percent of the targeting agent.
• the linking carrier may be a macromolecule, including a liposome, a polymerized vesicle, a dendrimer, and a block copolymer, among others.
• the linking carrier comprises a phosphatidylcholine derivative.
• the targeting entity may be associated with the lipid construct by covalent or non- covalent means.
• the targeting entity may target the lipid construct to a cell surface, or may have a vascular target, a tumor cell target.
• the targeting entity is an integrin-specific molecule, such as an RGD peptide, or RGD petidomimetic, including 3- ⁇ 4-[2-(3,4,5,6-tetrahydropyrimidin-2- ylamino)-ethyloxy]-benzoylamino ⁇ -2(S)-benzene-sulfonyl-aminopropionic acid.
• the present invention provides a targeted therapeutic agent comprising more than one 3- ⁇ 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino ⁇ - 2(S)-benzene-sulfonyl-aminopropionic acid moiety.
• the targeted therapeutic agent may include a compound of the formula:
• the targeting entity is a tyrosine kinase specific molecule, such as the compounds AG1433 or SU1498.
• the targeting entity has a target selected from the group consisting of P-selectin, E-selectin, pleiotropin, G-protein coupled receptors, endosialin, endoglin, VEGF receptors, PDGF receptor, EGF receptor, FGF receptors, the matrix metalloproteases including MMP2 and MMP9, and prostate specific membrane antigen
• the targeting entity is an enzyme modulator.
• the targeted therapeutic agent further comprises a stabilizing entity, such as natural polymer, a semi-synthetic polymer, and a synthetic polymer, such as dextran, modified dextran, and poly (ethylene imine).
• the stabilizing entity provides the capacity for multivalency.
• the targeted therapeutic agent may comprise a therapeutic entity which is present at a concentration of about 1% to about 20%.
• the therapeutic entity may be doxorubicin, daunorubicin, epirubin, or idarubicin., or a taxane compound, such as paclitaxel or docetaxel, or other agents, such as camptothecin or topotecan.
• the invention provides a targeted therapeutic agent, comprising doxorubicin and 3- ⁇ 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)-ethyloxy]- benzoylamino ⁇ -2(S)-benzene-sulfonyl-aminopropionic acid.
• the invention provides a method of preparing a targeted therapeutic agent, comprising providing a targeted lipid construct, said targeted lipid construct comprising more than one targeting entity, and associating a therapeutic entity within the lipid construct.
• the lipid construct selected from the group consisting of liposomes, micelles, vesicles, and polymerized liposomes.
• the targeting entity may be doxorubicin, daunorubicin, epirubin, idarubicin, a taxane compound, such as paclitaxel or docetaxel, or other therapeutic entity, such as camptothecin or topotecan.
• the invention provides a method of treating a patient in need thereof comprising administering an effective amount of a pharmaceutical composition comprising a linking carrier, said linking carrier comprising at least one targeting entity, and an associated therapeutic entity to a patient need thereof.
• 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 1A 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 IG 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 carier 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 ⁇ -caproylamine-DPPE hydrochloride.
• Figures 5-15 show exemplary lipids with a variety of functionalites for linking a lipid to a targeting entity or therapeutic entity, and showing various spacer groups.
• Figure 16 shows the synthesis of the integrin antagonist that contains a linker to attach to a lipid for incorporation into polymerized vesicles for multivalent display.
• the compound was designed to incorporate an ethylamine linker and retain the aminosulfonate that is necessary for binding to the integrins.
• Figure 17 shows the key monomeric lipids 12-16 for use in assembling the polymerized PVs PV1-PV6.
• the lipids were combined in the ratios as shown in the accompanying table. These compounds were then sonicated, cooled and polymerized by irradiation with UV light (254nm) for 2 hours and then sterile filtered (0.2 ⁇ M).
• Figures 17- 30 show lipids for the attachment of targeting agents. These lipids may be used to prepare vesicles for the attachment of targeting or therapeutic agents or both. Some of these lipids may be incorporated into vesicles and then further derivatized in aqueous solution with chemically reactive entities to which targeting agents may be attached.
• R is defined as any lipid, fatty acid, or di- or tri-block copolymer.
• Figure 18A-18B shows the binding of vesicles containing chelator lipid 15 to ⁇ v ⁇ 3 integrin-coated 96-well plates.
• vesicles were labled with europium, and time- resolved fluorescence was measured as described in EXAMPLE 5.
• Figure 19 shows the concentration of RGD mimetic 10 required to inhibit 50% of RGD mimetic polymerized vesicles contructs from Table Z. These results were obtained using the integrin-binding assay described in Example 5.
• Figure 20A-20E showe the use of PVs in imaging tumors in vivo.
• Figure 20A shows a schematic of the imaged animal and tumor.
• Figures 20B and 20C are images at 3 hours and 24 hours respectively, after injection of PV1 (targeted PV).
• Figures 20D and 20E are images at 3 hours and 24 hours respectively, after injection of PV4 (control PV).
• Figure 21 shows results from the treatment of endothelial cells and tumor cells in vitro with peptidomimetic- vesicle conjugates containingl% by weight doxorubicin (PM-V- l%Dox) vesicles containing 1% by weight doxorubicin (V-1% Dox), and free doxorubicin at concentrations identical to that used in the vesicles (1% Dox).
• the cells were treated as described in EXAMPLE 36.
• Figure 22 shows the inhibition of the binding of HRP-labeled fibronectin to the ⁇ v ⁇ 3 integrin by RGD peptidomimetic (PM) vesicles containing N-succinyl-DPPE (SDPPE), DMPC, DPPC, cholesterol (CH), BisT-PC, RGD peptidomimetic lipid (PML) 1, and paclitaxel (PTX).
• PM RGD peptidomimetic
• Figure 23 shows the efficacy of integrin-targeted vesicles labeled with yttrium 90 (IA- ⁇ P-Y90) in the mouse melanoma model as described in Example 29.
• Treatment groups include IA (the RGD peptidomimetic 10), IA-NP (RGD- peptidomimetic-polymerized vesicle conjugates), NP-Y90 (polymerized vesicles labeled with yttrium-90), and IA-NP- Y90 (RGD-peptidomimetic-polymerized vesicle conjugates labeled with yttrium-90).
• Figure 24 shows the normalized tumor volume 7 days post treatment sorted by treatment group for the study described in Example 29.
• Figure 25 shows the tumor growth delay data in mouse melanoma study described in example 29 as measured by tumor volume quadrupling time (TVQT).
• Figure 26 Treatment of solid tumors in a mouse melanoma model with integrin targeted dextran-coated polymerized vesicle conjugates labeled with yttrium-90 as described in Example 30.
• Figure 27 shows efficacy in the mouse colon cancer model as described in Example 31. Error bars indicate ⁇ one standard error.
• Treatment groups include buffer, PM (RGD peptidomimetic 10), PM-PV (RGD peptidomimetic-vesicle conjugates), PV-Y90 (polymerized vesicles labeled with yttrium-90), and PM-PV- Y90 (RGD peptidomimetic- vesicle conjugates labeled with yttrium-90).
• Figure 28 Plot of normalized tumor volume on day 8 sorted by group for the study in Example 31.
• Figure 29 shows the inhibition of the papain-catalyzed hydrolysis of substrate Ala- Phe-Lys-7-aminomethylcoumarm (Biochim. Biophys Acta 1190, 430, (1994)) by N-Acetyl- Leu-Val-Lys-aldehyde (LVK-CHO, J. Med. Chem 36, 1084, (1993)) and N-Acetyl-Leu-Val- Lys-aldehyde-vesicle conjugates (Vesicle-LVK-CHO) described in Example 44.
• Figure 30 shows the inhibition of the papain-catalyzed hydrolysis of substrate Z-Phe-
• Figure 31 shows the inhibition of the papain-catalyzed hydrolysis of substrate Ala- Phe-Lys-7-aminomethylcoumarin by vesicles by Gly-Phe-Gly-semicarbazone (GFGsc, J.
• Figure 32 shows the inhibition of the papain-catalyzed hydrolysis of substrate Z-Phe- Arg-7-aminomethylcoumarin by vesicles by Gly-Phe-Gly-semicarbazone (GFGsc) and Gly- Phe-Gly-semicarbazone-vesicle conjugates (Vesicle-dex-GFGsc and Vesicle-GFGsc) described in Example 44.
• Figure 33 shows the inhibition of the cathepsin-catalyzed hydrolysis of substrate Z- Arg-Arg-7-aminomethylcoumarin (Z-RRamc, Meth. Enzymol. 80, 535 (1981)) by inhibitor Leu-Val-Lys-aldehyde (LVK-CHO) and a vesicle conjugate (Vesicle-LVK-CHO).
• Figure 34 shows compound 18: Arginine-lipid
• Figure 35A and 35B shows the structures of AG1433 and SU1498, respectively.
• FIG. 36 Synthetic scheme for the preparation of compounds in Examples 39-43.
• FIG. 37 Synthetic scheme for the preparation of compounds in Examples 46-49.
• FIG 38 Shows the normalized tumor volumes after the treatment of subcutaneous tumors in a syngeneic murine tumor model with sucrose ( ⁇ ), Ldox (D, liposomal doxorubicin, 10 ⁇ g/g doxorubicin), ITL (O, integrin-targeted liposomes), ITLdoxl (•, integrin-targeted liposomes containing doxorubicin, 1 ⁇ g/g doxorubicin), and ITLdoxlO ( ⁇ , integrin-targeted liposomes containing doxorubicin, 10 ⁇ g/g doxorubicin) as described in EXAMPLE 36.
• Figure 39 Structure of a typical phosphatidylcholine lipid. 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 1A-1I which are comprised of a linking carrier, 50, a stabilizing agent, 52, a targeting entity 54, and/or a therapeutic or treatment entity, 56.
• the targeting and/or therapeutic entities may be associated with the lipid construct or the stabilizing entity.
• Figures 1A, IB, 1C, 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.
• the therapeutic entity may be encapsulated within the lipid construct, or may be associated with the surface of the lipid construct or stabilizing agent.
• 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.
• the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.
• 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.
• 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.
• linking carrier refers to any entity which A) serves to link the therapeutic entity and the targeting entity, and B) confers additional advantageous properties to the vascular-targeted therapeutic agents other than merely keeping the therapeutic entity and the targeting entity in close proximity.
• additional advantages include, but are not limited to: 1) multivalency, which is defined as the ability to attach either i) multiple therapeutic entities to the vascular-targeted therapeutic agents (i.e., several units of the same therapeutic entity, or one or more units of different therapeutic entities), which increases the effective "payload" of the therapeutic entity delivered to the targeted site; ii) multiple targeting entities to the vascular-targeted therapeutic agents (i.e., one or more units of different therapeutic entities, or, preferably, several units of the same targeting entity); or iii) both items i) and ii) of this sentence; and 2) improved circulation lifetimes, which can include tuning the size of the particle to achieve a specific rate of clearance by the reticuloendothelial system.
• the effective payload of therapeutic entity is the number of therapeutic entities delivered to the target site per binding event of the agent to the target.
• the payload will depend on the particular therapeutic entity and target. In some cases the payload will be as little as about 1 molecule delivered per binding event of the agent. In the case of a metal ion, the payload can be about one to 10 3 molecules delivered per binding event. It is contemplated that the payload can be as high as 10 4 molecules delivered per binding event.
• the payload can vary between about 1 to about 10 4 molecules per binding event.
• linking carriers are biocompatible polymers (such as dextran) or macromolecular assemblies of biocompatible components, such as lipid constructs, dendrimers, block copolymers, and the like. Components which may be used in the preparation of macromolecular assemblies are described herein. Examples of linking carriers include, but are not limited to, liposomes, micelles, di- and tri-block copolymers, polymerized liposomes, other lipid vesicles, dendrimers, polyethylene glycol assemblies, capped polylysines, poly(hydroxybutyric acid), dextrans, and coated polymers.
• a preferred linking carrier is a polymerized liposome. Polymerized liposomes are described in U.S. Patent Nos.
• a "lipid construct,” as used herein, is a structure containing lipids, phospholipids, or derivatives thereof comprising a variety of different structural arrangements which lipids are known to adopt in aqueous suspension. These structures include, but are not limited to, lipid bilayer vesicles, micelles, liposomes, emulsions, lipid ribbons or sheets, and may be complexed with a variety of drugs and components which are known to be pharmaceutically acceptable.
• the lipid construct is a liposome or polymerized vesicle. Liposomes
• 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.
• 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 39) 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.
• 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.
• 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.
• the composition of the liposomes may be altered to modulate the biodistribution and clearance properties of the resulting liposomes.
• 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.
• 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 barrier 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 ⁇ m (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.
• 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.
• the formulations preferably utilize UVs having a diameter of less than 200 nm, preferably less than 100 nm.
• Polymerized liposomes also referred to herein as “polymerized vesicles” and
• nanoparticles are self-assembled aggregates of lipid molecules which offer great versatility in particle size and surface chemistry.
• Polymerized liposomes are described in U.S. Patent Nos. 5,512,294 and 6,132,764, incorporated by reference herein in their entirety.
• the hydrophobic tail groups of polymerizable lipids are derivatized with polymerizable groups, such as diacetylene groups, which irreversibly cross-link, or polymerize, when exposed to ultraviolet light or other radical, anionic or cationic, initiating species, while maintaining the distribution of functional groups at the surface of the liposome.
• the resulting polymerized liposome particle is stabilized against fusion with cell membranes or other liposomes and stabilized towards enzymatic degradation.
• polymerized liposomes can be controlled by extrusion or other methods known to those skilled in the art.
• Polymerized liposomes may be comprised of polymerizable lipids, but may also comprise saturated and non-alkyne, unsaturated lipids.
• the polymerized liposomes can be a mixture of lipids which provide different functional groups on the hydrophilic exposed surface.
• some hydrophilic head groups can have functional surface groups, for example, biotin, amines, cyano, carboxylic acids, isothiocyanates, thiols, disulfides, ⁇ -halocarbonyl compounds, ⁇ , ⁇ -unsaturated carbonyl compounds and alkyl hydrazines.
• These groups can be used for attachment of targeting entities, such as antibodies, ligands, proteins, peptides, carbohydrates, vitamins, nucleic acids or combinations thereof for specific targeting and attachment to desired cell surface molecules, and for attachment of therapeutic entities, such as drugs, nucleic acids encoding genes with therapeutic effect or radioactive isotopes.
• targeting entities such as antibodies, ligands, proteins, peptides, carbohydrates, vitamins, nucleic acids or combinations thereof for specific targeting and attachment to desired cell surface molecules
• therapeutic entities such as drugs, nucleic acids encoding genes with therapeutic effect or radioactive isotopes.
• Other head groups may have an attached or encapsulated therapeutic entity, such as, for example, antibodies, hormones and drugs for interaction with a biological site at or near the specific biological molecule to which the polymerized liposome particle attaches.
• hydrophilic head groups can have a functional surface group of diethylenetriamine pentaacetic acid, ethylenedinitrile tetraacetic acid, tetraazocyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA), porphoryin chelate and cyclohexane-l,2,-diamino-N, N -diacetate, as well as derivatives of these compounds, for attachment to a metal, which provides for the chelation of radioactive isotopes or other materials that serve as the therapeutic entity.
• Examples of lipids with chelating head groups are provided in U.S. Patent No. 5,512,294, incorporated by reference herein in its entirety.
• the polymerized liposome particle can also contain groups to control nonspecific adhesion and reticuloendothelial system uptake.
• groups to control nonspecific adhesion and reticuloendothelial system uptake For example, PEGylation of liposomes has been shown to prolong circulation lifetimes; see International Patent Application WO 90/04384.
• the component lipids of polymerized liposomes can be purified and characterized individually using standard, known techniques and then combined in controlled fashion to produce the final particle.
• the polymerized liposomes can be constructed to mimic native cell membranes or present functionality, such as ethylene glycol derivatives, that can reduce their potential immunogenicity. Additionally, the polymerized liposomes have a well- defined bilayer structure that can be characterized by known physical techniques such as transmission electron microscopy and atomic force microscopy.
• Dendrimers are polymers with well-defined branching from a central core (e.g., "starburst polymers"). In contrast to conventional polymers, dendrimers tend to be highly branched, monodisperse macromolecules, i.e., the molecular weight tends to be very well-defined instead of a range as with conventional linear or branched polymers. Dendrimers are described in U.S. Patent Nos. 4,507,466, 4,558,120, 4,568,737, 4,587,329, 4,631,337, 4,694,064, 4,737,550, and 4,857,599, as well as numerous other patents and patent publications.
• Dendrimer structure, synthesis, and characteristics are reviewed in Kim and Zimmerman, "Applications of dendrimers in bio-organic chemistry,” Current Opinion In Chemical Biology (1998) 2(6):733-42; Tarn and Spetzler, "Chemoselective approaches to the preparation of peptide dendrimers and branched artificial proteins using unprotected peptides as building blocks," Biomedical Peptides, Proteins & Nucleic Acids (1995) l(3):123-32; Frechet, “Functional polymers and dendrimers: reactivity, molecular architecture, and interfacial energy," Science (1994) 263(5154):1710-5; Liu and Frechet, "Designing dendrimers for drug delivery," Pharmaceutical Science and Technology Today (1999) 2(10):393401; Verprek and Jezek “Peptide and glycopeptide dendrimers. Part I,” Journal of Peptide Science (1999) 5(l):5-23; Veprek and Jezek
• dendrimers typically uses reiterative synthetic cycles, allowing control over the dendrimer's size, shape, surface chemistry, flexibility, and interior topology.
• An example of a dendrimer suitable for use as a linking entity is described in Wu et al., "Metal-Chelate-Dendrimer-Antibody Constructs for Use in Radioimmunotherapy and Imaging," Bioorganic and Medicinal Chemistry Letters (1994) 4(3):449-454.
• Dendrimers can be readily used as linking carriers by employing a variety of chemical conjugation techniques to attach the targeting entity and therapeutic entity.
• U.S. Patent No. 6,020,457 which discloses a dendrimer having a disulfide (-S-S-) bond in its core
• the dendrimer can be constructed by the methods described in the patent.
• the final external layer of the dendrimer can be capped with a reactive group such as an amine or carboxyl group. These reactive groups can then be derivatized with either targeting entities or therapeutic entities (or, in some cases, a mixture of both).
• the core disulfide bond can then be reduced to a thiol, and the complementary entity attached via the thiol functionality.
• a targeting entity can be attached via the free -SH group.
• a targeting entity is an N-terminal-iodoacetylated peptide (the peptide may be a hormone or bioactive fragment of a larger protein), which is readily synthesized by standard solid-phase peptide techniques.
• the iodoacetyl group will react with the free thiol functionality, resulting in the conjugation of the therapeutic-entity-derivatized linking carrier with the targeting entity (the peptide).
• a block copolymer as used herein, is combination of two or more chains of constitutionally or configurationally different features.
• a block copolymer can be used as a linking carrier by employing a variety of chemical conjugation techniques to attach the targeting entity and therapeutic entity.
• Block copolymers include diblock, triblock, or multiblock copolymers.
• amphiphilic block copolymer micelles has recently been attracting much interest as a potentially effective drug carrier which is capable of solubilizing a hydrophobic drug in an aqueous environment.
• amphiphilic block copolymer micelles having surfactant-like properties there have been reported many studies on amphiphilic block copolymer micelles having surfactant-like properties, and particularly noteworthy are the attempts to incorporate hydrophobic drugs into block copolymer micelles stabilized due to the specific nature and properties of the copolymer.
• 0 397 307 A2 discloses polymeric micelles of an AB type amphiphilic diblock copolymer which contains poly(ethylene oxide) as the hydrophilic component and poly(amino acids) as the hydrophobic component, wherein therapeutically active agents are chemically bonded to the hydrophobic component of the polymer.
• EP No. 0583 955 A2 discloses a method for physically incorporating hydrophobic drugs into amphiphilic diblock copolymer micelles described in EP No. 0 397 307 A2.
• EP No. 0 552802 A2 discloses formation of chemically fixed micelles having poly(ethylene oxide) as the hydrophilic component and poly(lactic acid) as the hydrophobic component which can be crosslinked in an aqueous phase.
• 4,745,160 discloses a pharmaceutically or veterinary acceptable amphiphilic, non- cross linked linear, branched or graft block copolymer having polyethylene glycol as the hydrophilic component and poly(D-, L- and DL-lactic acids) as the hydrophobic components.
• U.S. Pat. No. 5,543,158 discloses nanoparticle or microparticle formed of a block copolymer consisting essentially of poly(alkylene glycol) and a biodegradable polymer, poly(lactic acid).
• the biodegradable moieties of the copolymer are in the core of the nanoparticle or microparticle and the poly(alkylene glycol) moieties are on the surface of the nanoparticle or microparticle in an amount effective to decrease uptake of the nanoparticle or microparticle by the reticuloendothelial system.
• U.S. Pat. No. 6,007,845 describes a multiblock copolymer-based composition prepared by covalently linking a multifunctional compound with one or more hydrophobic polymers and one or more hydrophilic polymers, and containing a biologically active material.
• 5,543,158 provides for block copolymer bas-ed particles that are not rapidly cleared from the blood stream by the macrophages of the reticuloendothelial system, and that can be modified as necessary to achieve variable release rates or to target specific cells or organs as desired.
• the particles have a biodegradable solid core containing a biologically active material and poly(alkylene glycol) moieties on the surface.
• the terminal hydroxyl group of the poly(alkylene glycol) can be used to covalently attach onto the surface antibodies targeted to specific cells or organs, or molecules affecting the charge, lipophilicity or hydrophilicity of the particle.
• biocompatible polymers suitable for use as linking carrier block copolymers in the present inveintion are poly(ethylene-covinyl acetate), and silicone rubber cross linked to poly (dimethyl siloxan sulfoxide) and derivatives thereof, polylactic acid, polyglycolic acid or polycaprolactone and their associated copolymers, e.g. poly (lactide-co- glycolide) at all lactide to glycolide ratios, and both L-lactide or D,L lactide.
• Additional hydrophilic polymers include polypyrrolidone, poly(amino acids), including short non-toxic and non-immunogenic proteins and peptides such as human albumin, fibrin, gelatin and fragments thereof, dextrans, and poly(vinyl alcohol).
• Other materials include a PluronicTM F68 (BASF Corporation), a copolymer of polyoxyethylene and polyoxypropylene, which is approved by the U.S. Food and Drug Administration (FDA).
• hydrophobic polymers can be polyanhydrides, polydioxanones, polyphosphazenes, polymers of ⁇ -hydroxy carboxylic acids, polyhydroxybutyric acid, polyorthoesters, polycaprolactone, polyphosphates, or copolymers prepared from the monomers of these polymers can be used to form the multiblock copolymers described herein.
• the variety of materials that can be used to prepare the block copolymers forming the particles significantly increases the diversity of release rate and profile of release that can be accomplished in vivo.
• a polyester of poly(lactic-co-glycolic)acid (PLGA) is used as a hydrophobic erodible polymer bound to the multifunctional compound.
• the block copolmers of the present invention are preferably composed of a polymeric-backbone having an interactive region for physically cross-linking with other entities, including targeting entities, therapeutic eritities, or other polymers.
• the backbone of the polymer comprises a plurality of interactive regions.
• the functional groups encompass conjugatable groups such as for example amines, hydroxyls, carbonyls, thiols, and carboxylic acids for covalently bonding of other bioactive molecules to the surface of the particle, as described in mre detail below.
• the linkages formed following conjugation of the bioactive molecules to the conjugatable groups include amides, esters, and thioethers. Examples of copolymers which have conjugatable functional groups include (poly) lysine, acetylated poly (lysine); poly (glutamic acid, and poly(oxyethylene)-poly (oxyproplene) copolymers.
• 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.
• 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 doxorubicin, 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- ⁇ 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.
• the therapeutic entity is an intracellular kinase inhibitor such as AG1433 or SU1498 ( Figure 35A and 35B, respectively) and the target is Flk-1/KDR.
• therapeutic entities such as AG1433 or SU1498 could also be classified as targeting entities; likewise, some targeting entities may also act as therapeutic entities.
• the therapeutic entity is encapsulated within a liposome or polymerized vesicle or associated by covalent or non- covalent means with the linking carrier or macromolecular assembly.
• these agents are encapsulated in amounts such that the dose of targeted therapeutic agents is effective to treat the disease.
• the therapeutic entity is associated with the surface of a liposome or polymerized vesicle.
• Stabilizing entities The agents of the present invention preferably contain a stabilizing entity.
• 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.
• 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.
• natural polymers include naturally occurring polysaccharides, such as, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, 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 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,
• suitable polymers include proteins, such as albumin, polyalginates, and polylactide-glycolide copolymers, cellulose, cellulose (microcrystalline), mefhylcellulose, 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, poly
• the stabilizing entity is dextran.
• the stabilizing entity is a modified dextran, such as amino dextran.
• the stabilizing entity is poly(ethylene imine) (PEI).
• PEI poly(ethylene imine)
• dextran may increase circulation times of liposomes in a manner similar to PEG.
• 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.
• polymers and their derivatives are used.
• copolymers including a monomer having at least one reactive site, and preferably multiple reactive sites, for the attachment of the cop
• the polymer may act as a hetero- or homobifunctional linking agent for the attachment of targeting agents, therapeutic entities, proteins or chelators such as DTPA and its derivatives.
• the stabilizing entity is associated with the vesicle by covalent means. In another embodiment, the stabilizing entity is associated with the vesicle by non- covalent 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.
• the stabilizing agent forms a coating on the liposome.
• targeting entity refers to a molecule, macromolecule, or molecular assembly which binds specifically to a biological target.
• 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.
• 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.
• 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.
• 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.
• the targeting entity may modulate the function of an enzyme target.
• 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.
• 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 polymerized liposomes, or linking carriers 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.
• small molecule ligands such as carbohydrates, and compounds such as those disclosed in U.S. Patent No. 5,792,783
• proteins such as antibodies and growth factors
• targeting entities can be used to control the biodistribution, non-specific adhesion, and blood pool half-life of the lipid constructs.
• ⁇ -D-Iactose 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.
• 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.
• the attachment is by covalent means. In another embodiment, the attachment is by non-covalent means.
• 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 polymerized liposome.
• 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, ⁇ v ⁇ 3 , ⁇ v ⁇ 5, a-s ⁇ i, a$ ⁇ , ⁇ -i ⁇ i, ⁇ 2 ⁇ 2 , or prostate specific membrane antigen (PSMA).
• MMPs matrix metalloproteases
• Targets include the CD family of cell surface antigens including CD1 through CD178, 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.
• 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.
• Conivaptan Yamanou
• the targeting entity is an integrin-specific molecule.
• the integrin specific molecule may be an RDG peptide or derivative thereor.
• 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.
• 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 ⁇ v ⁇ 3 .
• the integrin- specific molecule is Cilengitide.
• the targeting entity is a protease inhibitor such as N-acetyl-Leu-Val-Lys-aldehyde (Bachem ⁇ -1380) or Gly-Phe-Gly-aldehyde semicarbazone (Bachem C-3085) and the target is papain or cathespin B.
• a protease inhibitor such as N-acetyl-Leu-Val-Lys-aldehyde (Bachem ⁇ -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.
• 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.
• a vascular-targeted therapeutic agent is combined with an agent targeted directly towards tumor cells.
• This embodiment takes advantage of the fact that the neovasculature surrounding tumors is often highly permeable or "leaky,” allowing direct passage of materials from the bloodstream into the interstitial space surrounding the tumor.
• 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.
• 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.
• 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.
• 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.
• 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, multivalency).
• the particles can be sized at 30, 50, 100, 150, 200, 250, 300 or 350 nm in size, as desired.
• 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.
• 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.
• 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.
• 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,
• 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 neovasulature 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.
• the linking carrier 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 linking carrier 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 linking carrier and another member of the pair is on the therapeutic or targeting entity, e.g. a biotin- avidin interaction.
• FIG. 2 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.
• the chemical functionalities will be activated prior to forming the linkage between the targeting entity and the lipid, linking carrier, and/or optionally, the spacer group.
• chemical functionalities including hydroxy, amino, and carboxy groups
• a hydroxyl group of the ligand or lipid can be activated through treatment with phosgene to form the corresponding chloroformate.
• 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 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.
• the acyl halide is prepared through the reaction of the carboxy containing group with oxalyl chloride.
• the lipid or linking carrier is linked covalently to a targeting entity using standard chemical techniques through their respective chemical functionalities.
• 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.
• the lipid-targeting agent complex is prepared by linking a lipid to a targeting entity
• the lipid e.g., chemical functionality 1
• the targeting entity optionally via a spacer group, (e.g., chemical functionality
• 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.
• the targeting entity is attached to a carboxyl head group on the lipid.
• the targeting entity is attached to a maleimide or the alpha-methyl group of an acetamide.
• Exemplary lipids with a variety of functionalites 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 desrcribed in Hale, et al., U.S. Patent No. 5,607,691.
• 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.
• 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.
• 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-hexanedioI, beta-alanine, 2- aminoethanol, cysteamine (2-aminoethanethiol), 5-aminopentanoic acid, 6-aminohexanoic acid, 3-maleimidobenzoic acid, phthalide, alpha-substituted phthalides, the carbonyl group, 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 linking carrier-targeting entity complex. Generally, the additional mass 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, linking carrier-targeting entity complexes with a range of serum half -lives can be produced.
• linkage used to couple the spacer group to the chemical modifier or pharmaceutical agent may affect the serum half -life.
• linking carrier or the linking carrier-targeting entity complex.
• Other entities which can be covalently bound to the linking carrier-targeting entity complex will serve to affect or modify a chemical, physical, or biological property of the 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.
• 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.
• ком ⁇ онент is capable of extending the excretion half-life of a pharmaceutical agent. Typically, these entities will find use with peptide and protein drugs or other pharmaceutical agents with short excretion half -lives.
• this modifier will comprise a moiety capable of binding to a serum protein, such as human serum albumin. Typically those moieties will be bound to plasma more than 60%, preferably more than 70%, more preferably more than 80%, and most preferably more than 90%, as measured by the procedures known in the art.
• effector groups include naproxen, fluoxetine, oxazepam, nitrazepam, phenylbutazone, nortriptyline, methadone hydrochloride, lorazepam, imipramine, haloperidol, flurazepam, doxycycline, ditonin, diflunisal, diazoxide, diazepam, nordazepam, desipramine, dapsone, clofibrate, amantadine, chlorthalidone, clonazepam, chlorpropamide, chlorpromazine, chlorpheiramine, chloroquine, carbamazepine, auranofin, amitriptyline, amphotericin B, piroxicam, warfarin, pimozide, doxorubicin, pyrimethamine, amidoarone, protriptylene, desipramine, nortriptyline, oxazepam, nitraze
• a receptor crosslinking functionality modifier is essentially a targeting modifier.
• Crosslinking of cell surface receptors is a useful ability for a pharmaceutical agent in that crosslinking is often a required step before receptor internalization.
• the crosslinking modifier can be used as a means to incorporate a pharmaceutical agent into a cell.
• the presence of two receptor binding sites i.e., targeting modifiers gives the pharmaceutical agent increased avidity.
• 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.
• a functionality modifier may serve to prevent aggregation.
• 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.
• the hexameric form of insulin can be inhibited through the use of an appropriate functionality modifier and thus, result in greater diffusability of the mo ⁇ omeric form of insulin.
• therapeutic entities may be attached to one linking carrier that may also bear from several to about one thousand 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.
• multi-valent sialyl Lewis X derivatized liposomes can be used to block neutrophil binding, and antibodies against VCAM-1 on polymerized liposomes can be used to block lymphocyte binding, e.g. T-cells.
• lipids suitable for use in polymerized liposomes 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 a polymerizable group to stabilize the liposomes.
• Targeted polymerized 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 polymerized liposomes at the desired location.
• the high concentration of therapeutic entities in the polymerized liposomes render possible site-specific delivery of high concentrations of drugs or other therapeutic entities, while minimizing the burden on other tissues.
• the polymerized 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 polymerized 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.
• the polymerized liposomes were found to have blood pool half-lives of about 20 hours in rats.
• the site-specific polymerized liposomes having attached monoclonal antibodies for specific receptor targeting may be used to deliver therapeutic entities to cells expressing intercellular adhesion molecule-1, ICAM-1.
• ICAM-1 intercellular adhesion molecule-1
• compositions of the present invention can also include other components such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier.
• compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate.
• 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.
• compositions 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.
• 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.
• the excipient in a non-liquid formulation, can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
• 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 (Vaxcel.TM., Inc. Norcross, 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.
• a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• CSF cerebral spinal fluid
• 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-Iifes 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.
• a sufficient amount of the radiation synovectomy composition to provide adequate radiation synovectomy is injected into the knee-joint.
• 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.
• 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.
• 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.
• CNV choroidal neovascularization
• CNV 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 CNV 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.
• 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).
• 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.
• targets may be present on either side of the vasculature.
• the agents of the present invention can be in many forms, including intravenous, ophthalmic, and topical.
• 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.
• 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.
• the therapeutic agents of the present invention are useful for gene therapy or gene delivery.
• the phrases "gene therapy” or “gene delivery” refer to the transfer of genetic material (e.g., DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition.
• the genetic material of interest encodes a product (e.g., a protein polypeptide, peptide or functional RNA) whose production in vivo is desired.
• the genetic material of interest can encode a hormone, receptor, enzyme or polypeptide of therapeutic value.
• the subject invention utilizes a class of lipid molecules for use in non-viral gene therapy which can complex with nucleic acids as described in Hughes, et al., U.S. Patent No. 6,169,078, incorporated by reference herein in its entirety, in which a disulfide linker is provided between a polar head group and a lipophilic tail group of a lipid.
• These therapeutic compounds of the present invention effectively complex with DNA and facilitate the transfer of DNA through a cell membrane into the intracellular space of a cell to be transformed with heterologous DNA. Furthermore, these lipid molecules facilitate the release of heterologous DNA in the cell cytoplasm thereby increasing gene transfection during gene therapy in a human or animal.
• Cationic lipid-polyanionic macromolecule aggregates may be formed by a variety of methods known in the art. Representative methods are disclosed by Feigner et al., supra; Eppstein et al. supra; Behr et al. supra; Bangham, A. et al. M. Mol. Biol. 23:238, 1965;
• aggregates may be formed by preparing lipid particles consisting of either (1) a cationic lipid or (2) a cationic lipid mixed with a colipid, followed by adding a polyanionic macromolecule to the lipid particles at about room temperature (about 18 to 26 °C). In general, conditions are chosen that are not conducive to deprotection of protected groups. In one embodiment, the mixture is then allowed to form an aggregate over a period of about 10 minutes to about 20 hours, with about 15 to 60 minutes most conveniently used. Other time periods may be appropriate for specific lipid types. The complexes may be formed over a longer period, but additional enhancement of transfection efficiency will not usually be gained by a longer period of complexing.
• the compounds and methods of the subject invention can be used to intracellularly deliver a desired molecule, such as, for example, a polynucleotide, to a target cell.
• a desired polynucleotide can be composed of DNA or RNA or analogs thereof.
• the desired polynucleotides delivered using the present invention can be composed of nucleotide sequences that provide different functions or activities, such as nucleotides that have a regulatory function, e.g., promoter sequences, or that encode a polypeptide.
• the desired polynucleotide can also provide nucleotide sequences that are antisense to other nucleotide sequences in the cell.
• the desired polynucleotide when transcribed in the cell can provide a polynucleotide that has a sequence that is antisense to other nucleotide sequences in the cell.
• the antisense sequences can hybridize to the sense strand sequences in the cell.
• Polynucleotides that provide antisense sequences can be readily prepared by the ordinarily skilled artisan.
• the desired polynucleotide delivered into the cell can also comprise a nucleotide sequence that is capable of forming a triplex complex with double- stranded DNA in the cell.
• the desired polynucleotide delivered into the cell can interfere with biological pathways of the cell, thereby resulting in cell death.
• 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 as gadolinium, ultrasound imaging agents, or nuclear imaging agents, such as Tc-99m, In-Ill, Ga-67, Rh-105, 1-123, Nd -147, Pm-151,
• magnetic resonance imaging contrast agents such as gadolinium, ultrasound imaging agents, or nuclear imaging agents, such as Tc-99m, In-Ill, Ga-67, Rh-105, 1-123, Nd -147, Pm-151,
• This invention also provides a method of diagnosing abnormal pathology in vivo comprising, introducing a plurality of targeting image enhancing polymerized particles targeted to a molecule involved in the abnormal pathology into a bodily fluid contacting the abnormal pathology, the targeting image enhancing polymerized particles attaching to a molecule involved in the abnormal pathology, and imaging in vivo the targeting image enhancing polymerized particles attached to molecules involved in the abnormal pathology.
• Integrin-targeted PVs consist of a phosphocholine (PC) lipid for biocompatibility, a lipid derivative of diethylenetriamine pentaacetic acid (DTP A) to impart colloidal stability and allow for in vitro binding assays, and a targeting lipid with a head group derived from the ⁇ v ⁇ 3 integrin binding ligand 3- ⁇ 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)-ethyloxy]- benzoylamino ⁇ -2(S)-benzene-sulfonyl-aminopropionic acid, compound 10 in Figure 16.
• PC phosphocholine
• DTP A diethylenetriamine pentaacetic acid
• Figure 16 outlines the preparation of novel trivalent lipid-integrin antagnoist 12, used in the preparation targeting agents of the present invention.
• FIG 17 outlines the formation of the nanoparticles (PVs) by self-assembly and polymerization of the appropriate lipids as previously described in Storrs, et al., ibid.
• the trivalent lipid-integrin antagnoist 12 was combined with commercially available diacetylene phospholipid 13 and the europium-chelator lipid complex 14 in a chloroform solution.
• Compound 14 was added at one per cent to all formulations in order to visualize the particles using Fluorescence spectroscopy. Orellana, et al., Biochim. Biophys. Acta (1996) 1284:29-34.
• the anionic chelator lipid 15 or the cationic lipid 16 were added either the anionic chelator lipid 15 or the cationic lipid 16 in order to vary the surface charge and provide a surface to chelate radionuclides. Storrs, et al., 1995b.
• the surface density of the integrin antagonist on the PVs was controlled by varying the concentration of compound 12.
• the combined lipid solutions were evaporated to dryness and dried under high vacuum to remove any residual solvent.
• the dried lipid film was hydrated to a knows lipid density (30 mM) using deionized water.
• the resulting suspension was then sonicated at temperatures above the gel -liquid crystal phase transition (T m ⁇ 64 S C) using a probe-tip sonicator while maintaining the pH between 7.0 and 7.5.
• the mean diameter of the PVs were between 40 nm and 50 nm as determined by dynamic light scattering (DLS) and the zeta potential was between -42 and -53 mV for PV1 through PV4 and +35 and +43 mV for PV5 and PV6 respectively (Brookhaven Instruments, Holtsville, NY).
• the PVs were stable for months without significant changes in the physical and biological properties when formulated for in vivo applications using 150mM sodium chloride, 50 mM histidine, and 5% dextrose solutions. Properties of exemplary PVs are shown in Table Z. Table Z. Composition and physical properties of the PVs
• PVs were also prepared containing ⁇ v ⁇ 3 integrin agonist-lipid compound 12 at 1-30 mole percent along with l,2-bis(10,12-tricosadiynoyl)-5 , n-glycero-3-phosphocholine (BisT- PC, 13) at 99-70 mole percent.
• Liposomes and PVs containing agonist-lipid compound 12 are referred to herein as "integrin targeted liposomes" or ITLs.
• ITLs integrated liposomes
• vesicles were labeled with europium and binding was monitored in 96 well plates coated with the ⁇ v ⁇ 3 integrin by time resolved fluorescence (TRF), as described in EXAMPLE 5. TRF signal was 6 fold higher for PVs than signal for non-targeting liposomes. Specific targeting was also demonstrated in a competition assay where signal from ITL-Eu complexes was reduced by the integrin ligand without the lipid side chain.
• polymerized vesicles were constructed using 0.1, 1 and 10 mol% of integrin antagonist lipid complex compound 12 and compounds 13-16 as outlined in Table Z.
• the materials that contained 10 mol% of compound 12 (PVl and PV5) had the highest avidity for the integrin v ⁇ 3 .
• the PVs (PVl - PV5) were mixed with various concentration of 10 and then added to a 96 well plate previously coated with v ⁇ 3 integrin.
• the PVs had approximately 200 times increased avidity to the integrins when compared to the monomeric ligand. This demonstrates that a robust interaction occurs between the PV surface and the surface of the cell. This interaction is independent of surface charge on the PVs and is directly related to a specific receptor ligand interaction. Thus an increase of approximately two orders in magnitude of avidity can be achieved by multivalent presentation of an integrin antagonist on the surface of the PVs compared to the free ligand.
• the amount of compound 12 in the PV formulations was decreased by 10 fold and 100 fold to 1 mol% and 0.1 mol% to give PV2 and PV3 respectively, the capacity to block cell adhesion decreased by approximately one and two orders of magnitude (Table 3).
• the cell adhesion assay was also performed with plates coated with collagen. Collagen binds to collagen receptors ( ⁇ 2 ⁇ integrins) but not ⁇ v ⁇ 3 integrins. In this case, it was observed that the PV-integrin agonist inhibited cell adhesion, whereas neither the PV alone nor the agonist alone inhibited cell adhesion. Since neither component alone showed inhibition, it is clear that the individual components don't bind to collagen receptors. Without being bound by theory, it is believed that the observed inhibition of cell adhesion by PV-integrin agonist is due to the PV preventing interaction of collagen and its receptor by steric hindrance, due to the large size of the PV. Thus, not only does the PV targeted to a specific receptor bind to the receptor on the cell surface, but it blocks access to adjacent receptors due to its steric bulk.
• TRF time resolved fluorescence
• Paramagnetic PVs are useful for imaging tumors in vivo, as described in EXAMPLE 7. These materials can, therefore, serve as spatial and temporal imaging agents that have high avidity for the integrins in vivo.
• this effect is also observed in vivo by showing that a significant uptake of the PVs containing the integrin antagonist on the surface occurs in a melanoma tumor model and persists at the tumor site even after 24 hours, as shown in Figure 20A-E.
• Quantitative encapsulation of doxorubicin at 0.15 and 1.5 mg/mL was achieved in
• doxorubicin by ITLs was demonstrated with murine endothelial cells (MECs) in an in vitro cell proliferation assay described in , but the murine tumor cells were resistant to treatment under identical assay conditions.
• MECs murine endothelial cells
• incubation with ITLdox resulted in 4-fold higher reduction in cell density than untargeted Ldox.
• ITLs without doxorubicin had no effect on cell proliferation.
• the EXAMPLES section also describes a number of other procedures, including encapsulation of other therapeutic entities, association of other targeting entities, entities with differing lipid compositions, association of therapeutic radioisotopes and the like.
• MALDI-TOF mass spectrometry was performed on PerSeptive DE instrument (Mass Spectrometry, The Scripps Resea ch 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).
• 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 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 L), 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 %).
• the solution was then neutralized with 200 ⁇ L 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.
• DTPA-(COOH) 3 (11, 69 mg, 50 ⁇ mole) was dissolved in anhydrous CH 3 CN (5mL), anhydrous CH 2 C1 2 (2 mL) and Et 3 N (1 mL) in a 3-neck round bottomed flask, previously flame dried and filled with argon. To this solution was added the BOP reagent (134 mg, 150 ⁇ mol) and the solution was stirred well for 5 minutes. A solution of 10 (69 mg, 150 ⁇ mol) was prepared in a dry vial filled with argon, in a mixture of anhydrous CH 3 CN (5 mL) and anhydrous DMF (2 mL).
• lipid components (12, 13, 14, and 15) dissolved in organic solvents (CHC1 3 and CH 3 OH in a ratio 1:1) were combined. The solvents were evaporated and the residue dried in vacuo for 24h while shielded from light. Distilled and deionized water was added to yield a heterogeneous solution 30 mM in lipid concentration. The lipid/water mixture was then sonicated with a probe-tip sonicator for at least one hour and the solution became clear. Throughout sonication, the pH of the solution was maintained between 7.0 and 7.5 with IN NaOH solution, and the temperature was maintained above the gel-liquid crystal phase transition point (T m ) with the heat generated from sonication.
• T m gel-liquid crystal phase transition point
• the liposome solution was transferred to a petri dish resting on a bed of wet ice, cooled to 0 °C, and irradiated at 254 nm for at least one hour with a hand-held UV lamp placed - 1 cm above the petri dish, yielding PVs.
• the PVs were then filtered through a 0.2 ⁇ m filter and collected. Composition and physical properties of the PVs are shown in Table Z:
• PVs were constructed as outlined in Table Z and labeled with europium. Integrin binding was determined by coating purified ⁇ v ⁇ 3 onto 96 well plates and then PVs were added with incubated at room temperature. The unbound PVs were removed by washing with buffers and the bound PVs were measured using time resolved fluorescence of the europium in the PVs (Wallac, Gaitherburg, MD 20877 USA). The materials that contained 10 mol% of compound 12, (PVl and PV5) had the highest avidity for the integrin ⁇ v ⁇ 3 .
• PVs (PV1-PV5) were mixed with various concentrations of 10 to inhibit 50% of binding of the PVs to ⁇ v ⁇ 3 .
• the reported values are average of quadruplicate values and have a maximum standard error ⁇ 3.
• a schematic of this assay is shown in Figure 18A-18B.
• a cell adhesion inhibition study was done on plates coated with vitronectin (Wu, et al., In Methods in Molecular Biology: Integrin Protocols; Howlett, Ed.; Humana Press: Totowa, NJ, 1999; vol. 129, pp 211-217), using a human melonoma cell line M21.
• the multivalent particle complex PV1-PV6 as well as the monomeric ligand 10 were separately incubated with M21 cells and applied onto the 48 well plates coated with vitronectin. After lh incubation, the wells were washed and the cells that adhered were stained with a solution of crystal violet and the OD at 590nm was measured.
• the OD measured was proportional to the number of cells bound to the vitronectin plate and was plotted against the concentration of 10 on the surface of the PVs in different formulations to calculate the IC 5 0.
• the reported values are average of quadruplicate values and have a maximum standard error of 0.05.
• the multivalency effect was calculated by dividing the IC 50 for free ligand 10 by the IC 50 of the concentration of 10 on the PVs.
• mice aged 10 to 12 weeks were anesthetized (Nembutal (58 mg/kg)), and their right flanks were shaved and an average of 2 x 10 5 tumor cells (mouse MK504 melanoma cells) in Hanks' solution (0.5 mL) were injected intradermally in the right flank region of each mouse with a 27 G needle. Mice were monitored for tumor growth. Approximately 2 weeks were required for tumors to grow to 1 cm in size. Two mice with tumors were divided into two groups. Both the PVs were labeled with radioactive indium ( n ⁇ In) as previously described, Storrs, et al., 1995a; Haubner, et al., Cancer Res.
• radioactive indium n ⁇ In
• BisT-PC 13 (500 mg, 546.9 ⁇ mole, 95 mole %) was weighed into a clean 100 ml round bottom flask.
• Chelator lipid 15 (3.15 ml, 31.5 mg, 23 ⁇ mole, 4 mole %), and RGD peptidomimetic lipid 12 (1.54 ml, 15.4 mg, 5.74 ⁇ mole, 1 mole %) were added to the flask by glass syringe. Chloroform was removed by rotary evaporation. The lipid film was hydrated with 20 ml of 250 mM ammonium sulfate and 190 ⁇ l 0.5 N NaOH while rotating the flask in the 65°C water bath.
• the lipid suspension was briefly sonicated in the 100 ml flask to reduce the size of the aggregates and then transferred to the extruder.
• the lipid suspension was extruded through a series of successively smaller pore size polycarbonate (PC) membranes.
• PC polycarbonate
• the 10 ml thermal barrel extruder maintained at 90 °C was fitted with 2 stacked membranes and the lipid suspension was extruded through 100 nm membranes, then 50 nm membranes, and finally 30 nm membranes using argon at 300-600 p.s.i.
• the vesicles were transferred to dialysis cassettes and dialyzed against 10 % sucrose (2 X 1800 ml, 4 h). The size determined by dynamic light scattering was approximately 60 nm.
• BisT-PC 13 (91.4 mg, 99.96 ⁇ moles, 95 mole %) chelator lipid 15 (5.8 mg, 4.24 ⁇ moles, 4 mole %), and RGD peptidomimetic lipid 12 (2.8 mg, 1.04 ⁇ moles, 1 mole %) were added to a 100 mL flask and dissolved in 10 ml of chloroform. Chloroform was removed by rotary evaporation for 60 minutes at 65°C, and 10 ml of 0.3 M sodium citrate at pH 4 was added to the evaporated lipid. The heterogeneous solution was frozen on acetone/dry ice and thawed in a 65 °C water bath.
• Non-targeting liposomes were prepared exactly as described in EXAMPLE 8, except no integrin-targeting lipid was used, and the mole percent of chelator lipid 15 was 5%.
• non-targeting vesicles containing 1 mole % of the tri-arginine lipid 18 were prepared exactly as described in EXAMPLE 8, except the RGD peptidomimetic lipid 12 was omitted.
• Integrin-targeted liposomes containing ammonium sulfate from EXAMPLE 8 (2 mL, 60 mg) were placed in a 12 x 100 mm glass culture tube and 600 ⁇ l (6 mg) doxorubicin in 10% sucrose was added. The mixture was incubated for 5 minutes at 65°C and size exclusion chromatography (SEC) showed that the loading of doxorubicin was quantitative. SEC analysis was performed with 10 mM HEPES buffer containing 200 mM NaCl pH 7.4 by adding a 100 ⁇ l sample from the doxorubicin loading mixture to a Sepharose CL 4B column (1.5 x 6 cm). The mixture was diluted with 10% sucrose to give a final vesicle concentration of 15 mg/ml. These vesicles contain 10% doxorubicin by weight. The size measured by dynamic light scattering was 60-65 nm.
• EXAMPLE 12 Preparation of integrin-targeted vesicles containing 1% doxorubicin Integrin-targeted liposomes containing ammonium sulfate from EXAMPLE 8 (2 mL,
• doxorubicin solution 60 mg were placed in a 12 x 100 mm glass culture tube and 60 ⁇ l (0.6 mg) doxorubicin solution added. The tube was immersed in a water bath maintained at 65°C for 5 minutes. The mixture was diluted with 10 % sucrose to give a final vesicle concentration of 15 mg/ml. SEC analysis was performed as described in EXAMPLE 11 and showed that all doxorubicin added was encapsulated in the liposome. These vesicles contain 1% doxorubicin by weight. The size measured by dynamic light scattering was 60-65 nm.
• the solution of the integrin-targeted vesicles containing citrate from EXAMPLE 9 was adjusted to pH 8 with 1 M HEPES buffer at pH 7.4 and sodium hydroxide.
• To this solution was added 200 ⁇ l of doxorubicin (10 mg/ml in 10 % sucrose) to 1 ml (10 mg) of vesicles at pH 8 and the solution was incubated for 7 min at 65°C in a water bath.
• SEC analysis was performed as described in EXAMPLE 12 and showed that all doxorubicin added was encapsulated in the liposome. The size measured by dynamic light scattering was 93 nm.
• EXAMPLE 14 Preparation of non-targeting liposomes containing 10% doxorubicin
• Vesicles (60 mg, 2 ml) from EXAMPLE 10 were placed in a 12 x 100 mm glass culture tube and 600 ⁇ l doxorubicin solution (10 mg/ml in 10 % sucrose) was added. The tube was immersed in a water bath maintained at 65°C for 5 minutes. The mixture was diluted with 10 % sucrose to give a final vesicle concentration of 15 mg/ml. SEC analysis was performed as described in EXAMPLE 12 and showed that all doxorubicin added was encapsulated in the liposome. These vesicles contain 10% doxorubicin by weight.
• EXAMPLE 15 Preparation of vesicles containing N-succinyl-DPPE and ammonium sulfate BisT-PC (1 g, 1093.7 ⁇ mole, 95 mole %) and N-succinyl-DPPE (47 mg, 57.6 ⁇ mole,
• the vesicles were transferred to dialysis cassettes and dialyzed against 10 % sucrose. The size determined by dynamic light scattering was approximately 68 nm. This procedure was also used without the addition of sodium hydroxide to prepare vesicles containing 10 mole percent of the N-succinyl-DPPE lipid, 50 mole percent of dimyristoyI-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC), or distearoyl-sn-glycero-3-phosphocholine (DSPC), and 40 mole percent cholesterol.
• DMPC dimyristoyI-sn-glycero-3-phosphocholine
• DPPC dipalmitoyl-sn-glycero- 3-phosphocholine
• DSPC distearoyl-sn-glycero-3-phosphocholine
• EXAMPLE 16 Preparation of vesicles containing N-succinyl-DPPE Vesicles identical to those in EXAMPLE 15 were prepared without ammonium sulfate.
• Vesicles prepared with 95 mole percent l,2-bis(10, 12-tricosadiynoyl)-5/ ⁇ -glycero- phosphocholine (BisT-PC 13, Avanti Polar Lipids) and 5 mole percent of the DTPA lipid derivative 13 were coated with aminodextran as follows: Vesicles (10 ml, 250 mg) were added dropwise to stirred aminodextran (amine modified 10,000 MW dextran, Molecular Probes, product D-1860, 500 mg, 3 amino groups per dextran polymer) in 5 ml of 50 mM HEPES buffer at pH 8.
• the peak fractions (2 thru 6) were pooled and filtered through a 0.45 ⁇ m filter (Nalgene 25 mm syringe filter, product 190-2545) followed by a 0.2 ⁇ m filter (Nalgene 25 mm syringe filter, product 190-2520).
• the concentration of coated vesicle was determined by drying a sample to constant weight in an oven maintained at 90°C.
• Aminodextran-coated vesicles from EXAMPLE 17 (15 ml, 465 mg) in 10 mM HEPES buffer at pH 7.4 were diluted with an equal volume of 200 mM HEPES buffer and the pH was adjusted to 8 with 1 N NaOH.
• Succinic anhydride (Aldrich product 23,969-0, 278 mg) was dissolved in 1 ml DMSO (dimethyl sulfoxide (Aldrich product 27685-5) and 100 ⁇ l aliquots were added to the coated-vesicle suspension with rapid stirring. The pH was monitored and adjusted as necessary to maintain the pH between 7.5 and 8 by the addition of I N NaOH. After the final addition of succinic anhydride, the mixture was stirred for 1 hour at room temperature and then transferred to dialysis cassettes and dialyzed against 10 mM HEPES buffer at pH 7.4.
• EXAMPLE 19 Coupling of an RGD peptidomimetic to succinylated, dextran-vesicle conjugates
• the succinylated aminodextran-coated vesicles from EXAMPLE 18 200 mg in 6.9 ml water
• ⁇ v ⁇ 3 integrin-targeting agent 12 40 mg in 1 ml of water
• water 6.1 ml
• 1 M NaCl 3 ml
• 500 mM MES buffer pH 6 2 ml
• EDAC (19.2 mg, 1 ml) was added.
• the solution was mixed and incubated at room temperature for 18 h. Analysis of the reaction mixture by size exclusion chromatography showed that the coupling yield was approximately 30-50%.
• Integrin-targeted paclitaxel particles containing integrin-targeting lipid 12 are made as described in EXAMPLE 8, but without ammonium sulfate.
• the preparation of 100 mg of vesicles containing BisT-PC 13, chelator lipid 15, integrin- targeting lipid 12, and 4.5% w/w paclitaxel was achieved using 90.7 mg BisT-PC, 6.5 mg PDA-DTP A, 2.8 mg integrin-targeting lipid, and 4.5mg paclitaxel.
• HPLC analysis also showed that this process did not result in the degradation of paclitaxel, and the size was 63 nm.
• DMPC in chloroform (42.5 mg, 62.7 umole; Avanti), N-succinyl-DPPE in 1:1 chloroform/methanol (5 mg, 6.1 umol; Avanti), and paclitaxel in chloroform (2.5 mg, 2.9 ⁇ mole; Sigma) were placed in a round bottom flask. The total volume was 5 mL. The solvent was removed at 48°C by rotory evaporation. The vacuum-dried lipid was hydrated with 5 ml of 50 mM HEPES buffer pH 7.4 while mixing in a 48°C water bath.
• the mixture was extruded through a Lipex 10 ml thermal barrel extruder at 48°C using 50 nm polycarbonate track-etched filters (Osmonics) by applying 700 psi of pressure of argon. The process was repeated 5 times, followed by extrusion 5 times through 50 nm filters. The size measured by dynamic light scattering was 73 nm.
• RGD peptidomimetic 10 was attached to the vesicles containing taxol by activation of the carboxyl group of the ⁇ -succinyl-DPPE lipid in the vesicles with EDC in the presence of the peptidomimetic.
• the vesicles may be activated with EDC, followed by the addition of the peptidomimetic, or the vesicles may be activated with EDC, followed by removal of remaining EDC by size exclusion chromatography, followed by the addition of the peptidomimetic to the activated vesicles.
• EDC electrospray diluent
• Vesicles (15 mg, 1 mM carboxyl group), peptidomimetic 10 (2 mM) and EDC (5 mM) are incubated in a volume of 1.5 mL at room temperature in a 1.5 mL polypropylene tube.
• the conjugate was dialyzed against 50 mM HEPES buffer at pH 7.4 (10K MWCO dialysis cassette) to remove unreacted peptidomimetic.
• the attachments were monitored by SEC analysis, and the RGD peptidomimetic-vesicle conjugates containing paclitaxel inhibit the binding of biotinylated fibrinogen, as shown in Figure 22.
• EXAMPLE 22 Preparation of RGD peptidomimetic vesicles containing paclitaxel, DMPC, and cholesterol Vesicles identical to those in EXAMPLE 21 were prepared, except the components were DMPC (30.8 mg, 45.4 umole), N-succinyl-DPPE (5 mg, 6.1 umol; Avanti), cholesterol (11.7 mg, 30.3 umol), and paclitaxel (2.5 mg, 2.9 ⁇ mole; Sigma). The size measured by dynamic light scattering was 85.3 nm.
• Vesicles identical to those in EXAMPLE 21 were prepared, except the components were DPPC (42.5 mg, 57.9 umole), N-succinyl-DPPE (5 mg, 6.1 umol; Avanti), and paclitaxel (2.5 mg, 2.9 ⁇ mole; Sigma).
• the size measured by dynamic light scattering was 80.0 nm.
• Vesicles identical to those in EXAMPLE 21 were prepared, except the components were DPPC (31.4 mg, 42.8 umole), N-succinyl-DPPE (5 mg, 6.1 umol; Avanti), cholesterol (ll.lmg, 28.6 umol), and paclitaxel (2.5 mg, 2.9 ⁇ mole; Sigma).
• the size measured by dynamic light scattering was 91.1 nm.
• a dried lipid film containing BisT-PC (1 g, 1093.7 ⁇ mole, 95 mole %) andN- succinyl-DPPE (47 mg, 57.6 ⁇ mole, 5 mole %) was prepared by rotary evaporation of a chloroform solution.
• the dried film was hydrated by addition of 250 mM ammonium sulfate and warming in a 65 °C water bath for 30 minutes.
• the hydrated lipid suspension was then extruded through a series of successively smaller pore sized polycarbonate track etched filter membranes using a thermal barrel extruder maintained at 65°C. Extrusion was initiated with a 100 nm pore size filter and terminated with a 30 nm pore size filter.
• EXAMPLE 26 Preparation of RGD peptidomimetic-dextran-vesicle conjugates containing doxorubicin by process 2 Succinylated dextran-coated vesicles containing BisT-PC (1 g, 1093.7 ⁇ mole, 95 mole
• RGD mimetic 10 was coupled to these vesicles as described in EXAMPLE 19.
• the resulting RGD mimetic-dextran vesicle conjugates were suspended in 250 mM ammonium sulfate solution and heated to 65°C for 30 minutes. Excess ammonium sulfate was removed by dialysis with 10 % sucrose solution. Doxorubicin was loaded into the vesicles by mixing with a sucrose solution of doxorubicin and warming the mixture to 65°C for 5 minutes.
• doxorubicin in a typical preparation, doxorubicin at 10 mg/ml in 10% sucrose solution was added to 1 ml of vesicles containing ammonium sulfate. Uptake of the added doxorubicin was confirmed by SEC on a column of Sepharose CL 4B equilibrated and eluted with 10 mM HEPES, 200 mM ⁇ aCl pH 7.4.
• the ⁇ v ⁇ 3 integrin-binding RGD peptidomimetic 10 was attached to liposomes containing ammonium sulfate (EXAMPLE 15) using the method described in EXAMPLE 19.
• the peptidomimetic was attached in 50 mM HEPES buffer at pH 7 to ammonium sulfate loaded vesicles containing N-succinyl-DPPE, DMPC, and cholesterol in mole ratios of 10/50/40 by adding ED AC to a final concentration of 5 mM, followed by 2 equivalents of the peptidomimetic 10 to generate vesicles containing approximately 14 ⁇ g of the peptidomimetic per mg of lipid.
• EXAMPLE 28 Attachment of 90 Y to peptidomimetic-vesicle complexes
• the peptidomimetic-vesicle complexes containing chelator lipid 15 are labeled with
• Yttrium-90 chloride in 50 mM HC1 was diluted to a working solution containing approximately 20 mCi/mL.
• the percent 90 Y bound to the therapeutic vesicle was determined by adding 100 ⁇ L of the 90 Y-vesicle complex to a 100k MWCO NanosepTM (Pall Filtron) filter.
• the filter assembly was spun in a microfuge at 4000 rpm for 1 hr or until all of the solution has passed through the filter.
• the "total 90 Y" in the assembly was determined with the Capintec CRC-15R dosimeter.
• the filter portion of the assembly was removed and discarded. Using the dosimeter, the remaining part of the assembly containing the "unbound 90 Y" that passed through the filter was counted.
• "Bound 90 Y" was determined by subtracting the "unbound 90 Y" from the "total 90 Y”.
• Percent 90 Y bound was determined by dividing the "bound 90 Y" by the "total 90 Y” and multiplying by 100.
• EXAMPLE 29 Study of antitumor efficacy of 90 Y-peptidomimetic-vesicle complexes in a mouse melanoma model
• the K1735-M2 mouse melanoma model was prepared by subcutaneous injection of tumor cells as previously described (X. Li, et al. Invasion Metastasis 1998, 18, 1-14). Animals received a single i.v. injection of placebo or therapeutic agent and tumor volume was measured until the tumors had quadrupled in size. Tumors were induced in the mice as follows: tumors were implanted by subcutaneous injection of approximately 1x10° K1735 M2 melanoma cells (X. Li, B. Chen, S. D. Blystone, K. P. McHugh, F. P. Ross, D. M. Ramos, Differential expression of alpha v integrins in K1735 melanoma cells.
• Figure 23 shows the normalized tumor volume data obtained in this study.
• the seventh day post treatment is the last day that all animals in the study were alive.
• Figure 24 on the following page shows the normalized tumor volumes for each animal sorted by treatment group on the seventh day post treatment.
• Normalized tumor volume seven days post treatment was compared using analysis of variance (ANOVA) and Kruskal-Wallis statistical tests. These tests determine if the observed differences between treatment groups are due to chance alone.
• the ANOVA tests the equality of the treatment means.
• the ANOVA is most reliable when there are no significant outliers in the data.
• the Kruskal Wallis test 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.
• Tumor growth delay was also used to monitor efficacy in this study. Tumor growth delay is defined as the time required for a given tumor to show a fourfold increase in volume when compared with the tumor volume measured on the day of treatment (Tumor Volume Quadrupling Time or TVQT). The exact time for four-fold growth is extrapolated by drawing a line between the two nearest time points.
• Figure 23 summarizes the growth delay data for this study.
• Table III Summary of P-values obtained using Tukey's pairwise comparisons with tumor volume quadrupling time data.
• Buffer IA IA-NP (2.5uCi/g) (5uCi/g) (2.5uCi/g)
• IA-NP >0.05 >0.05
• IA-NP- Y90 (2.5uCi/g) >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05
• IA-NP-Y90 (5uCi/g) ⁇ 0.01 ⁇ 0.01 >0.05 ⁇ 0.01 ⁇ 0.01 >0.05
• TUNEL assay results indicate that Hist/Cit Buffer, IA, and NP-Y90 2.5 ⁇ Ci/g treatment result in mostly healthy cells, while, IA-NP, IA-NP-Y90 2.5 ⁇ Ci/g, and IA-NP-Y905 ⁇ Ci/g, show inceasing amounts of apotosis and cell death.
• IA-NP- Y90 Treatment with IA-NP- Y90 at 5 ⁇ Ci/g significantly reduces tumor growth in this tumor model (significance was established at the 95% confidence level).
• the average TVQT for tumors treated with IA-NP- Y90 at 5 ⁇ Ci/g is 15.0 days compared to 6.4 days for tumors treated with buffer. Histological study of tumor samples confirms this result.
• melanoma cells are known to be relatively resistant to radiotherapy. This type of targeted therapy relies only on the presence of neovascular cell surface markers on the endothelial cells that are terminally differentiated and genetically stable.
• EXAMPLE 30 Study of antitumor efficacy of peptidomimetic-dextran-vesicle 90 Y complexes in a mouse melanoma model
• Dextran coated vesicles were also tested in the mouse melanoma model as described in EXAMPLE 29. Results are shown in Figure 26. For these studies, dextran-coated vesicles containing BisT-PC and chelator lipid 15 were used, and they were prepared as described in Examples 17-19, and labled with yttrium-90 as described in Example 28.
• EXAMPLE 31 Study of antitumor efficacy of peptidomimetic- vesicle- 90 Y complexes in a mouse colon cancer model.
• a CT-26 colon cancer cell line implanted by subcutaneous injection in female BALB/c mice as previously reported (H.N., Moehler, T., Siang, R., Jonczyk, A., Gillies, S.D., Cheresh, D.A., Reisfeld, R.A., Proc. Natl. Acad. Sci. USA, 96: 1591-1596, 1999), was used to assess the anti-tumor activity of Targesome's radiopharmaceutical agent.
• the purpose of this study was to investigate the potential anti-tumor effects with a single intravenous administration of the IA-NP-Y90 complex.
• CT-26 tumor cells were implanted by subcutaneous injection of approximately 1x106 CT-26 cells.
• the CT-26 tumor cells were grown in tissue culture flasks in Dulbeco's medium with 10% fetal calf serum (FCS). Cells are harvested using Trypsin-EDTA solution (containing 0.05% trypsin), resuspended in PBS at 10,000,000/ml, and kept on ice.
• FCS fetal calf serum
• Figure 27 summarizes the normalized tumor volume data.
• Day eight is the last day that all animals in the study were still alive. Differences between treatment groups were compared using analysis of variance (ANOVA) and Kruskal-Wallis statistical tests. In the case of the normalized tumor volume on day 8, the P-value for both the ANOVA and the Kruskal-Wallis tests is below 0.0005. It is reasonable to conclude that there are significant differences between treatments in this study. None of the treatment groups contained large outliers that might skew the results of an ANOVA analysis. For this reason Tukey's W procedure was used to determine which treatments show significantly different normalized tumor volumes on the eighth day post treatment.
• Table III Summary of P-values obtained using Tukey's Pairwise Comparisons with Normalized Tumor Volume Measurements Eight Days Post treatment.
• Tumor growth delay was also used to monitor efficacy in this study. Tumor growth delay is defined as the time required for a given tumor to show a four-fold increase in volume when compared with tumor volume measured on the day of treatment. The exact time for fourfold growth is extrapolated by drawing a line between the two nearest time points. Figure 28 summarizes the growth delay data for this study.
• IA compared with IA-NP, NP-Y90 and IA-NP-Y90
• Integrin binding of RGD peptidomimetic-liposome conjugates containing the chelator lipid 15 was demonstrated in vitro using a radiometric binding assay. Briefly, 96- well plates coated with the ⁇ v ⁇ 3 integrin were blocked with BSA. Samples of rabbit serum or buffer containing 0-100 micrograms/mL of the agonist-liposome- 90 Y complex were added and incubated for one hour at room temperature. The plate was washed 3X with PBST buffer and the 90 Y was measured using a Wallac Microbeta scintillation counter. EXAMPLE 32. Preparation of lipid-based particles containing paclitaxel
• vesicles containing 1-10 weight percent paclitaxel Weigh out 93.4 mg of BisT-PC 13, 6.6 mg of chelator lipid 15, and 1 mg paclitaxel (Sigma). Place in a round bottom flask and add 5 ml chloroform. Swirl to dissolve lipids and paclitaxel. Attach the round bottom to a rotary evaporator equipped with a dry ice/acetone cold trap and lower the flask into a 48°C water bath. Pull a vacuum while the flask is rotating to remove the chloroform and continue the vacuum for one hour. Remove the flask from the rotary evaporator and add 10 ml of 50 mM HEPES, pH 7.4.
• EXAMPLE 34 Preparation of lipid-based, integrin-targeted particles containing tyrosine kinase inhibitors Integrin-targeted particles containing AG1433 and integrin-targeting lipid 12 are made as described in EXAMPLE 33. For example, the preparation of 100 mg of vesicles containing BisT-PC 13, chelator lipid 15, integrin-targeting lipid 12, and 12.7 weight percent AG1433 was achieved with 90.7 mg BisT-PC, 6.5 mg chelator lipid 4, 2.8 mg integrin- targeting lipid 12, and 12.7 mg AG1433.
• EXAMPLE 35 Treatment of a the K1735-M2 mouse melanoma model
• the K1735-M2 mouse melanoma model was prepared by subcutaneous injection of tumor cells as previously described (X. Li, et al. Invasion Metastasis 1998, 18, 1-14). Animals received a single i.v. injection of placebo or therapeutic agent and tumor volume was measured until the tumors had quadrupled in size. Tumors were induced in the mice as follows: tumors were implanted by subcutaneous injection of approximately 1x10 K1735 M2 melanoma cells (X. Li, B. Chen, S. D. Blystone, K. P. McHugh, F. P. Ross, D. M. Ramos, Differential expression of alpha v integrins in K1735 melanoma cells.
• EXAMPLE 36 In vitro cell toxicity measured in a cell proliferation assay Targeted drug delivery was assessed in vitro by incubating vesicles with MSI mouse endothelial pancreatic islet cells and the K1735-M2 murine melanoma tumor cells. The effect of free doxorubicin or liposome-encapsulated doxorubicin on the cells was assayed colorimetrically by crystal violet staining method with slight modification. Mouse endothelial cells (ATCC# CRL-2279) and mouse melanoma cells (M2) were seeded in 96- well flat-bottomed microtitre plates. The effect of the vesicles on cell proliferation was determined with cells near confluence (about 80%).
• the medium in each well was replaced with 100 ul of culture medium containing 250ug/ml of vesicles containing doxorubicin.
• the mouse endothelial and melanoma cells were incubated for 1 hour at 37°C and 5% CO 2 . After 1 hour incubation, the drug was removed, and compete medium that was lacking drug was added, and the cells were incubated for 48 hours at 37°C and 5% CO 2 . The experiment was performed in duplicate. At the end of the incubation period, the cells were washed once with PBS and the cultures were fixed by 70% ethanol overnight. Next, the cells were stained with 100 ul of 0.1% crystal violet in 10% ethanol for 10 minutes at room temperature, and the cells were gently washed with water for 5 times.
• EXAMPLE 37 Preparation of RGD peptidomimetic vesicles containing paclitaxel, DPPC, and cholesterol Vesicles identical to those in EXAMPLE 21 were prepared, except the components were
• DPPC (31.4 mg, 42.8 umole), N-succinyl-DPPE (5 mg, 6.1 umol; Avanti), cholesterol (ll.lmg, 28.6 umol), and paclitaxel (2.5 mg, 2.9 ⁇ mole; Sigma).
• the size measured by dynamic light scattering was 91.1 nm.
• EXAMPLE 38 Inhibition of fibronectin binding to the ⁇ v ⁇ 3 integrin in vitro
• EXAMPLE 40 N-(8'-amino-3',6'-dioxaoctyl)-10,12-tricosadiynamide (TA-PEG3 amine) ( Figure 36, Compound 41)
• 10,12-tricosadiynoic acid, ⁇ HS ester from EXAMPLE 39 (30 g) in dichloromethane was added from a dropping funnel, slowly, to a stirred solution of l,8-diamino-3,6-dioxaoctane (PEG3, Jeffamine, Texaco Chemical Co, 28 g, 187 mmol) in dichloromethane (100 mL).
• the mixture was stirred at room temparature, shielded from light, for 40 h.
• the resulting emulsion was chromatographed on a silica gel column (9 cm x 18 cm) using a gradient of dichloromethane/methanol (25/1 to 8/1). The homogeneous fractions were pooled and concentrated to give 4.8 g of a bluish solid. Proton and carbon NMR, and mass spectrum analysis were consistent with the desired compound.
• TA-PEG3 amine from EXAMPLE 40 (4.79 g, 9.9 mmol) were dissolved in pyridine (50 mL). Some insoluble material was removed by filtration. The solution was concentrated to 25 ml, and succinic anhydride (0.99 g, 9.9 mmol, Aldrich) was added. The mixture was stirred overnight at room temperature, shielded from light. The solution was filtered and concentrated under reduced pressure, followed by evaporation to dryness with methanol (50 mL) twice. The residue was dissolved in acetone (200 ml), and the mixture precipitated upon storage at 4°C overnight. A reddish powder was isolated by filtration (3.2 g), and the product was recrystallized from methanol (100 ml) to give 2.9 g of a solid. Proton and carbon ⁇ MR were consistent with the desired structure.
• EXAMPLE 42 N-succinamido-PEG3-TA, ⁇ HS ester ( ⁇ HS-SP-TA) ( Figure 36, Compound 43)
• N- succinamido-PEG3-TA from EXAMPLE 41 (1.6 g, 2.77 mmol), N-hydroxysuccinimide (Aldrich, 0.53 g, 4.7 mmol), and EDC (0.7 g, 3.4 mmol).
• the mixture in dichloromethane (50 ml) was stirred at room temperature, shielded from light for 15 h. The resulting clear solution was washed with water (40 ml).
• A. Coupling of a protease inhibitor to polymer-coated vesicles Succinylated aminodextran-coated vesicles (120 mg in 0.414 ml water), water (0.546 ml) MOPS buffer (120 ⁇ l of 500 mM) and Ac-LVK-aldehyde (120 ⁇ l of 25 mM; Bachem) were added to a 2 ml polypropylene tube with cap and the solution was mixed. To 1 ml of solution, ED AC (0.96 mg, lO ⁇ l) was added. The solution was mixed and incubated at room temperature for 18 hr.
• the conjugate was dialyzed twice in a 10K MWCO cassette in 3.5 L of 50 mM Histidine buffer containing 5 mM citrate at pH 7.4. Analysis of the conjugate mixture by size exclusion chromatography showed that the coupling yield was approximately 82%
• GFG-aldehyde semicarbazone was attached to vesicles in the same manner as Ac-LVK-aldehyde.
• A. Papain Activity Assay Add substrate (20 ul of 3 mM AFK-7AMC or 2 mM Z-FR-AMC; Bachem) to 3 ml of buffer (50 mM potassium phosphate/lmM EDTA.5%DMSO pH 6.8) in a 4.5 ml methyl acrylate cuvette (VWR). Add peptide (10 ⁇ l of 25 mM GFGsc or 0.25 mM LVK-ald) or inhibitor-vesicle conjugate (20 ⁇ l of 0 to320 ⁇ g/ml dilutions in water) to cuvette. Add papain (20 ⁇ l of 2 ⁇ M Papain in 50 mM potassium phosphate/lmM
• Cathepsin Activity Assay Add cathepsin (15 ⁇ l of l ⁇ M in 50 mM Acetate buffer at pH 5.5 and 5 mM DTT) to 15 ⁇ l of peptide inhibitor (2 ⁇ M Ac-LVK-cho) or peptide- vesicle conjugate (0 to 80 ⁇ g/ml dilutions in water) in a 1.5 ml polypropylene tube and incubate at room temperature for 15 min. Add substrate (10 ⁇ l of 4 mM Z-RR-amc; Bachem) to 3 ml buffer (50 mM Acetate buffer at pH 5.5) in 4.5 ml cuvette immediately before adding inhibitor soulution.
• EXAMPLE 46 Preparation of 10,12-Pentacosadiynoic acid N-hydroxysuccinimide ester (PDA-CONHS 32) ( Figure 37) 10,12-Pentacosadiynoic acid (PDA 30) (Lancaster, FW: 374.61, 374mg, 1 mmole) was dissolved in methylene chloride (Aldrich, 10 mL)) (under argon).
• the reaction mixture was diluted with methylene chloride (lOOmL), washed with 0.1 N HC1 (25mL), water (25 mL), and finally with brine (25 mL).
• the organic layer was dried over anhydrous sodium sulfate and the solvent then removed by spin evaporation.
• the crude product thus obtained (401mg, 85% yield) was used without further purification.
• EXAMPLE 47 EXAMPLE Preparation of 10,12-Pentacosadiynoic polyethyleneglycolamide (PDA-CONH-PEG33 36) ( Figure 37)
• PDA-CONHS from EXAMPLE 46 (401 mg, 0.85 mmole) was dissolved in methylene chloride (Aldrich, 10 mL)under argon. To this solution was added PEG33 (Huntsman, FW: 2000, 2.55 g, 1.28 mmole) using a syringe pump during a period of 5h.
• the CH 2 C1 2 layer was separated, dried over anhydrous sodium sulfate, filtered to remove the sodium sulfate, and the solvent removed by spin evaporation.
• the product 36 was dried under high vacuum.
• EXAMPLE 48 Preparation of 4-[2-(3,4,5,6-Tetrahydropyrimidin-2- y lamino) ethy Ioxy] benzoy l-2-(S) - (10 ' ,11 ' -Pentacosadiynoic amidoethy lsulfony lamino) - ⁇ -alanine (PDA-PM 34) ( Figure 37) PDA-CONHS from EXAMPLE 46 (141.3 mg, 300 ⁇ mol) and compound 10 ( Figure
• EXAMPLE 49 Preparation of vesicles containing 10% PDA-PM, 10% PDA-DTPA, 1% PDA-DTPA-Eu, and 79% PC ( Figure 37).
• the above lipids (114.82 mg, 4 mL) were mixed in test tubes, dissolved in 0.5 mL of CHC1 3 , spin evaporated to dryness, and dried under high vacuum overnight.
• the effective concentration of PM was 3 mM (1.37/mg/mL)
• the dried residue was suspended in 4mL of water and sonicated for about one hour while checking the pH frequently.
• the formed vesicles were polymerized by first cooling the solution in a petri dish in an ice bath and then placing under an UV lamp for 120 min. The solution was then dialyzed in 50 mM histidine, 5 mM sodium citrate, pH 7.4 overnight.
• PVs polymerized vesicles
• the solution was removed from the dialysis cassette using a 30mL naked syringe.
• the needle was removed from the syringe and was fitted with a 0.2 ⁇ filter and the particles were filtered into a vial. Size and zeta potential were measured by diluting 25 ⁇ L of the PV with 2 mL of water.

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