WO2003011345A1 - Lipid constructs as therapeutic and imaging agents - Google Patents

Lipid constructs as therapeutic and imaging agents Download PDF

Info

Publication number
WO2003011345A1
WO2003011345A1 PCT/US2002/023947 US0223947W WO03011345A1 WO 2003011345 A1 WO2003011345 A1 WO 2003011345A1 US 0223947 W US0223947 W US 0223947W WO 03011345 A1 WO03011345 A1 WO 03011345A1
Authority
WO
WIPO (PCT)
Prior art keywords
lipid
entity
construct
lipid construct
therapeutic
Prior art date
Application number
PCT/US2002/023947
Other languages
French (fr)
Inventor
Charles Aaron Wartchow
Neal Edward Dechene
John S. Pease
Original Assignee
Targesome, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/976,254 external-priority patent/US20020071843A1/en
Application filed by Targesome, Inc. filed Critical Targesome, Inc.
Priority to EP02756736A priority Critical patent/EP1420831A1/en
Priority to JP2003516575A priority patent/JP2005519861A/en
Priority to CA002455598A priority patent/CA2455598A1/en
Publication of WO2003011345A1 publication Critical patent/WO2003011345A1/en

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12Preparations 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
    • A61K51/1217Dispersions, suspensions, colloids, emulsions, e.g. perfluorinated emulsion, sols
    • A61K51/1234Liposomes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/02Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00Drugs for disorders of the senses
    • A61P27/02Ophthalmic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00Drugs for disorders of the senses
    • A61P27/02Ophthalmic agents
    • A61P27/06Antiglaucoma agents or miotics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system

Definitions

  • This invention relates generally to lipid constructs for radiotherapy and imaging. More specifically, this invention relates to liposomes containing a chelator, a targeting entity, a detectable entity, and/or a therapeutic entity, and methods relating to their use.
  • the therapeutic or treatment entity may be associated with the lipid construct by covalent or non- covalent means. In some cases, the therapeutic or treatment entity is a radioisotope.
  • Cancer remains one of the leading causes of death in the industrialized world. In the United States, cancer is the second most common cause of death after heart disease, accounting for approximately one-quarter of the deaths in 1997. Clearly, new and effective treatments for cancer will provide significant health benefits. Among the wide variety of treatments proposed for cancer, targeted therapeutic agents hold considerable promise. In principle, a patient could tolerate much higher doses of a cytotoxic agent if the cytotoxic agent is targeted specifically to cancerous tissue, as healthy tissue should be unaffected or affected to a much smaller extent than the pathological tissue.
  • Solid tumors in particular, express certain antigens, on both the transformed cells comprising the tumor and the vasculature supplying the tumors, which are either unique to the tumor cells and vasculature, or overexpressed in tumor cells and vasculature in comparison to normal cells and vasculature.
  • an antibody specific for a tumor antigen, or a tumor vasculature antigen, to a cytotoxic agent should provide high specificity to the site of pathology.
  • One group of such antigens is a family of proteins called cell adhesion molecules (CAMS), expressed by endothelial cells during a variety of physiological and disease processes.
  • 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 tumor metastasis, solid tumor growth (neoplasia), osteoporosis, Paget's disease, humoral hypercalcemia of malignancy, angiogenesis, including tumor angiogenesis, retinopathy, 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 ⁇ 3 would be beneficial for treating such conditions.
  • RGD Arg-Gly-Asp
  • fibrinogen Bosset et al., Proc. Natl. Acad. Sci. USA, Vol. 80 (1983) 2417
  • fibronectin Ginsberg et al., J. Clin. Invest., Vol. 71 (1983) 619-624
  • von Willebrand factor Ruggeri et al., Proc.
  • RGD peptides in general are non- selective for RGD dependent integrins.
  • RGD peptides that bind to v ⁇ 3 also bind to ⁇ v ⁇ 5 , Oy ⁇ i, and ⁇ b ⁇ a- Antagonism of platelet ⁇ nb ⁇ ma (also known as the fibrinogen receptor) is known to block platelet aggregation in humans.
  • Ginsberg et al. U.S. Pat. No. 5,306,620 discloses 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 ct 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 discloses 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 .
  • U.S. Patent No. 6,171,588 describes monoclonal antibodies which can be used in a method for blocking ⁇ v ⁇ 3 -mediated events such as cell adhesion, osteoclast- mediated bone resorption, restenosis, ocular neovascularization and growth of hemangiomas, as well as neoplastic cell or tumor growth and dissemination.
  • ⁇ v ⁇ 3 -mediated events such as cell adhesion, osteoclast- mediated bone resorption, restenosis, ocular neovascularization and growth of hemangiomas, as well as neoplastic cell or tumor growth and dissemination.
  • Other uses described are antibody-mediated targeting and delivery of therapeutics for disrupting or killing ⁇ v ⁇ 3 bearing neoplasms and tumor-related vascular beds.
  • the inventive monoclonal antibodies can be used for visualization or imaging of ⁇ v ⁇ 3 bearing neoplasms or tumor related vascular beds by NMR or immunoscintigraphy.
  • 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 examples include SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane- 1-carboxylate) or the linkers disclosed in U.S. Patent No. 4,880,935, and oligopeptide spacers.
  • Carbodiimides and N-hydroxysuccinimide reagents have been used to directly join therapeutic and targeting entities with the appropriate reactive chemical groups.
  • the use of cationic organic molecules to deliver heterologous genes in gene therapy procedures has been reported in the literature. Not all cationic compounds will complex with DNA and facilitate gene transfer. Currently, a primary strategy is routine screening of cationic molecules.
  • cationic polymers such as polyethyleneamine, ethylene diamine cascade polymers, and polybrene. Proteins, such as polylysine with a net positive charge have also been used.
  • 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.
  • liposomes 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.
  • U.S. Patent Numbers 5,077,057 and 5,277,914 teach preparation of liposome or lipidic particle suspensions having particles of a defined size, particularly lipids soluble in an aprotic solvent, for delivery of drugs having poor aqueous solubility.
  • U.S. Patent No. 4,544,545 teaches phospholipid liposomes having an outer layer including a modified, cholesterol derivative to render the liposome more specific for a preselected organ.
  • U.S. Patent No. 5,246,707 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.
  • 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 TI (longitudinal) and T2 (transverse) relaxation values and the proton density, which generally corresponds to the free water content, of the tissues.
  • TI longitudinal
  • T2 transverse relaxation values
  • proton density which generally corresponds to the free water content
  • a contrast medium may be designed to change either the TI, 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 (TI) and transverse (T2) relaxation.
  • Paramagnetic contrast agents typically comprise metal ions, for example, transition metal ions, which provide a source of unpaired electrons. However, these metal ions are also generally highly toxic.
  • Gd-DTPA gadolinium ion
  • 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. Ultrasound provides certain advantages over other diagnostic techniques.
  • diagnostic techniques involving nuclear medicine and X-rays generally involves 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. In addition, ultrasound is relatively 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, generally referred to as backscatter or reflectivity, forms the basis for developing an ultrasound image. In this connection, sound waves reflect differentially from different body tissues.
  • 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 discloses 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.
  • a presentation was made by Moseley et al., at a 1991 Napa, California meeting of the Society for Magnetic Resonance in Medicine, which is summarized in an abstract entitled "Microbubbles: A Novel
  • MR Susceptibility Contrast Agent The microbubbles described by Moseley et al. comprise air coated with a shell of human albumin.
  • 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.
  • Liposomal imaging agents should ideally be prepared with a simple and efficient labeling procedure with an affordable labeling group, should be stable in vivo with no release of free label, and be stable upon storage.
  • Conjugation of radionuclides to liposomes has been investigated primarily to develop of advanced imaging and diagnostic modalities for ⁇ - scintigraphy, single photon emission tomography (SPECT) or positron emission tomography (PET).
  • SPECT single photon emission tomography
  • PET positron emission tomography
  • Liposomes have been labeled with radionuclides, most commonly 99m-Tc, 111-In, and 67-Ga, by conjugation to a chelator or other suitable anchoring molecule contained within the aqueous interior of the liposome or within the lipid bilayer.
  • Efficient labeling (> 95%) with 99m-Tc is now routine, using the bifunctional chelator HYNIC conjugated to lipids, such as distearoylphosphatidylethanolamine (DSPE).
  • DSPE distearoylphosphatidylethanolamine
  • liposomes include simple entrapment within the liposome interior. Entrapment may occur using ionophores, such as A23187, or during liposome formation.
  • A23187 for the encapsulation of an isotope in a liposome includes indium-111 labeled VesCan ((Kubo et al, Eur. J. Nucl. Med (1993), 20(2), 107-113). Entrapment requires purification of the liposomes containing the encapsulated isotope from non-encapsulated isotope by chromatography. Surface labeling of liposomes with Tc99m has been reported by Ahkong & Tilcock, Nucl. MedBiol.
  • Tilcock reference is that labeling the DTTA chelator, a derivative of DTP A, with Tc99m is that chromatography is required to separate free and colloidal forms of technetium from the liposome-Tc99m complexes.
  • Sterically stabilized (GM-coated) liposomes are taken up by the muscle tumor tissue more readily than are SUVs.
  • GM-coated liposomes are taken up by the muscle tumor tissue more readily than are SUVs.
  • 211-At and 188-Re deliver higher tumor doses when combined with the former, but 67-Cu, 90-Y and 131-1 are more effective with the latter.
  • Kostarelos & Emfietzoglou conclude that the importance of liposome size and steric barrier when designing effective radionuclide-carrier systems, as well as optimal matching between the radionuclide half-life and the time of maximum liposome accumulation ratio between tumor and normal tissue, are important considerations.
  • a description of the use of 90 Y-liposome complexes for therapy was not provided in this theoretical report. Bard, et al., Gin. Exp.
  • the liposomes were prepared by combining 3-cholesteryl 6- [N'-iminobis(ethylenenitrilo)tetraacetic acid acidjhexyl ether (Chol-DTTA) with DSPC and a radioactive isotope, either 51-Cr or 177-Lu.
  • Chol-DTTA N'-iminobis(ethylenenitrilo)tetraacetic acid acidjhexyl ether
  • the treatment of rheumatoid arthritis by radiosynovectomy has been restricted by the difficulty of preventing leakage of the radioisotope from the joint cavity.
  • liposomes were prepared with 3-cholesteryl 6-[N'-iminobis(ethylenenitrilo)-tetraacetic acidjhexyl ether (Chol-DTTA) which can complex with a.variety of beta-emitting radionuclides.
  • Chol-DTTA N'-iminobis(ethylenenitrilo)-tetraacetic acidjhexyl ether
  • 51-Cr was used as the radioisotope.
  • the liposomes were injected into the knee joint cavity of rabbits with expertimentally induced arthritis.
  • the present invention provides a lipid construct comprising a chelator, a targeting entity, and a detectable entity or a therapeutic entity.
  • the lipid construct may include lipids, including phosphatidylcholine derivatives.
  • the lipid constructs can also include additional components, such as cholesterol or stabilization agent, such as polyethylene glycol.
  • the therapeutic entity is a radionuclide, such as Y-90, 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.
  • the therapeutic agent may be associated with the surface of the lipid construct or encapsulated within the lipid construct.
  • the chelator is part of a chelating lipid, such as 1,2- dimyristoyl-5 «-glycero-3-phosphoethanolamidotriamine tetraacetic acid (BisM-PE-DTPA4).
  • DOTA 1, 4,7,10-tetraazacyclododecane- N,N',N",N" '-tetraacetic acid
  • the chelating lipid contains a diacetylene lipid or a polymerizable lipid such as l,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine or The lipid construct of Claim 17 or 18, wherein the polymerizable lipid is [PDA-PEG3] 2 -
  • the present invention further provides lipid constructs containing chelating lipids that comprise an ionizable group such as carboxyl, phosphate, phosphonate, sulfate, sulfonate, or sulfinate, ionizable groups generating a surface capable of binding an isotope or metal with a valency of +2 or greater, and ionizable groups generating a surface capable of binding an isotope or metal with a valency of +3 or greater.
  • an ionizable group such as carboxyl, phosphate, phosphonate, sulfate, sulfonate, or sulfinate
  • ionizable groups generating a surface capable of binding an isotope or metal with a valency of +2 or greater
  • ionizable groups generating a surface capable of binding an isotope or metal with a valency of +3 or greater.
  • the targeting entity of the lipid construct may include a small molecule ligand and a protein, and in some embodiments targets the lipid construct to a cell surface.
  • the targeting entity is associated with a carboxyl head group of said lipid, maleimide group of a lipid, the alpha-methyl group of an acetamide of a lipid, or other covalent means, such as amine, cyano, carboxylic acid, isothiocyanate, thiol, disulfide, ⁇ - halocarbonyl, ⁇ , ⁇ -unsaturated carbonyl or alkyl hydrazine.
  • the detectable entity is a radionuclide, such as Tc-99m, In-111, Ga-67, Rh-105, Nd -147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, or Tl- 201.
  • a radionuclide such as Tc-99m, In-111, Ga-67, Rh-105, Nd -147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, or Tl- 201.
  • the targeting entity is associated with the lipid construct by non-covalent means such as a biotin-avidin biotinylated antibody sandwich.
  • the targeting entity is an antibody such as an antibody having a target selected from the group consisting of P-selectin, E-selectin, pleiotropin, G-protein coupled receptors, endosialin, endoglin, VEGF receptor, PDGF receptor, EGF receptor, the matrix metalloproteases, and prostate specific membrane antigen (PSMA).
  • a target selected from the group consisting of P-selectin, E-selectin, pleiotropin, G-protein coupled receptors, endosialin, endoglin, VEGF receptor, PDGF receptor, EGF receptor, the matrix metalloproteases, and prostate specific membrane antigen (PSMA).
  • PSMA prostate specific membrane antigen
  • the targeting entity has a vascular target, such as the targeting entities Vitaxin or LM609.
  • the targeting entity is an anti-VCAM-1 antibody, an anti- ICAM-1 antibody, or an anti-integrin antibody. In other embodiments, the targeting entity has a targeting entity having a tumor cell target.
  • the present invention also provides a therapeutic agent comprising a lipid construct, said lipid construct comprising a chelating lipid, a targeting entity, and a therapeutic entity, wherein the therapeutic entity is associated with the chelating lipid at the surface of said lipid construct.
  • the therapeutic agent may include a therapeutic entity that is a metal ion, including a radioactive metal ion such as Y-90, 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, or Re-188, a radiation synovectomy agent.
  • the present invention also provides an imaging agent comprising a lipid construct, the lipid construct comprising a chelating lipid, a targeting entity, and a detectable entity, wherein the detectable entity is associated with the chelating lipid at the surface of said lipid construct.
  • the imaging agent may include an imaging entity that is a metal ion, including a radioactive metal ion, such as Tc-99m, In-I ll, Ga-67, Rh-105, Nd -147, Pm-151, Sm-153, Gd-159, Tb- 161, Er-171, Re-186, Re-188, and Tl-201.
  • the present invention also provides a method for preparing a lipid construct comprising preparing a liposome comprising a chelating lipid, and contacting the liposome with a metal ion, including a radioactive metal ion, including Y-90, 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 (first group of metal ions), Tc-99m, In-I ll, Ga-67, Rh-105, 1-
  • the method may further include contacting the liposome with a second metal ion, including a method where the first metal ion is a metal ion from the first group, and the second metal ion is a metal ion from the second group.
  • the two metal ions may reside in the same mixture.
  • the method may further comprising removing unbound metal ion.
  • the present invention also provides a method of imaging a patient comprising administering an imaging agent to a patient in need thereof, said imaging agent comprising a lipid construct, said lipid construct comprising a chelating lipid and a detectable entity, and imaging the patient.
  • the imaging may include magnetic resonance imaging or nuclear scintigraphy.
  • the present invention further 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 lipid construct, said lipid construct comprising a chelating lipid and a therapeutic entity.
  • the patient suffers from rheumatoid arthritis.
  • the present invention also provides a lipid construct useful for therapy and imaging, comprising a chelating lipid, a targeting entity, a detectable entity, and a therapeutic entity, wherem said detectable entity and therapeutic entity are associated with said chelating lipid at the surface of said lipid construct, hi some embodiments, the therapeutic entity is a metal ion from the first group of metal ions listed above, and the detectable entity is a metal ion from the the second group of metal ions listed above.
  • Figure 1 shows the purity of an antibody-liposome conjugate by size exclusion chromatography and ELISA.
  • the signal from an ELIS A performed on each fraction is plotted as a function of fraction number after purification of the conjugate using a Sepharose CL-4B column.
  • the larger antibody-liposome complex elutes in fractions 2-6 and the unbound antibody elutes in fractions 7-12 (data not shown).
  • Figure 2 shows the response in an ⁇ v ⁇ 3 -specific radioimmunoassay for the anti- v ⁇ 3 antibody-liposome-yttrium-90 complex in serum. This assay generates signal when the antibody and the yttrium-90 are associated with the same vesicle and no signal originates from unbound yttrium-90.
  • Figure 3 shows some of the lipid structures used to prepare liposomes.
  • Figure 4 shows stability of Vitaxin-liposome- 90 Y conjugates in rabbit serum.
  • Figure 5 shows additional lipid structures used to prepare liposomes. These lipids are prepared from tricosadiynoic acid and the hydrophilic linker l,8-diamino-3,6-dioxaoctane, except for BisT-PE-DTTA4, which does not contain the linker.
  • Figure 6 shows the biodistribution of Vitaxin-liposome- 90 Y conjugates in healthy New
  • Figure 7 shows the efficacy of integrin-targeted vesicles labeled with yttrium 90 (IA-NP-Y90) in the mouse melanoma model as described in Example 29.
  • Treatment groups include LA (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 8 shows the normalized tumor volume 7 days post treatment sorted by treatment group for the study described in Example 8.
  • Figure 9. 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 9.
  • Figure 10 shows efficacy in the mouse colon cancer model as described in Example 10. Error bars indicate ⁇ one standard error.
  • Treatment groups include buffer, PM (RGD peptidomimetic alone), 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 11 Plot of normalized tumor volume on day 8 sorted by group for the study in Example 10.
  • the present invention is directed toward a lipid construct comprising a lipid chelator, a targeting entity, and a therapeutic and/or a detectable entity.
  • the present invention is also directed toward methods of preparation of the lipid constructs of the present invention.
  • the present invention is further directed towards lipid constructs containing one or more chemically distinct lipids.
  • the present invention is also directed toward therapeutic agents comprising the lipid constructs of the present invention.
  • the present invention is further directed toward a method of administering a therapeutic agent of the present invention to a patient in need thereof.
  • the present invention is yet further directed towards the treatment of diseases where the vasculature associated with the disease may be treated or targeted with a targeted therapeutic agent.
  • Suitable targets are ICAM, VCAM, integrins, P-selectin, E-selectin, pleiotropin, endosialin, endoglin, VEGF receptor, PDGF receptor, EGF receptor, the matrix metalloproteases, G-protein coupled receptors, and prostate specific membrane antigen (PSMA).
  • PSMA prostate specific membrane antigen
  • the present invention is also directed toward imaging agents comprising the lipid constructs of the present invention, and a method of imaging a patient comprising administering an imaging agent of the present invention to a patient, and imaging the patient.
  • the present invention is also directed towards agents which may be used for both therapy and imaging.
  • the present invention is further directed to methods and reagents for diagnosis using the lipid constructs of the present invention.
  • a lipid construct 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.
  • Common additional components in lipid constructs include cholesterol and alpha-tocopherol, among others.
  • the lipid constructs may be used alone or in any combination which one skilled in the art would appreciate to provide the characteristics desired for a particular application.
  • the technical aspects of lipid constructs and liposome formation are well known in the art and any of the methods commonly practiced in the field may be used with the present invention.
  • 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 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.
  • Lipid bilayer vesicle refers to a closed, fluid-filled microscopic sphere which is formed principally from individual molecules having polar (hydrophilic) and non-polar (lipophilic) portions.
  • the hydrophilic portions may comprise phosphate, glyceryophosphate, carboxy, sulfate, amino, hydroxy, choline and other polar groups and derivatives thereof.
  • non-polar groups are saturated or unsaturated hydrocarbons such as alkyl, alkenyl or other lipid groups.
  • Sterols e.g., cholesterol
  • other pharmaceutically acceptable components including anti-oxidants like alpha-tocopherol
  • lipids to which a targeting agent, such as a ligand, peptidomimetic, peptide, or other synthetic molecule may be incorporated into liposomes by preparing mixtures of the targeting lipid or lipids with additional chemically distinct lipids.
  • One or more targeting lipid may be mixed with other chemically distinct lipids.
  • 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,
  • 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 present invention includes lipid derivatives containing carboxyl, phosphate, phosphonate, sulfate, sulfonate, and sulfinate groups.
  • 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.
  • PEG polyethylene glycol
  • 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.
  • composition of the liposomes may be altered to modulate the biodistribution and clearance properties of the resulting liposomes.
  • the present invention is also directed toward methods of preparation of the lipid constructs of the present invention.
  • the lipids may first be dissolved and mixed in an organic solvent, for example, chloroform or chloroform:methanol mixtures, to assure a homogeneous mixture of lipids.
  • organic solvent for example, chloroform or chloroform:methanol mixtures
  • lipid solutions are prepared at 10-20 mg lipid ml organic solvent, although higher concentrations may be used if the lipid solubility and mixing are acceptable.
  • the solvent is removed to yield a lipid film, either by using a dry nitrogen or argon stream in a fume hood or rotary evaporation, followed by removal of residual organic solvent by placing the vial or flask on a vacuum pump overnight.
  • the lipids may be dissolved in a solvent that may be frozen and lyophilized.
  • the lipid solution is transferred to containers and frozen by, for example, placing the containers on a block of dry ice or swirling the container in a dry ice-acetone or alcohol (ethanol or methanol) bath. After freezing completely, the frozen lipid cake is placed on a vacuum pump and lyophilized until dry, typically one to three days depending on volume.
  • Hydration of the dry lipid film is accomplished simply by adding an aqueous medium to the container of dry lipid and agitating.
  • the temperature of the hydrating medium should be above the gel-liquid crystal transition temperature (T c or T m ) of the lipid with the highest T c before adding to the dry lipid.
  • the hydration medium is generally determined by the application of the lipid vesicles. Suitable hydration media include distilled water, buffer solutions, saline, and nonelectrolytes such as sugar solutions. Physiological osmolality (290 mOsm/kg) is recommended for in vivo applications. Generally accepted solutions which meet these conditions are 0.9% saline, 5% dextrose, and 10% sucrose.
  • the product of hydration is a large, multilamellar vesicle (LMV) analogous in structure to an onion, with each lipid bilayer separated by a water layer.
  • LMV multilamellar vesicle
  • the spacing between lipid layers is dictated by composition with polyhydrating layers being closer together than highly charged layers which separate based on electrostatic repulsion.
  • the particles can be downsized by a variety of techniques, including sonication, extrusion, or high-shear homogenization.
  • the present invention is directed toward a method for preparing lipid constructs comprising preparing a lipid mixture, preparing a lipid construct from the lipid mixture, and curing the lipid construct.
  • Preparing a lipid construct may mean any technique or techniques suitable for preparing the desired lipid construct. For example, a freeze/thaw procedure performed upon hydration is known to produce multilamellar vesicles.
  • Extrusion may be used to create particles of a particular size range.
  • extrusion is a preferred method of downsizing.
  • Curing refers to heating the lipid constmct to a temperature effective to impart increased stability in the lipid construct for a time effective to impart increased stability to the lipid construct, hi one embodiment, curing comprises heating at about 80-90 °C for about 16-18 hours. Curing provides some stability to the vesicles in the presence of proteins and salt.
  • MLVs may be prepared by repeated freeze- thaw cycles, or by the reversed-phase evaporation procedure (see Liposome Technology, Volume I, "Liposome Preparation and Related Techniques", G.
  • Micelles may be prepared using lipid tails consisting of less than 10 carbon atoms, and these tails may contain alkynes, alkenes, or alkanes.
  • the liposome may be converted to a polymerized liposome.
  • 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
  • the particles can be micelles, as well as particles which 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 1 nanometer (nm) to about 400 nm in diameter, and all combinations and subcombinations of ranges therein. More preferably, the vesicles have diameters from about 1 nm to about 500 nm, with diameters from about 40 nm to about 120 nm being even more preferred. In connection with particular uses, for example, intravascular use, including magnetic resonance imaging of the vasculature, it may be preferred that the vesicles be no larger that about 500 nm in diameter, with smaller vesicles being no larger than about 60-80 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.
  • 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 associated with the outside of the liposome for gene therapy 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 through endocytosis.
  • the invention may also utilize vesicles that bind to the target site and deliver a therapeutic agent to the desired site without internalization.
  • the therapeutic agent may be a radioisotope that irradiates surrounding cells and cell layers.
  • the therapeutic agent may also be a drug or pro-drug that is released while the invention is bound to the desired site.
  • the formulations preferably utilize UVs having an average diameter of less than 200 nm, more preferably less than 100 nm, and even more preferably about 60-80 nm.
  • the liposomes of the present invention comprise egg or soy phosphatidylcholine and cholesterol.
  • liposomes comprise stabilization agents such as polyethylene glycol
  • Lipid constructs of the present invention also comprise a chelator.
  • the chelator is part of a chelating lipid.
  • a chelating lipid is one in which a chelator is chemically associated with a lipid of which the lipid construct is comprised.
  • the chelating lipid is l,2-dimyristoyl-5 , «-glycero-3- phosphoethanolamidotriamine tetraacetic acid (BisM-PE-DTPA4).
  • This name is a common name that refers to the tetraacetic acid containing, phosphoethanolamine lipid derivative that is associated with DTPA by an amide bond where DTPA is the organic molecule diethylenetriaminepentaacetic acid.
  • Further embodiments of the present invention comprise lipis constructs containing an encapsulated chelator, such as DTPA or DOTA.
  • Lipid constructs of the present invention also optionally include polymerizable lipids, which result in a lipid construct that is a polymerized liposome.
  • Some preferred polymerizable lipids are [PDA-PEG3 J2 -DTTA3, described as N,N-bis[[[[(13 l5'- pentacosadiynamido-3,6-doxaoctyl)carbamoyl]methyl](carboxymethyl)amino]ethyl]glycine (compound 8a in J4CS 1995, 117(28), 7301-7306) and l,2-bis(10,12-tricosadiynoyl)-OT- glycero-3-phosphocholine (BisT-PC).
  • the present invention also contemplates lipid constructs of the present invention further comprising compounds, such as, for example, drugs or other therapeutic agents, or imaging agents, encapsulated within the lipid constructs of the present invention.
  • compounds such as, for example, drugs or other therapeutic agents, or imaging agents.
  • Agents which may be encapsulated include radionuclides which are discussed elsewhere herein. The radionuclide could be chelated inside the liposome by encapsulated DOTA, DTPA, or an encapsulated macromolecule that has a chelator associated with it.
  • Another method for encapsulation is analogous to the use of salt gradients used to encapsulate doxorubicin as described in co- pending united states patent application serial no.
  • Radionuclide to a solution of lipid constructs containing encapsulated salts, where the bulk solution has a much lower concentration of the salts. This concentration difference may be used to load the liposome by precipitation of the radionuclide-salt complex inside the liposome.
  • salts may be phosphates, sulfates, sulfites, phosphonates, carbonates.
  • Polyanionic polymers or ionophores may also be encapsulated to generate the appropriate gradient.
  • 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, such as doxorubicin and other chemotherapy agents; toxins such as ricin; radioactive isotopes; 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,
  • the therapeutic or treatment entity may be associated with the lipid construct by covalent or non-covalent means.
  • associated means associated with the liposome by covalent or noncovalent interactions.
  • the present invention is also directed toward a therapeutic entity comprising the lipid constructs of the present invention.
  • the therapeutic agent is a radionuclide.
  • a therapeutic radionuclide is a nuclide which undergoes spontaneous transformation (nuclear decay) with an energy transfer sufficient to impart cytotoxic amounts of radiant energy to nearby cells.
  • radionuclides useful for diagnosis emit radiation capable of penetrating tissue with minimal cell damage. Such radiation may be detected using a suitable scintigraphic imager.
  • Therapeutic radionuclides of the present invention include, but are not limited to 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. Diagnostic or imaging nuclides of the present invention include, but are not limited to Tc-99m, In-Ill, Ga-67, Rh-105, 1-123, Nd -147, Pm-151, Sm- 153, Gd-159, Tb-161, Er-171, Re-186, Re-188, and Tl-201.
  • a therapeutic radionuclide is associated with the lipid construct by non-covalent means.
  • the therapeutic radionuclide is associated with a chelating lipid.
  • yttrium-90 is the therapeutic radionuclide, and l,2-dimyristoyl-5 «-glycero-3- phosphoethanolamidotriamine tetraacetic acid as defined above is the chelating lipid.
  • chelating lipids which are preferred are lipid derivatives of diethylenetriaminepentaacetic acid including diethylenetriaminetetraacetic acids and diethylenetriaminetriacetic acids, derivatives of ethylaminediaminetetracetic acid, and derivatives of 1,4,7, 10-tetraazacyclododecane- N,N',N",N" '-tetraacetic acid (DOTA).
  • lipid derivatives of diethylenetriaminepentaacetic acid including diethylenetriaminetetraacetic acids and diethylenetriaminetriacetic acids, derivatives of ethylaminediaminetetracetic acid, and derivatives of 1,4,7, 10-tetraazacyclododecane- N,N',N",N" '-tetraacetic acid (DOTA).
  • lipids containing ionizable groups including carboxyls (such as nitrilotriacetic acid iminodiacetic acid, for example), phosphates, phospho
  • lipids containing a single ionizable group may self assemble to generate a surface capable of binding an isotope or metal with a valency of +2 or greater.
  • lipids containing two ionizable groups may self assemble to generate a surface capable of binding an isotope or metal with a valency of +3 or greater.
  • the present invention also provides methods for the preparation of liposomes of the present invention.
  • the method comprises preparation of a liposome of the present invention, attachment of a targeting agent, and chelation of an isotope primarily to the surface of the liposome.
  • the method of the present invention overcomes the deficiencies of the prior art by attaching a targeting agent to the liposome and by generating liposomes containing both a targeting agent and a therapeutic isotope.
  • the therapeutic isotope may be attached to the targeting agent-liposome conjugate with high efficiency and without the need for the removal of unassociated isotope.
  • the therapeutic isotope of the present invention may be attached to the liposomes of the present invention without the use of extreme temperatures, e.g., at room temperature.
  • the resulting targeting agent-liposome-isotope complex binds to a target in the presence of serum in-vitro where the targeting agent binds to its target and the isotope is detected using the appropriate detection method and apparatus.
  • the present invention is also directed towards a lipid construct comprising both a therapeutic entity and an imaging entity. Imaging or diagnostic agents are described in detail below in the sections entitled “Imaging” and "Diagnostics.”
  • the therapeutic entity is a therapeutic radionuclide
  • the imaging entity is an imaging radionuclide.
  • the therapeutic isotope is yttrium-90 and the imaging isotope is indium- 111 or a technetium isotope.
  • the lipid constructs may be prepared by providing the therapeutic isotope and the imaging isotope in the in the same mixture, followed by contacting the mixture containing the lipid constructs.
  • the present invention is further directed towards a therapeutic entity or imaging agent consisting of a targeting agent, a carrier, and an encapsulated therapeutic or imaging isotope.
  • targeting entity refers to a molecule, macromolecule, or molecular assembly which binds specifically to a biological target.
  • the targeting entity may be of natural, synthetic, or semi-synthetic origin.
  • Examples of targeting entities include, but are not limited to, antibodies (including antibody fragments and other antibody-derived molecules which retain specific binding, such as Fab, F(ab')2, Fv, and scFv derived from antibodies); receptor-binding ligands, such as hormones or other molecules that bind specifically to a receptor; cytokines, which are polypeptides that affect cell function and modulate interactions between cells associated with immune, inflammatory or hematopoietic responses; molecules that bind to enzymes, such as enzyme inhibitors; nucleic acid ligands or aptamers, and one or more members of a specific binding interaction such as biotin or iminobiotin and avidin or streptavidin.
  • preferred targeting entities are molecules which specifically bind to receptors or antigens found on vascular cells. More preferred are molecules which specifically bind to receptors, antigens or markers found on cells of angiogenic neovasculature or receptors, antigens or markers associated with tumor vasculature.
  • the receptors, antigens or markers associated with tumor vasculature can be expressed on cells of vessels which penetrate or are located within the tumor, or which are confined to the inner or outer periphery of the tumor.
  • 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 system 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.
  • preferred targeting entities are molecules which specifically bind to receptors, antigens, or markers on cells that circulate within the vasculature, such as malignant B cells, or cells expressing antigens as a result of viral infection.
  • Targeting entities attached to the liposomes 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 1000 daltons or less, which serve as ligands for a vascular target or vascular cell marker), such as the integrin-binding molecules described in copending United States Patent Application Serial No.
  • Targeted Multivalent Macromolecules 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.
  • 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.
  • head groups can be used to control the biodistribution, non
  • ⁇ -D-lactose has been attached on the surface to target the asialoglycoprotein (ASG) found in liver cells which are in contact with the circulating blood pool.
  • the targeting entity is an integrin-specific molecule, such as an
  • RGD peptide see above, or an 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 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
  • Glycolipids can be derivatized for use as targeting entities, for example, by converting the commercially available lipid (DAGPE) or the pentadicosanoic acid derivative _V-(8'- amin ⁇ -3',6'-dioxaoctyl)-10,12-pentacosadiynamide (PEG-PDA amine) into its isocyanate followed by treatment with triethylene diamine spacer l,8-diamino-3,6-dioxaoctane to produce the amine terminated thiocarbamate lipid which by treatment with the para- isothiocyanophenyl glycoside of the carbohydrate ligand produces the desired targeting glycolipids.
  • DAGPE commercially available lipid
  • PEG-PDA amine pentadicosanoic acid derivative _V-(8'- amin ⁇ -3',6'-dioxaoctyl)-10,12-pentaco
  • This synthesis provides a water soluble flexible spacer molecule spaced between the lipid that will form the internal structure or core of the liposome and the ligand that binds to cell surface receptors, allowing the ligand to be readily accessible to the protein receptors on the cell surfaces.
  • the carbohydrate ligands can be derived from reducing sugars or glycosides, such as para-nitrophenyl glycosides, a wide range of which are commercially available or easily constructed using chemical or enzymatic methods. Liposomes coated with carbohydrate ligands can be produced by mixing appropriate amounts of individual lipids followed by sonication, extrusion, polymerization if polymerizable lipids are used, and filtration as described above.
  • Suitable carbohydrate derivatized liposomes have about 1 to about 30 mole percent of the targeting glycolipid and filler lipid, such as PDA, DAPC, DAPE, or other phosphocholine based lipid, with the balance being metal-chelated lipid or metal- chelating lipid.
  • Other lipids may be included in the liposomes to assure liposome formation and provide high contrast and recirculation.
  • 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 Drags, Adv. Drug Del. Rev. (1999)
  • the targeting entity is attached to a carboxyl head group on the lipid. In another preferred embodiment, the targeting entity is attached to a maleimide or the alpha-methyl group of an acetamide. In one embodiment, 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 liposome.
  • lipid head groups for the attachment of targeting agents include 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 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, and for attachment of therapeutic entities, such as drugs or radioactive isotopes.
  • head groups may have an attached therapeutic entity, such as, for example, antibodies, peptidomimetics, and hormones and drugs for interaction with a biological site at or near the specific biological molecule to which the polymerized liposome particle attaches.
  • the 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
  • the vascular-targeted therapeutic agent itself can induce permeability in the tumor vasculature.
  • 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 invention provides a vascular targeted therapeutic agent that comprises an integrin targeting agent and a 90 Y therapeutic entity.
  • 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 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 therapy agent, but targeted against the tumor cell rather than the vasculature.
  • a conventional antitumor therapy such as cisplatin
  • antibodies directed against tumor markers such as anti-Her2/neu antibodies (e.g., Herceptin)
  • tripartite agents such as those described herein for vascular-targeted therapy agent, 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 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 antibody-liposome-radioisotope agent can be administered to spinal fluid, where the antibody targets a site of pathology accessible from the spinal fluid.
  • the agent may also be injected subcutaneously for administration to the lymphatic system.
  • 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.
  • One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal.
  • 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 anti-tumor 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 compostions 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.
  • the concentration of the radiation synovectomy agent varies with the particular use, but a sufficient amount is present to provide satisfactory radiation synovectomy. For example, in radiation synovectomy of the hip, the concentration of the agent will generally be higher than when used for the radiation synovectomy of the wrist joints.
  • the radiation synovectomy composition is administered so that preferably it remains substantially in the joint for 20 half-lifes of the isotope although shorter residence times are acceptable as long as the leakage of the radionuclide is small and the leaked radionuclide is rapidly cleared from the body.
  • the radiation synovectomy compositions may be used in the usual way for such procedures.
  • a sufficient amount of the radiation synovectomy composition to provide adequate radiation synovectomy is injected into the knee-joint.
  • 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.
  • macular degeneration The most common form of macular degeneration is called “dry” or involutional macular degeneration and results from the thinning of vascular and other structural or nutritional tissues underlying the retina in the macular region. A more severe form is termed
  • choroidal neovascularization CNV
  • 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.
  • histoplasmosis syndrome a series of events occur in the choroidal layer of the inside lining of the back of the eye resulting in localized inflammation of the choroid and consequent scarring with loss of function of the involved retina and production of a blind spot (scotoma).
  • the choroid layer is provoked to produce new blood vessels that are much more fragile than normal blood vessels. They have a tendency to bleed with additional scarring, and loss of function of the overlying retina.
  • Diabetic retinopathy involves retinal rather than choroidal blood vessels resulting in hemorrhages, vascular irregularities, and whitish exudates. Retinal neovascularization may occur in the most severe forms. When the vasculature of the eye is targeted, it should be appreciated that targets may be present on either side of the vasculature.
  • 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.
  • the phrase "gene therapy” refers 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 (poly) peptide 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; Olson, F. et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, F. et: al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, E. et al. Biochim. Biophys. Acta 775: 169, 1984; Kim, S. et al. Biochim.
  • 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).
  • room temperature about 18 to 26 °C
  • conditions are chosen that are not conducive to deprotection of protected groups.
  • 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. 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 invention provides therapeutic agents to treat inflamed synovia of people afflicted with rheumatoid arthritis. This type of therapeutic agent is a radiation synovectomy agent.
  • the concentration of the radiation synovectomy agent varies with the particular use, but a sufficient amount is present to provide satisfactory radiation synovectomy. For example, in radiation synovectomy of the hip, the concentration of the agent will generally be higher than when used for the radiation synovectomy of the wrist joints.
  • the radiation synovectomy composition is administered so that preferably it remains substantially in the joint for 20 half- lifes of the isotope although shorter residence times are acceptable as long as the leakage of the radionuclide is small and the leaked radionuclide is rapidly cleared from the body.
  • the radiation synovectomy compositions may be used in the usual way for such procedures.
  • 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.
  • 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-111, Ga-67, Rh-105, 1-123, Nd -147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, and Tl-201.
  • the imaging agents of the present invention are useful in imaging a patient generally, and/or in specifically diagnosing the presence of diseased tissue in a patient.
  • the imaging process may be carried out by administering an imaging agent of the invention to a patient, and then scanning the patient using ultrasound or magnetic resonance imaging to obtain visible images of an internal region of a patient and/or of any diseased tissue in that region.
  • region of a patient it is meant the whole patient, or a particular area or portion of the patient.
  • the imaging contrast agent may be employed to provide images of the vasculature, heart, liver, and spleen, and in imaging the gastrointestinal region or other body cavities, or in other ways as will be readily apparent to those skilled in the art, such as in tissue characterization, blood pool imaging, etc. Any of the various types of ultrasound or magnetic resonance imaging devices can be employed in the practice of the invention, the particular type or model of the device not being critical to the method of the invention.
  • Targeting agent-conjugated lipid constructs of this invention achieve in vitro and in vivo targeting of specific molecules associated with specific body tissues and specific molecules associated with specific bodily functions and pathologies to provide sufficient signal enhancement for detection by imaging methods such as magnetic resonance imaging or nuclear scintigraphy.
  • imaging methods such as magnetic resonance imaging or nuclear scintigraphy.
  • Characterization of these responses in individual animals simplifies assessment of the interventions, since expression and regression of the target can be confirmed as it relates to disease outcomes.
  • this technique detects disease at early stages, thereby enabling more effective treatment.
  • the lipid constructs of this invention are suitable for combination of imaging and delivery of drugs for therapeutic treatments.
  • Various agents can be encapsulated or attached to the surface of liposomes for delivery to specific sites in vivo.
  • target-specific drug/liposomes of this invention the drug delivery can be simultaneously visualized by magnetic resonance imaging.
  • the site-specific liposome having attached monoclonal antibodies for specific receptor targeting may be used to visualize abnormal pathology related to solid tumors, inflammation, rheumatoid arthritis, and osteoporosis using cell surface markers including the integrins, VEGF receptors, PDGF receptors, matrix metalloproteases, selectins, PSMA, endosialin, G-protein coupled receptors, and endoglin.
  • the present invention further provides methods and reagents for diagnostic purposes.
  • Diagnostic assays contemplated by the present invention include, but are not limited to, receptor-binding assays, antibody assays, immunohistochemical assays, flow cytometry assays, genomics and nucleic acid detection assays. High-throughput screening arrays and assays are also contemplated.
  • antibody- conjugated liposomes provide an ultra-sensitive diagnostic assay for specific antigens in solution.
  • Liposomes of this invention having a chelator head group chelated to spectroscopically distinct ions provide high sensitivity for assays involving protein-protein, ligand-protein , drug-protein, nucleic-acid protein, and nucleic acid-nucleic acid interactions.
  • Liposomes of this invention having a fluorophore head group provide a method for detection of glycoproteins on cell surfaces.
  • the invention provides an agent which may be used for both therapy and imaging.
  • the dual function agent in one embodiment comprises, a lipid construct, a therapeutic agent and an imaging agent.
  • the dual function agent may also comprise a targeting agent.
  • This invention further provides a method of assaying abnormal pathology in vitro comprising, introducing a plurality of liposomes of the present invention to a molecule involved in the abnormal pathology into a fluid contacting the abnormal pathology, the targeting liposome attaching to a molecule involved in the abnormal pathology, and detecting in vitro the liposome attached to molecules involved in the abnormal pathology.
  • PC/Chol/BisM-PE-DTPA4 liposomes Phosphatidylcholine from egg (PC, Avanti, 232 mg), cholesterol (Choi, Avanti, 50 mg), and l,2-dimyristoyl-,s7z-glycero- 3-phosphoethanolamidotriamine tetraacetic acid (Avanti, 25 mg) were dissolved in chloroform and the solvent was removed by rotary evaporation. The mixture was hydrated with water (10 mL) and 0.5 M NaOH (87 microliters) was added followed by five freeze-thaw cycles where the mixture was repeatedly frozen in a dry ice/acetone bath and thawed at approximately 65°C.
  • the solution was passed through a thermal barrel extruder heated at 95°C (Lipex Biomembranes Inc.) containing 30 nm polycarbonate filters to give a clear solution containing 60 nm liposomes as determined by dynamic light scattering with
  • the antibody-liposome conjugate is present in fractions 2-6 as determined by ELISA.
  • the unbound antibody also detected by ELISA, elutes in fractions 7-12 (data not shown). For this preparation, unbound antibody was not detected.
  • 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 is determined with the Capintec CRC-15R dosimeter.
  • the filter portion of the assembly is removed and discarded. Using the dosimeter, the remaining part of the assembly containing the "unbound
  • 90 Y that passed through the filter is counted.
  • "Bound 90 Y” is determined by subtracting the "unbound 90 Y” from the "total 90 Y”.
  • Percent 90 Y bound is calculated by dividing the "bound 90 Y” by the “total 90 Y” and multiplying by 100.
  • 90 Y binding is greater than 95% for lipid based DTPA chelators. D.
  • Liposomes containing diacetylene lipids are prepared as described in Example 1 A, except l,2-bis(10,12-tricosadiynoyl)-i'7 ⁇ -glycero-3-phosphocholine and chelating lipid [PDA- PEG3] 2 -DTTA3 ( Figure 3) or [TDA-PEG3] 2 -DTTA3 ( Figure 5) or BisT-PE-DTTA4 ( Figure 5) may be used instead of the lipids described in 1 A.
  • Antibody or peptide or peptidomimetic may be attached as described in IB, and yttrium-90 or indium-111 may be attached as described in IC.
  • Example 1 B An antibody specific to the human ⁇ v ⁇ 3 integrin was attached by the method in Example 1 B. and yttrium-90 was attached using the method in Example 1 C. Targeting was demonstrated in-vitro using a radioimmunoassay with the ot v ⁇ 3 integrin that is specific for the three component complex. Briefly, 96 well plates coated with the ⁇ v ⁇ 3 integrin (Chemicon International, Inc.) were blocked with BSA. Samples of rabbit serum containing 0-100 micrograms/mL of the anti- ⁇ v ⁇ 3 integrin antibody-liposome-yttrium 90 complex were added and incubated for 1 hour at room temperature. The plate was washed three times with PBST buffer and the yttrium 90 was measured using a Microbeta scintillation counter (Wallac). The standard curve is shown in Figure 2.
  • EXAMPLE 3 Preparation of polymerized vesicles containing a succinylated lipid and the attachment of antibody and yttrium-90 l,2-bis(10,12-tricosadiynoyl)-5n-glycero-3-phosphocholine (Avanti 870016, 400 mg, 0.44 mmol) and l,2-dipalmitoyl-5n-glycero-3-phosphoethanolamine-N-succinate (Avanti 870225, 19 mg, 0.02 mmol) were dissolved in chloroform and the solvent was removed by rotary evaporation.
  • the mixture was hydrated with water (8.3 mL) and 0.5 M NaOH (100 microliters) was added followed by five freeze-thaw cycles where the mixture was repeatedly frozen in a dry ice/acetone bath and thawed at approximately 65°C.
  • the solution was passed through a thermal barrel extruder heated at 95°C (Lipex Biomembranes Inc.) containing 30 nm polycarbonate filters to give a clear solution containing 68 nm vesicles as determined by dynamic light scattering with Brookhaven Instruments ZetaPals particle sizer. A portion of the solution was heated at 90°C overnight.
  • the solution was placed in a 1 x 15 cm round plastic dish and illuminated with UV light at 12.5°C for 3 h using a hand held lamp.
  • the resulting vesicles where 65 nm as determined by dynamic light scattering analysis.
  • Yttrium binding was determined as described in Example 1 by adding approximately 110 microcuries of yttrium-90 to 0.1 mg of lipid vesicle. The binding was found to be 80%.
  • the vesicle- 90 Y complex was incubated in the presence of 1 mM
  • control vesicles containing only l,2-bis(10,12-tricosadiynoyl)->s'7 ⁇ -glycero-3-phosphocholine do not bind yttrium-90 under identical conditions.
  • V2-liposomes from egg PC consist of the anti- ⁇ v ⁇ 3 integrin antibody Vitaxin covalently coupled to the carboxyl groups present on liposomes composed of egg phosphatidylcholine (PC), 1 ,2-dimyristoyl-5w-glycero-3 -phosphoethanolamidotriamine tetraacetic acid (BisM-PE-DTTA4), and cholesterol (65/5/30 mole percent) or Vitaxin coupled to the carboxyl groups on non-UV radiated liposomes prepared from l,2-bis(l 0, 12- tricosadiynoyl)-5 «-glycero-3-phosphocholine (BisT-PC) and a diethylenetriaminetriacetic acid-diacetylene lipid derivative (compound 8a, Journal of the American Chemistry Society, 1995, pp 7301-7306).
  • PC egg phosphatidylcholine
  • BisM-PE-DTTA4 1 ,2-dimyristoy
  • V2-liposomes were labeled with yttrium-90 and incubated at 37°C in rabbit serum for 1, 30, 60, and 180 minutes. At each time point, samples were removed and added to 96-well plates coated with human ⁇ v ⁇ 3 integrin and blocked with BSA prior to the addition of the sample. After a 1 hour incubation at room temperature, the plate was washed three times with PBST buffer and the yttrium signal was determined using a Wallac MicroBeta reader. Results are shown in Figure 3. Liposomes lacking Vitaxin do not generate significant signal in this assay.
  • Rabbits that were selected for treatment were immobilized using a rabbit restrainer and the ear prepared with alcohol (70% isopropyl) for intravenous administration of test samples via the marginal ear vein.
  • a 22-gauge catheter was used for ease of test article administration.
  • Test samples containing antibody-liposome-conjugate or test samples containing this conjugate that are labeled with 90 Y were properly drawn in sterile syringes and injected using a small needle (22-24 gauge). Intravenous injection was performed at a rate of no greater than 0.2 cc/sec. Upon delivery, gauze was applied with pressure to minimize bleeding. Biodistribution data is shown in Figure 6.
  • EXAMPLE 6 Preparation of integrin-targeting liposomes containing an integrin-targeting lipid and ammonium sulfate.
  • RGD peptidomimetic lipid is a lipid having three integrin-binding small molecules having the formula 3- ⁇ 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)-ethyloxy]- benzoylamino ⁇ -2(S)-benzene-sulfonyl-aminopropionic acid covalently attached.
  • RGD peptidomimetic lipid has the following structure:
  • RGD peptidomimetic The preparation of RGD peptidomimetic is described in commonly owned, copending application serial number 10/158,777, filed May 30, 2002, and entitled Targeted Multivalent Macromolecules .
  • l,2-bis(10,12-tricosadiynoyl)-OT-glycero-3-phosphocholine (BisT-PC) 500 mg, 546.9 ⁇ mole, 95 mole %) was weighed into a clean 100 ml round bottom flask.
  • Chelator lipid [PDA-PEG3] 2 DTTA 3 (3.15 ml, 31.5 mg, 23 ⁇ mole, 4 mole %), and RGD peptidomimetic lipid (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.
  • EXAMPLE 7 Attachment of 90 Y to peptidomimetic-vesicle complexes
  • the peptidomimetic-vesicle complexes containing chelator lipid [PDA-PEG3] 2 DTTA 3 are labeled with 90 Y in 50 mM histidine buffer containing 5 mM citrate at pH 7.4 by the following procedure.
  • Yttrium-90 chloride in 50 mM HC1 (NEN Life Science Products) was diluted to a working solution containing approximately 20 mCi/mL.
  • EXAMPLE 8 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.
  • the K1735 M2 tumor cells were grown in tissue culture flasks in Dubelco's medium with 10% fetal calf serum (FCS). Cells were harvested using Trypsin- EDTA solution (containing 0.05%> trypsin), resuspended in PBS at 10,000,000/ml, and kept on ice. Animals with tumors between 100 and 200 mm 3 were selected for treatment as described in Table I.
  • Figure 7 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 8 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 7 summarizes the growth delay data for this study. Again ANOVA and Kruskal-Wallis tests were used to compare TVQT values from different treatment groups. The P-values associated with both tests were highly significant (0.001 for the Kruskal-Wallis test and ⁇ 0.0005 for the ANOVA).
  • Pairwise comparisons of the different treatment groups indicate that treatment with IA-NP-Y90 at higher radiation doses (5 ⁇ Ci./g) is significantly different from buffer, IA and both low and high IA-NP treatments.
  • Table III on the following page shows the results of Tukey's W pairwise comparison procedure. These results were confirmed by non-parametric statistical tests as well.
  • 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 >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- Y90 5 ⁇ Ci g, show inceasing amounts of apotosis and cell death.
  • IA-NP-Y90 at 5 ⁇ Ci/g significantly reduces tumor growth in this tumor model (significance was established at the 95% confidence level).
  • the normalized tumor volume for tumors treated with IA-NP-Y90 at 5 ⁇ Ci/g were less than half the volume when compared to tumors treated with buffer.
  • 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 9 Study of antitumor efficacy of peptidomimetic-dextran-vesicle Y complexes in a mouse melanoma model
  • Dextran coated vesicles were also tested in the mouse melanoma model as described in EXAMPLE 8. Results are shown in Figure 9.
  • dextran-coated vesicles containing BisT-PC and chelator lipid [PDA-PEG3] 2 DTTA 3 were used. They were prepared as follows, and labeled with yttrium-90 as described in Example 7.
  • 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 atpH 8.
  • ED AC Aldrich 16146-2, ethyldimethylaminodipropyl carbodimimide HC1 salt, 6 mg
  • the clear reaction mixture was purified by size exclusion chromatography on a Sepharose CL 4B column (2.5 x 30 cm, Amersham Pharmacia Biotech AB product 17-0150- 01) equilibrated with 10 mM HEPES containing 200 mM NaCl at pH 7.4.
  • 4 ml fractions were collected.
  • 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.
  • EXAMPLE 10 Study of antitumor efficacy of peptidomimetic-vesicle- 90 Y complexes in a mouse colon cancer model.
  • 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 10 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

Abstract

Lipid constructs containing a chelator, a targeting entity, a detectable entity, and/or a therapeutic entity are provided, as well as methods relating to their use. The therapeutic or treatment entity may be associated with the lipid construct by covalent or non-covalent means. In some cases, the therapeutic or treatment entity is a radioisotope.

Description

LIPID CONSTRUCTS AS THERAPEUTIC AND IMAGING AGENTS
FIELD OF THE INVENTION
This invention relates generally to lipid constructs for radiotherapy and imaging. More specifically, this invention relates to liposomes containing a chelator, a targeting entity, a detectable entity, and/or a therapeutic entity, and methods relating to their use. The therapeutic or treatment entity may be associated with the lipid construct by covalent or non- covalent means. In some cases, the therapeutic or treatment entity is a radioisotope.
BACKGROUND OF THE INVENTION
Cancer remains one of the leading causes of death in the industrialized world. In the United States, cancer is the second most common cause of death after heart disease, accounting for approximately one-quarter of the deaths in 1997. Clearly, new and effective treatments for cancer will provide significant health benefits. Among the wide variety of treatments proposed for cancer, targeted therapeutic agents hold considerable promise. In principle, a patient could tolerate much higher doses of a cytotoxic agent if the cytotoxic agent is targeted specifically to cancerous tissue, as healthy tissue should be unaffected or affected to a much smaller extent than the pathological tissue.
Due to the high specificity of monoclonal antibodies, antibodies coupled to cytotoxic agents have been proposed for targeted cancer treatment therapies. Solid tumors, in particular, express certain antigens, on both the transformed cells comprising the tumor and the vasculature supplying the tumors, which are either unique to the tumor cells and vasculature, or overexpressed in tumor cells and vasculature in comparison to normal cells and vasculature. Thus, linking an antibody specific for a tumor antigen, or a tumor vasculature antigen, to a cytotoxic agent, should provide high specificity to the site of pathology. One group of such antigens is a family of proteins called cell adhesion molecules (CAMS), expressed by endothelial cells during a variety of physiological and disease processes. Reisfeld, "Monoclonal Antibodies in Cancer hnmunotherapy," Laboratory Immunology II, (1992) 12(2):201-216, and Archelos et al., "Inhibition of Experimental Autoimmune Encephalomyelitis by the Antibody to the Intercellular Adhesion Molecule
ICAM-1," Arm. of Neurology (1993) 34(2): 145-154. Reisfeld, Archelos, et al, and all other publications, patents, and patent applications referred to herein, are incorporated herein by reference in their entirety. Multiple endothelial ligands and receptors, including CAMs, are known to be upregulated during various pathologies, such as inflammation and neoplasia, and hence are attractive candidates for targeting strategies. Other potential targets are integrins. hitegrins are a group of cell surface glycoproteins that mediate cell adhesion and therefore are mediators of cell adhesion interactions that occur in various biological processes. Integrins are heterodimers composed of noncovalently linked α and β polypeptide subunits. Currently at least eleven different α subunits have been identified and at least six different β subunits have been identified. The various α subunits can combine with various β subunits to form distinct integrins. The integrin identified as αvβ3 (also known as the vitronectin receptor) has been identified as an integrin that plays a role in various conditions or disease states including tumor metastasis, solid tumor growth (neoplasia), osteoporosis, Paget's disease, humoral hypercalcemia of malignancy, angiogenesis, including tumor angiogenesis, retinopathy, arthritis, including rheumatoid arthritis, periodontal disease, psoriasis and smooth muscle cell migration (e.g., restenosis). Additionally, it has been found that such integrin inhibiting agents would be useful as antivirals, antifungals and antimicrobials. Thus, therapeutic agents that selectively inhibit or antagonize θγβ3 would be beneficial for treating such conditions. It has been shown that the αvβ3 integrin binds to a number of Arg-Gly-Asp (RGD) containing matrix macromolecules, such as fibrinogen (Bennett et al., Proc. Natl. Acad. Sci. USA, Vol. 80 (1983) 2417), fibronectin (Ginsberg et al., J. Clin. Invest., Vol. 71 (1983) 619-624), and von Willebrand factor (Ruggeri et al., Proc. Natl. Acad. Sci. USA, Vol. 79 (1982) 6038). Compounds containing the RGD sequence mimic extracellular matrix ligands so as to bind to cell surface receptors. However, it is also known that RGD peptides in general are non- selective for RGD dependent integrins. For example, most RGD peptides that bind to vβ3 also bind to αvβ5, Oyβi, and απbβ a- Antagonism of platelet αnbβma (also known as the fibrinogen receptor) is known to block platelet aggregation in humans.
A number of anti-integrin antibodies are known. Doerr, et al., J. Biol. Chem. 1996 271 :2443 reported that a blocking antibody to αvβ5 integrin in vitro inhibits the migration of
MCF-7 human breast cancer cells in response to stimulation from IGF-1. Gui et al., British J. Surgery 1995 82:1192, report that antibodies against θvβ5 and Ovβi and Oyβs inhibit in vitro chemoinvasion by human breast cancer carcinoma cell lines Hs578T and MDA-MB-231. Lehman et al., Cancer Research 1994 54:2102 show that a monoclonal antibody (69-6-5) reacts with several αv integrins including αvβ3 and inhibited colon carcinoma cell adhesion to a number of substrates, including vitronectin. Brooks et al., Science 1994 264:569 show that blockade of integrin activity with the anti-αvβ monoclonal antibody LM609 inhibits tumor- induced angiogenesis of chick chorioallantoic membranes by human M21-L melanoma fragments. Gutheil et al., Clinical Cancer Research 2000 6:3056 describes a Phase 1 clinical trial with a humanized version of this antibody, Vitaxin, for the treatment of cancer patients . Chuntharapai, et al., Exp. Cell. Res. 1993 205:345 discloses monoclonal antibodies 9G2.1.3 and IOC4.1.3 which recognize the αvβ3 complex, the latter monoclonal antibody is said to bind weakly or not at all to tissues expressing θγβ3 with the exception of osteoclasts and was suggested to be useful for in vivo therapy of bone disease. The former monoclonal antibody is suggested to have potential as a therapeutic agent in some cancers.
Ginsberg et al., U.S. Pat. No. 5,306,620 discloses 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 ctvβ3 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 discloses a protein homologous to the RGD epitope of integrin β subunits and a monoclonal antibody that inhibits integrin-ligand binding by binding to the β3 subunit. That action is said to be of use in therapies for adhesion-initiated human responses such as coagulation and some inflammatory responses .
Carron, U.S. Patent No. 6,171,588, describes monoclonal antibodies which can be used in a method for blocking αvβ3-mediated events such as cell adhesion, osteoclast- mediated bone resorption, restenosis, ocular neovascularization and growth of hemangiomas, as well as neoplastic cell or tumor growth and dissemination. Other uses described are antibody-mediated targeting and delivery of therapeutics for disrupting or killing αvβ3 bearing neoplasms and tumor-related vascular beds. In addition, the inventive monoclonal antibodies can be used for visualization or imaging of αvβ3 bearing neoplasms or tumor related vascular beds by NMR or immunoscintigraphy.
Examples of the targeted therapeutic approach have been described in various patent publications and scientific articles. International Patent Application WO 93/17715 describes antibodies carrying diagnostic or therapeutic agents targeted to the vasculature of solid tumor masses through recognition of tumor vasculature-associated antigens. International Patent Application WO 96/01653 and U.S. Patent No. 5,877,289 describe methods and compositions for in vivo coagulation of tumor vasculature through the site-specific delivery of a coagulant using an antibody, while International Patent Application WO 98/31394 describes the use of
Tissue Factor compositions for coagulation and tumor treatment. International Patent Application WO 93/18793 and U.S. Patent Nos. 5,762,918 and 5,474,765 describe steroids linked to polyanionic polymers which bind to vascular endothelial cells. International Patent Application WO 91/07941 and U.S. Patent No. 5,165,923 describe toxins, such as ricin A, bound to antibodies against tumor cells. U.S. Patent Nos. 5,660,827, 5,776,427, 5,855,866, and 5,863,538 also disclose methods of treating tumor vasculature. International Patent Application WO 98/10795 and WO 99/13329 describe tumor homing molecules, which can be used to target drugs to tumors. hi Tabata, et ah, Int. J. Cancer 1999 82:737-42, antibodies are used to deliver radioactive isotopes to proliferating blood vessels. Ruoslahti & Rajotte, Annu. Rev. Immunol.
2000 18:813-27; Ruoslahti, Adv. Cancer Res. 1999 76:1-20, review strategies for targeting therapeutic agents to angiogenic neovasculature, while Arap, et al., Science 1998 279:377-80 describe selection of peptides which target tumor blood vessels.
It should be noted that the typical arrangement used in such systems is to link the targeting entity to the therapeutic entity via a single bond or a relatively short chemical linker.
Examples of such linkers include SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane- 1-carboxylate) or the linkers disclosed in U.S. Patent No. 4,880,935, and oligopeptide spacers. Carbodiimides and N-hydroxysuccinimide reagents have been used to directly join therapeutic and targeting entities with the appropriate reactive chemical groups. The use of cationic organic molecules to deliver heterologous genes in gene therapy procedures has been reported in the literature. Not all cationic compounds will complex with DNA and facilitate gene transfer. Currently, a primary strategy is routine screening of cationic molecules. The types of compounds which have been used in the past include cationic polymers such as polyethyleneamine, ethylene diamine cascade polymers, and polybrene. Proteins, such as polylysine with a net positive charge have also been used. The largest group of compounds, cationic lipids; includes DOTMA, DOTAP, DMPJE, DC-chol, and DOSPA. All of these agents have proven effective but suffer from potential problems such as toxicity and expense in the production of the agents. Cationic liposomes are currently the most popular system for gene transfection studies. Cationic liposomes serve two functions: protect DNA from degradation and increase the amount of DNA entering the cell.
While the mechanisms describing how cationic liposomes function have not been fully delineated, such liposomes have proven useful in both in vitro and in vivo studies. However, these liposomes suffer from several important limitations. Such limitations include low transfection efficiencies, expense in production of the lipids, poor colloidal stability when complexed to DNA, and toxicity.
Although conjugates of targeting entities with therapeutic entities via relatively small linkers have attracted much attention, far less attention has been focused on using large particles as linkers. Typically, the linker functions simply to connect the therapeutic and targeting entities, and consideration of linker properties generally focuses on avoiding interference with the entities linked, for example, avoiding a linkage point in the antigen binding site of an immunoglobulin.
Large particulate assemblies of biologically compatible materials, such as liposomes, have been used as carriers for administration of drugs and paramagnetic contrast agents. U.S. Patent Numbers 5,077,057 and 5,277,914 teach preparation of liposome or lipidic particle suspensions having particles of a defined size, particularly lipids soluble in an aprotic solvent, for delivery of drugs having poor aqueous solubility. U.S. Patent No. 4,544,545 teaches phospholipid liposomes having an outer layer including a modified, cholesterol derivative to render the liposome more specific for a preselected organ. U.S. Patent No. 5,213,804 teaches liposome compositions containing an entrapped agent, such as a drug, which are composed of vesicle-forming lipids and 1 to 20 mole percent of a vesicle-forming lipid derivatized with hydrophilic biocompatible polymer and sized to control its biodistribution and recirculatory half life. U.S. Patent No. 5,246,707 teaches phospholipid coated microcrystalline particles of bioactive material to control the rate of release of entrapped water soluble biomolecules, such as proteins and polypeptides. U.S. Patent No. 5,158,760 teaches liposome encapsulated radioactive labeled proteins, such as hemoglobin. U.S. Patent Nos. 5,512,294 and 6,090,408, and 6,132,764 (the contents of which are hereby incorporated by reference herein), describe the use of polymerized liposomes for various biological applications. One listed embodiment is to targeted polymerized liposomes which may be linked to or may encapsulate a therapeutic compound, (e.g., proteins, hormones or drugs) for directed delivery of a treatment agent to specific biological locations for localized treatment. Other publications describing liposomal compositions include U.S. Patent Nos. 5,663,387, 5,494,803, and 5,466,467, to liposomes containing polymerized lipids for non-covalent immobilization of proteins and enzymes; 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; Wu et al., "Metal-Chelate-Dendrimer-Antibody Constructs for Use in Radioimmunotherapy and Imaging," Bioorganic and Medicinal Chemistry Letters (1994) 4(3):449-454 is a publication directed to dendrimer-based compounds.
The need for recirculation of therapeutic agents in the body, that is avoidance of rapid endocytosis by the reticuloendothelial system and avoidance of rapid filtration by the kidney, to provide sufficient concentration at a targeted site to afford necessary therapeutic effect has been recognized. Experience with magnetic resonance contrast agents has provided useful information regarding circulation lifetimes. Small molecules, such as gadolinium diethylenetriaminepentaacetic acid, tend to have limited circulation times due to rapid renal excretion while most liposomes, having diameters greater than 800 nm, are quickly cleared by the reticuloendothelial system. Attempts to solve these problems have involved use of macromolecular materials, such as gadolinium diethylenetriaminepentaacetic acid derived polysaccharides, polypeptides, and proteins. These agents have generally not demonstrated the versatility in chemical modification to provide for both long recirculation times and active targeting.
Imaging Magnetic resonance imaging (MRI) 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 TI (longitudinal) and T2 (transverse) relaxation values and the proton density, which generally corresponds to the free water content, of the tissues. To change the signal intensity in a region of a patient by the use of a contrast medium, several possible approaches are available. For example, a contrast medium may be designed to change either the TI, the T2 or the proton density. Generally speaking, 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. In the past, attention has focused primarily on paramagnetic contrast agents for MRI. 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 (TI) and transverse (T2) relaxation. Paramagnetic contrast agents typically comprise metal ions, for example, transition metal ions, which provide a source of unpaired electrons. However, 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. In an effort to decrease toxicity, 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. Ultrasound provides certain advantages over other diagnostic techniques. For example, diagnostic techniques involving nuclear medicine and X-rays generally involves 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. In addition, ultrasound is relatively 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, generally referred to as backscatter or reflectivity, forms the basis for developing an ultrasound image. In this connection, 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. As with the diagnostic techniques discussed above, 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, discloses stabilized microbubble-type ultrasonic imaging agents produced from heat-denaturable biocompatible protein, for example, albumin, hemoglobin, and collagen.
The reflection of sound from a liquid-gas interface is extremely efficient. Accordingly, liposomes or vesicles, including gas-filled bubbles, are useful as contrast agents. As discussed more fully hereinafter, the effectiveness of liposomes as contrast agents depends upon various factors, including, for example, the size and/or elasticity of the bubble.
Many of the liposomes disclosed in the prior art have undesirably poor stability. Thus, the prior art liposomes are more likely to rupture in vivo resulting, for example, in the untimely release of any therapeutic and/or diagnostic agent contained therein. Various studies have been conducted in an attempt to improve liposome stability. Such studies have included, for example, the preparation of liposomes in which the membranes or walls thereof comprise proteins, such as albumin, or materials which are apparently strengthened via crosslinking. See, e.g., Klaveness et al., WO 92/17212, in which there are disclosed liposomes which comprise proteins crosslinked with biodegradable crosslinking agents. A presentation was made by Moseley et al., at a 1991 Napa, California meeting of the Society for Magnetic Resonance in Medicine, which is summarized in an abstract entitled "Microbubbles: A Novel
MR Susceptibility Contrast Agent." The microbubbles described by Moseley et al. comprise air coated with a shell of human albumin. Alternatively, membranes can comprise compounds which are not proteins but which are crosslinked with biocompatible compounds. See, e.g., Klaveness et al., WO 92/17436, WO 93/17718 and WO 92/21382. Liposomal imaging agents should ideally be prepared with a simple and efficient labeling procedure with an affordable labeling group, should be stable in vivo with no release of free label, and be stable upon storage. Conjugation of radionuclides to liposomes has been investigated primarily to develop of advanced imaging and diagnostic modalities for γ- scintigraphy, single photon emission tomography (SPECT) or positron emission tomography (PET). Liposomes have been labeled with radionuclides, most commonly 99m-Tc, 111-In, and 67-Ga, by conjugation to a chelator or other suitable anchoring molecule contained within the aqueous interior of the liposome or within the lipid bilayer. Efficient labeling (> 95%) with 99m-Tc is now routine, using the bifunctional chelator HYNIC conjugated to lipids, such as distearoylphosphatidylethanolamine (DSPE). Other techniques for labeling liposomes include simple entrapment within the liposome interior. Entrapment may occur using ionophores, such as A23187, or during liposome formation. An example of the use of A23187 for the encapsulation of an isotope in a liposome includes indium-111 labeled VesCan ((Kubo et al, Eur. J. Nucl. Med (1993), 20(2), 107-113). Entrapment requires purification of the liposomes containing the encapsulated isotope from non-encapsulated isotope by chromatography. Surface labeling of liposomes with Tc99m has been reported by Ahkong & Tilcock, Nucl. MedBiol. 1992, 19:831-840, using the lipophilic chelator dipalmitoylphosphatidylethanolamine-DTTA. A disadvantage of the Tilcock reference is that labeling the DTTA chelator, a derivative of DTP A, with Tc99m is that chromatography is required to separate free and colloidal forms of technetium from the liposome-Tc99m complexes.
Utilization of liposomes as carriers of radionuclides for therapeutic applications, has not been widely reported. One major hurdle in this area is the efficient labeling of liposomes with therapy nuclides. One strategy is described in Hafeli, et al., Nucl. Med. Biol. (1991) 18:449-54. In Hafeli, et al., liposomes with a 70 nm diameter were made by the detergent removal technique on a gel filtration column, and a radioactive Re complex was incorporated into the bilayer of the liposomes during liposome formation. The stablility of these radioactive liposomes was tested by dialysis, and a loss of 40% of the radioactivity identified as perrhenate was observed after 8 days. Addition of the antioxidant ascorbic acid diminished the loss to 20%. Hafeli, et al., suggest that liposomes carrying the lipophilic radioactive Re- complex can potentially be used in beta-radiotherapy.
Another report, Utkhede, et al., J Liposome Res. (1994) 4: 1049-1061, describes 90-Y entrapment into SUV's and PEG-coated liposomes via the cation ionophore A23187. After transport across the lipid bilayer, 90-Y was chelated in the vesicle interior by DTPA. No loading occurs at 40°C, and 89.2-95.9% loading occurs at 41-50°C. No in vivo biodistribution studies were reported, nor any dosimetry studies to assess the therapuetic potential of the liposomes.
Although various liposome systems have been presented that exhibit preferential tumor localization, efficient for chemotherapeutic and imaging purposes, very little work has been described investigating the possible therapeutic effects of liposome delivered particle- emitting radionuclides to tumors. Kosterelos, et al., J. Liposome Res. (1999) 9:407-24, reviewed the use of liposomes for imaging and therapy. The report states that "there has not been a single study in the literature utilizing liposomes as carriers of radionuclides for therapeutic applications" (although this is not strictly true) and further suggests that the success or failure of any radiotherapeutic modality will be critically dependent on its proper dosimetry assessment. In a more recent theoretical publication by Kostarelos & Emfietzoglou, Anticancer Res. (2000) 20:3339-45, dosimetry estimates for liposomes containing various isotopes were calculated from previously reported biodistribution data for liposome-isotope complexes. Multilamellar (MLV), small unilamellar (SUV) and sterically stabilized (GMl- and PEG-coated) liposomes were examined in combination with the particle emitting radionuclides 67-Cu, 188-Re and 211-At, 90-Y and 131-1. Regardless of radionuclide, the poorest values were obtained for the MLV liposomes. Sterically stabilized (GM-coated) liposomes are taken up by the muscle tumor tissue more readily than are SUVs. As a result, 211-At and 188-Re deliver higher tumor doses when combined with the former, but 67-Cu, 90-Y and 131-1 are more effective with the latter. Kostarelos & Emfietzoglou conclude that the importance of liposome size and steric barrier when designing effective radionuclide-carrier systems, as well as optimal matching between the radionuclide half-life and the time of maximum liposome accumulation ratio between tumor and normal tissue, are important considerations. A description of the use of 90Y-liposome complexes for therapy was not provided in this theoretical report. Bard, et al., Gin. Exp. Rheumatol. (1985) 3:237-42, have studied the effect of the intra-articular injection of lutetium-177 in chelator liposomes on the progress of an experimental arthritis in rabbits. The liposomes were prepared by combining 3-cholesteryl 6- [N'-iminobis(ethylenenitrilo)tetraacetic acid acidjhexyl ether (Chol-DTTA) with DSPC and a radioactive isotope, either 51-Cr or 177-Lu. The treatment of rheumatoid arthritis by radiosynovectomy has been restricted by the difficulty of preventing leakage of the radioisotope from the joint cavity. In this study, liposomes were prepared with 3-cholesteryl 6-[N'-iminobis(ethylenenitrilo)-tetraacetic acidjhexyl ether (Chol-DTTA) which can complex with a.variety of beta-emitting radionuclides. In a previous study, Bard, et al., Gin. Exp. Rheumatol. (1983) 1:113-7, 51-Cr was used as the radioisotope. The liposomes were injected into the knee joint cavity of rabbits with expertimentally induced arthritis. For the 51-Cr liposomes, greater than 99% of the radioactivity was retained in the joint after 24 hours, with 93% of the radioactivity associated with the synovium (the membrane that covers synovial joints and secretes synovial fluid, and lubricates the joints), hi the case of 177-Lu, reported losses of radioactivity averages less than 1% over 47 days, and that low radiation dose resulted in very little synovitis with no damage to the knee cartilage.
The preliminary results of Kosterelos, et al. and Bard, et al., are encouraging, but in general, therapeutic applications of therapy radionuclides in conjunction with liposomes have been ignored. Accordingly, there remains a need for methods of preparation of stable liposomes suitable for delivery of therapeutic radionuclides in a variety of applications.
Furthermore, there remains a need for methods of preparation of stable liposomes containing a targeting agent for the delivery of therapeutic radionuclides. There is a further need for methods of preparation of stable liposomes containing a targeting agent and an imaging agent along with a therapeutic isotope.
SUMMARY OF THE INVENTION
The present invention provides a lipid construct comprising a chelator, a targeting entity, and a detectable entity or a therapeutic entity. The lipid construct may include lipids, including phosphatidylcholine derivatives. The lipid constructs can also include additional components, such as cholesterol or stabilization agent, such as polyethylene glycol. In preferred embodiments, the therapeutic entity is a radionuclide, such as Y-90, 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. The therapeutic agent may be associated with the surface of the lipid construct or encapsulated within the lipid construct. In preferred embodiments the chelator is part of a chelating lipid, such as 1,2- dimyristoyl-5«-glycero-3-phosphoethanolamidotriamine tetraacetic acid (BisM-PE-DTPA4). Y-90, or N,N-bis[[[[(13',15'-ρentacosadiynamido-3,6- doxaoctyl)carbamoyl]methyl] (carboxymethyl) amino] ethyl] glycine ([PD A-PEG3 ]2-DTTA3 ), derivatives of diethylenetriaminepentaacetic acid, or derivatives of ethylaminediaminetetracetic acid, and a derivative of 1, 4,7,10-tetraazacyclododecane- N,N',N",N" '-tetraacetic acid (DOTA).
In some embodiments the chelating lipid contains a diacetylene lipid or a polymerizable lipid such as l,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine or The lipid construct of Claim 17 or 18, wherein the polymerizable lipid is [PDA-PEG3]2-
DTTA3.
The present invention further provides lipid constructs containing chelating lipids that comprise an ionizable group such as carboxyl, phosphate, phosphonate, sulfate, sulfonate, or sulfinate, ionizable groups generating a surface capable of binding an isotope or metal with a valency of +2 or greater, and ionizable groups generating a surface capable of binding an isotope or metal with a valency of +3 or greater.
The targeting entity of the lipid construct may include a small molecule ligand and a protein, and in some embodiments targets the lipid construct to a cell surface. In other embodiments, the targeting entity is associated with a carboxyl head group of said lipid, maleimide group of a lipid, the alpha-methyl group of an acetamide of a lipid, or other covalent means, such as amine, cyano, carboxylic acid, isothiocyanate, thiol, disulfide, α- halocarbonyl, α,β-unsaturated carbonyl or alkyl hydrazine.
In some embodiments, the detectable entity is a radionuclide, such as Tc-99m, In-111, Ga-67, Rh-105, Nd -147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, or Tl- 201.
In other embodiments, the targeting entity is associated with the lipid construct by non-covalent means such as a biotin-avidin biotinylated antibody sandwich.
In some embodiments, the targeting entity is an antibody such as an antibody having a target selected from the group consisting of P-selectin, E-selectin, pleiotropin, G-protein coupled receptors, endosialin, endoglin, VEGF receptor, PDGF receptor, EGF receptor, the matrix metalloproteases, and prostate specific membrane antigen (PSMA).
In other embodiments, the targeting entity has a vascular target, such as the targeting entities Vitaxin or LM609.
In other embodiments, the targeting entity is an anti-VCAM-1 antibody, an anti- ICAM-1 antibody, or an anti-integrin antibody. In other embodiments, the targeting entity has a targeting entity having a tumor cell target.
The present invention also provides a therapeutic agent comprising a lipid construct, said lipid construct comprising a chelating lipid, a targeting entity, and a therapeutic entity, wherein the therapeutic entity is associated with the chelating lipid at the surface of said lipid construct. The therapeutic agent may include a therapeutic entity that is a metal ion, including a radioactive metal ion such as Y-90, 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, or Re-188, a radiation synovectomy agent. The present invention also provides an imaging agent comprising a lipid construct, the lipid construct comprising a chelating lipid, a targeting entity, and a detectable entity, wherein the detectable entity is associated with the chelating lipid at the surface of said lipid construct. The imaging agent may include an imaging entity that is a metal ion, including a radioactive metal ion, such as Tc-99m, In-I ll, Ga-67, Rh-105, Nd -147, Pm-151, Sm-153, Gd-159, Tb- 161, Er-171, Re-186, Re-188, and Tl-201.
The present invention also provides a method for preparing a lipid construct comprising preparing a liposome comprising a chelating lipid, and contacting the liposome with a metal ion, including a radioactive metal ion, including Y-90, 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 (first group of metal ions), Tc-99m, In-I ll, Ga-67, Rh-105, 1-
123, Nd -147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, and Tl-201 (second group of metal ions).
The method may further include contacting the liposome with a second metal ion, including a method where the first metal ion is a metal ion from the first group, and the second metal ion is a metal ion from the second group. The two metal ions may reside in the same mixture. The method may further comprising removing unbound metal ion.
The present invention also provides a method of imaging a patient comprising administering an imaging agent to a patient in need thereof, said imaging agent comprising a lipid construct, said lipid construct comprising a chelating lipid and a detectable entity, and imaging the patient. The imaging may include magnetic resonance imaging or nuclear scintigraphy.
The present invention further 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 lipid construct, said lipid construct comprising a chelating lipid and a therapeutic entity. In some embodiments, the patient suffers from rheumatoid arthritis.
The present invention also provides a lipid construct useful for therapy and imaging, comprising a chelating lipid, a targeting entity, a detectable entity, and a therapeutic entity, wherem said detectable entity and therapeutic entity are associated with said chelating lipid at the surface of said lipid construct, hi some embodiments, the therapeutic entity is a metal ion from the first group of metal ions listed above, and the detectable entity is a metal ion from the the second group of metal ions listed above.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the purity of an antibody-liposome conjugate by size exclusion chromatography and ELISA. In this figure, the signal from an ELIS A performed on each fraction is plotted as a function of fraction number after purification of the conjugate using a Sepharose CL-4B column. The larger antibody-liposome complex elutes in fractions 2-6 and the unbound antibody elutes in fractions 7-12 (data not shown).
Figure 2 shows the response in an αvβ3 -specific radioimmunoassay for the anti- vβ3 antibody-liposome-yttrium-90 complex in serum. This assay generates signal when the antibody and the yttrium-90 are associated with the same vesicle and no signal originates from unbound yttrium-90. Figure 3 shows some of the lipid structures used to prepare liposomes.
Figure 4 shows stability of Vitaxin-liposome-90Y conjugates in rabbit serum.
Figure 5 shows additional lipid structures used to prepare liposomes. These lipids are prepared from tricosadiynoic acid and the hydrophilic linker l,8-diamino-3,6-dioxaoctane, except for BisT-PE-DTTA4, which does not contain the linker. Figure 6 shows the biodistribution of Vitaxin-liposome-90Y conjugates in healthy New
Zealand rabbits.
Figure 7 shows the efficacy of integrin-targeted vesicles labeled with yttrium 90 (IA-NP-Y90) in the mouse melanoma model as described in Example 29. Treatment groups include LA (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 8 shows the normalized tumor volume 7 days post treatment sorted by treatment group for the study described in Example 8. Figure 9. 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 9.
Figure 10 shows efficacy in the mouse colon cancer model as described in Example 10. Error bars indicate ± one standard error. Treatment groups include buffer, PM (RGD peptidomimetic alone), 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 11 : Plot of normalized tumor volume on day 8 sorted by group for the study in Example 10.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is directed toward a lipid construct comprising a lipid chelator, a targeting entity, and a therapeutic and/or a detectable entity.
The present invention is also directed toward methods of preparation of the lipid constructs of the present invention. The present invention is further directed towards lipid constructs containing one or more chemically distinct lipids. The present invention is also directed toward therapeutic agents comprising the lipid constructs of the present invention. The present invention is further directed toward a method of administering a therapeutic agent of the present invention to a patient in need thereof. The present invention is yet further directed towards the treatment of diseases where the vasculature associated with the disease may be treated or targeted with a targeted therapeutic agent. Suitable targets are ICAM, VCAM, integrins, P-selectin, E-selectin, pleiotropin, endosialin, endoglin, VEGF receptor, PDGF receptor, EGF receptor, the matrix metalloproteases, G-protein coupled receptors, and prostate specific membrane antigen (PSMA). The present invention is even further directed towards the treatment of cancer, arteriosclerosis, rheumatoid arthritis, and osteoporosis.
The present invention is also directed toward imaging agents comprising the lipid constructs of the present invention, and a method of imaging a patient comprising administering an imaging agent of the present invention to a patient, and imaging the patient.
The present invention is also directed towards agents which may be used for both therapy and imaging. The present invention is further directed to methods and reagents for diagnosis using the lipid constructs of the present invention.
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. In the preferred embodiment, the lipid construct is a liposome. Common additional components in lipid constructs include cholesterol and alpha-tocopherol, among others. The lipid constructs may be used alone or in any combination which one skilled in the art would appreciate to provide the characteristics desired for a particular application. In addition, the technical aspects of lipid constructs and liposome formation are well known in the art and any of the methods commonly practiced in the field may be used with the present invention.
Liposomes
As used herein, lipid refers to an agent exhibiting amphipathic characteristics causing it to spontaneously adopt an organized structure in water wherein the hydrophobic portion of the molecule is sequestered away from the aqueous phase. A lipid in the sense of this invention is any substance with characteristics similar to those of fats or fatty materials. As a rule, molecules of this type possess an extended apolar region and, in the majority of cases, also a water-soluble, polar, hydrophilic group, the so-called head-group. Phospholipids are lipids which are the primary constituents of cell membranes. Typical phospholipid hydrophilic groups include phosphatidylcholine 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. Lipid bilayer vesicle, as used herein, refers to a closed, fluid-filled microscopic sphere which is formed principally from individual molecules having polar (hydrophilic) and non-polar (lipophilic) portions. The hydrophilic portions may comprise phosphate, glyceryophosphate, carboxy, sulfate, amino, hydroxy, choline and other polar groups and derivatives thereof.
Examples of non-polar groups are saturated or unsaturated hydrocarbons such as alkyl, alkenyl or other lipid groups. Sterols (e.g., cholesterol) and other pharmaceutically acceptable components (including anti-oxidants like alpha-tocopherol) may also be included to improve vesicle stability or confer other desirable characteristics. Additionally, lipids to which a targeting agent, such as a ligand, peptidomimetic, peptide, or other synthetic molecule, may be incorporated into liposomes by preparing mixtures of the targeting lipid or lipids with additional chemically distinct lipids. One or more targeting lipid may be mixed with other chemically distinct lipids.
Bilayer structures can also be formed into closed spherical shell-like structures which are called vesicles or liposomes. The liposomes employed in the present invention can be prepared using any one of a variety of conventional liposome preparatory techniques. As will be readily apparent to those skilled in the art, such conventional techniques include sonication, chelate dialysis, homogenization, solvent infusion coupled with extrusion, freeze- thaw extrusion, microemulsification, as well as others. These techniques, as well as others, are discussed, for example, in U.S. Pat. No. 4,728,578, U.K. Patent Application G.B. 2193095 A,
U.S. Pat. No. 4,728,575, U.S. Pat. No. 4,737,323, International Application PCT/US85/01161, Mayer et al., Biochimica et Biophysica Acta, Vol. 858, pp. 161-168 (1986), Hope et al., Biochimica et Biophysica Acta, Vol. 812, pp. 55-65 (1985), U.S. Pat. No. 4,533,254, Mahew et al., Methods In Enzymology, Vol. 149, pp. 64-77 (1987), Mahew et al., Biochimica et Biophysica Acta, Vol. 75, pp. 169-174 (1984), and Cheng et al., Investigative
Radiology, Vol. 22, pp. 47-55 (1987), and U.S. Ser. No. 428,339, filed Oct. 27, 1989. The disclosures of each of the foregoing patents, publications and patent applications are incorporated by reference herein, in their entirety. A solvent free system similar to that described in International Application PCT/US85/01161, or U.S. Ser. No. 428,339, filed Oct. 27, 1989, may be employed in preparing the liposome constructions. By following these procedures, one is able to prepare liposomes having encapsulated therein a gaseous precursor or a solid or liquid contrast enhancing agent.
The materials which may be utilized in preparing the liposomes of the present invention include any of the materials or combinations thereof known to those skilled in the art as suitable in liposome construction. The lipids used may be of either natural or synthetic origin. Such materials include, but are not limited to, lipids such as cholesterol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, lysolipids, fatty acids, sphingomyelin, glycosphingolipids, glucolipids, glycolipids, sulphatides, lipids with amide, ether, and ester- linked fatty acids, polymerizable lipids, and combinations thereof. Additionally, the present invention includes lipid derivatives containing carboxyl, phosphate, phosphonate, sulfate, sulfonate, and sulfinate groups. As one skilled in the art will recognize, the liposomes may be synthesized in the absence or presence of incorporated glycolipid, complex carbohydrate, protein or synthetic polymer, using conventional procedures. The surface of a liposome may also be modified with a polymer, such as, for example, with polyethylene glycol (PEG), using procedures readily apparent to those skilled in the art. Lipids may contain functional surface groups for attachment to a metal, which provides for the chelation of radioactive isotopes or other materials that serve as the therapeutic entity. Any species of lipid may be used, with the sole proviso that the lipid or combination of lipids and associated materials incorporated within the lipid matrix should form a bilayer phase under physiologically relevant conditions.
As one skilled in the art will recognize, the composition of the liposomes may be altered to modulate the biodistribution and clearance properties of the resulting liposomes.
The present invention is also directed toward methods of preparation of the lipid constructs of the present invention. When preparing liposomes having a mixed lipid composition, the lipids may first be dissolved and mixed in an organic solvent, for example, chloroform or chloroform:methanol mixtures, to assure a homogeneous mixture of lipids. Typically lipid solutions are prepared at 10-20 mg lipid ml organic solvent, although higher concentrations may be used if the lipid solubility and mixing are acceptable. Once the lipids are thoroughly mixed in the organic solvent, the solvent is removed to yield a lipid film, either by using a dry nitrogen or argon stream in a fume hood or rotary evaporation, followed by removal of residual organic solvent by placing the vial or flask on a vacuum pump overnight. Alternatively, the lipids may be dissolved in a solvent that may be frozen and lyophilized. The lipid solution is transferred to containers and frozen by, for example, placing the containers on a block of dry ice or swirling the container in a dry ice-acetone or alcohol (ethanol or methanol) bath. After freezing completely, the frozen lipid cake is placed on a vacuum pump and lyophilized until dry, typically one to three days depending on volume.
Hydration of the dry lipid film is accomplished simply by adding an aqueous medium to the container of dry lipid and agitating. The temperature of the hydrating medium should be above the gel-liquid crystal transition temperature (Tc or Tm) of the lipid with the highest Tc before adding to the dry lipid. The hydration medium is generally determined by the application of the lipid vesicles. Suitable hydration media include distilled water, buffer solutions, saline, and nonelectrolytes such as sugar solutions. Physiological osmolality (290 mOsm/kg) is recommended for in vivo applications. Generally accepted solutions which meet these conditions are 0.9% saline, 5% dextrose, and 10% sucrose. The product of hydration is a large, multilamellar vesicle (LMV) analogous in structure to an onion, with each lipid bilayer separated by a water layer. The spacing between lipid layers is dictated by composition with polyhydrating layers being closer together than highly charged layers which separate based on electrostatic repulsion. Once a stable, hydrated MLV suspension has been produced, the particles can be downsized by a variety of techniques, including sonication, extrusion, or high-shear homogenization. In one embodiment, the present invention is directed toward a method for preparing lipid constructs comprising preparing a lipid mixture, preparing a lipid construct from the lipid mixture, and curing the lipid construct. Preparing a lipid construct may mean any technique or techniques suitable for preparing the desired lipid construct. For example, a freeze/thaw procedure performed upon hydration is known to produce multilamellar vesicles.
Extrusion may be used to create particles of a particular size range. In the present invention, extrusion is a preferred method of downsizing. Curing, as defined herein, refers to heating the lipid constmct to a temperature effective to impart increased stability in the lipid construct for a time effective to impart increased stability to the lipid construct, hi one embodiment, curing comprises heating at about 80-90 °C for about 16-18 hours. Curing provides some stability to the vesicles in the presence of proteins and salt. MLVs may be prepared by repeated freeze- thaw cycles, or by the reversed-phase evaporation procedure (see Liposome Technology, Volume I, "Liposome Preparation and Related Techniques", G. Gregoriadis (ed), CRC Press, Boca Raton, 1993). Micelles may be prepared using lipid tails consisting of less than 10 carbon atoms, and these tails may contain alkynes, alkenes, or alkanes. In a further embodiment, where the liposome is prepared with polymerizable lipids, the liposome may be converted to a polymerized liposome.
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). Accordingly, the particles can be micelles, as well as particles which can be sized at 30, 50, 100, 150, 200, 250, 300 or 350 nm in size, as desired. In addition, the size of the particles can be chosen so as to permit a first administration of particles of a size that cannot pass through the permeable vasculature, followed by one or more additional administrations of particles of a size that can pass through the permeable vasculature. The size of the vesicles may preferably range from about 1 nanometer (nm) to about 400 nm in diameter, and all combinations and subcombinations of ranges therein. More preferably, the vesicles have diameters from about 1 nm to about 500 nm, with diameters from about 40 nm to about 120 nm being even more preferred. In connection with particular uses, for example, intravascular use, including magnetic resonance imaging of the vasculature, it may be preferred that the vesicles be no larger that about 500 nm in diameter, with smaller vesicles being no larger than about 60-80 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.
Either as MLVs or UVs, liposomes have proven valuable as vehicles for drug delivery in animals and in humans. Active drugs, including small hydrophilic molecules and polypeptides, can be trapped in the aqueous core of the liposome, while hydrophobic substances can be dissolved in the liposome membrane. Other molecules, such as DNA or RNA, may be associated with the outside of the liposome for gene therapy 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 through endocytosis. The invention may also utilize vesicles that bind to the target site and deliver a therapeutic agent to the desired site without internalization. In this case, the therapeutic agent may be a radioisotope that irradiates surrounding cells and cell layers. The therapeutic agent may also be a drug or pro-drug that is released while the invention is bound to the desired site. For these requirements the formulations preferably utilize UVs having an average diameter of less than 200 nm, more preferably less than 100 nm, and even more preferably about 60-80 nm.
In preferred embodiments, the liposomes of the present invention comprise egg or soy phosphatidylcholine and cholesterol. In other preferred embodiments, liposomes comprise stabilization agents such as polyethylene glycol
Lipid constructs of the present invention also comprise a chelator. In a preferred embodiment, the chelator is part of a chelating lipid. As used here, a chelating lipid is one in which a chelator is chemically associated with a lipid of which the lipid construct is comprised. In a preferred embodiment, the chelating lipid is l,2-dimyristoyl-5,«-glycero-3- phosphoethanolamidotriamine tetraacetic acid (BisM-PE-DTPA4). This name is a common name that refers to the tetraacetic acid containing, phosphoethanolamine lipid derivative that is associated with DTPA by an amide bond where DTPA is the organic molecule diethylenetriaminepentaacetic acid. Further embodiments of the present invention comprise lipis constructs containing an encapsulated chelator, such as DTPA or DOTA.
Lipid constructs of the present invention also optionally include polymerizable lipids, which result in a lipid construct that is a polymerized liposome. Some preferred polymerizable lipids are [PDA-PEG3J2-DTTA3, described as N,N-bis[[[[(13 l5'- pentacosadiynamido-3,6-doxaoctyl)carbamoyl]methyl](carboxymethyl)amino]ethyl]glycine (compound 8a in J4CS 1995, 117(28), 7301-7306) and l,2-bis(10,12-tricosadiynoyl)-OT- glycero-3-phosphocholine (BisT-PC).
The present invention also contemplates lipid constructs of the present invention further comprising compounds, such as, for example, drugs or other therapeutic agents, or imaging agents, encapsulated within the lipid constructs of the present invention. Methods for encapsulation of such entities are well known in the prior art. Agents which may be encapsulated include radionuclides which are discussed elsewhere herein. The radionuclide could be chelated inside the liposome by encapsulated DOTA, DTPA, or an encapsulated macromolecule that has a chelator associated with it. Another method for encapsulation is analogous to the use of salt gradients used to encapsulate doxorubicin as described in co- pending united states patent application serial no. 10/158,777, filed May 30, 2002, and entitled Targeted Multivalent Macromolecules. In general, it is possible to add radionuclide to a solution of lipid constructs containing encapsulated salts, where the bulk solution has a much lower concentration of the salts. This concentration difference may be used to load the liposome by precipitation of the radionuclide-salt complex inside the liposome. Examples of salts may be phosphates, sulfates, sulfites, phosphonates, carbonates. Polyanionic polymers or ionophores may also be encapsulated to generate the appropriate gradient.
Therapeutic Entities The term "therapeutic entity" refers to any molecule, molecular assembly or macromolecule that has a therapeutic effect in a treated subject, where the treated subject is an animal, preferably a mammal, more preferably a human. The term "therapeutic effect" refers to an effect which reverses a disease state, arrests a disease state, slows the progression of a disease state, ameliorates a disease state, relieves symptoms of a disease state, or has other beneficial consequences for the treated subject. Therapeutic entities include, but are not limited to, drugs, such as doxorubicin and other chemotherapy agents; toxins such as ricin; radioactive isotopes; 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. The therapeutic or treatment entity may be associated with the lipid construct by covalent or non-covalent means. As used herein, associated means associated with the liposome by covalent or noncovalent interactions.
The present invention is also directed toward a therapeutic entity comprising the lipid constructs of the present invention. In a preferred embodiment, the therapeutic agent is a radionuclide. As used herein, a therapeutic radionuclide is a nuclide which undergoes spontaneous transformation (nuclear decay) with an energy transfer sufficient to impart cytotoxic amounts of radiant energy to nearby cells. In contrast, radionuclides useful for diagnosis emit radiation capable of penetrating tissue with minimal cell damage. Such radiation may be detected using a suitable scintigraphic imager. Therapeutic radionuclides of the present invention include, but are not limited to 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. Diagnostic or imaging nuclides of the present invention include, but are not limited to Tc-99m, In-Ill, Ga-67, Rh-105, 1-123, Nd -147, Pm-151, Sm- 153, Gd-159, Tb-161, Er-171, Re-186, Re-188, and Tl-201.
In a preferred embodiment, a therapeutic radionuclide is associated with the lipid construct by non-covalent means. In a particularly preferred embodiment, the therapeutic radionuclide is associated with a chelating lipid. In another particularly preferred embodiment, yttrium-90 is the therapeutic radionuclide, and l,2-dimyristoyl-5«-glycero-3- phosphoethanolamidotriamine tetraacetic acid as defined above is the chelating lipid. Other chelating lipids which are preferred are lipid derivatives of diethylenetriaminepentaacetic acid including diethylenetriaminetetraacetic acids and diethylenetriaminetriacetic acids, derivatives of ethylaminediaminetetracetic acid, and derivatives of 1,4,7, 10-tetraazacyclododecane- N,N',N",N" '-tetraacetic acid (DOTA). Additionally, other lipids containing ionizable groups including carboxyls (such as nitrilotriacetic acid iminodiacetic acid, for example), phosphates, phosphonates, sulfates, sulfonates, and sulfmates may be preferred. In another preferred embodiment, lipids containing a single ionizable group may self assemble to generate a surface capable of binding an isotope or metal with a valency of +2 or greater. In another preferred embodiment, lipids containing two ionizable groups may self assemble to generate a surface capable of binding an isotope or metal with a valency of +3 or greater. In either of these embodiments, a single metal ion can bind to 2 or more lipid head groups. Additional chelators suitable for use in the present invention include those disclosed in Liu and Edwards, "Bifunctional Chelators for Therapeutic Lanthanide Radiopharmaceuticals," Bioconj. Chem. 12:7-34 (2001). The present invention also provides methods for the preparation of liposomes of the present invention. In a preferred embodiment, the method comprises preparation of a liposome of the present invention, attachment of a targeting agent, and chelation of an isotope primarily to the surface of the liposome. The method of the present invention overcomes the deficiencies of the prior art by attaching a targeting agent to the liposome and by generating liposomes containing both a targeting agent and a therapeutic isotope. The therapeutic isotope may be attached to the targeting agent-liposome conjugate with high efficiency and without the need for the removal of unassociated isotope. Additionally, the therapeutic isotope of the present invention may be attached to the liposomes of the present invention without the use of extreme temperatures, e.g., at room temperature. The resulting targeting agent-liposome-isotope complex binds to a target in the presence of serum in-vitro where the targeting agent binds to its target and the isotope is detected using the appropriate detection method and apparatus.
The present invention is also directed towards a lipid construct comprising both a therapeutic entity and an imaging entity. Imaging or diagnostic agents are described in detail below in the sections entitled "Imaging" and "Diagnostics." In a preferred embodiment, the therapeutic entity is a therapeutic radionuclide, and the imaging entity is an imaging radionuclide. In a particularly preferred embodiment the therapeutic isotope is yttrium-90 and the imaging isotope is indium- 111 or a technetium isotope. In this embodiment, the lipid constructs may be prepared by providing the therapeutic isotope and the imaging isotope in the in the same mixture, followed by contacting the mixture containing the lipid constructs.
The present invention is further directed towards a therapeutic entity or imaging agent consisting of a targeting agent, a carrier, and an encapsulated therapeutic or imaging isotope.
Targeting Entities
The term "targeting entity" refers to a molecule, macromolecule, or molecular assembly which binds specifically to a biological target. The targeting entity may be of natural, synthetic, or semi-synthetic origin. Examples of targeting entities include, but are not limited to, antibodies (including antibody fragments and other antibody-derived molecules which retain specific binding, such as Fab, F(ab')2, Fv, and scFv derived from antibodies); receptor-binding ligands, such as hormones or other molecules that bind specifically to a receptor; cytokines, which are polypeptides that affect cell function and modulate interactions between cells associated with immune, inflammatory or hematopoietic responses; molecules that bind to enzymes, such as enzyme inhibitors; nucleic acid ligands or aptamers, and one or more members of a specific binding interaction such as biotin or iminobiotin and avidin or streptavidin. In one embodiment of the present invention, preferred targeting entities are molecules which specifically bind to receptors or antigens found on vascular cells. More preferred are molecules which specifically bind to receptors, antigens or markers found on cells of angiogenic neovasculature or receptors, antigens or markers associated with tumor vasculature. The receptors, antigens or markers associated with tumor vasculature can be expressed on cells of vessels which penetrate or are located within the tumor, or which are confined to the inner or outer periphery of the tumor. In one embodiment, the invention takes advantage of pre-existing or induced leakage from the tumor vascular bed; in this embodiment, tumor cell antigens can also be directly targeted with agents that pass from the circulation system into the tumor interstitial volume.
Other targeting entities target endothelial receptors, tissue or other targets accessible through a body fluid or receptors or other targets upregulated in a tissue or cell adjacent to or in a bodily fluid. For example, targeting entities attached to carriers designed to deliver drugs to the eye can be injected into the vitreous, choroid, or sclera; or targeting agents attached to carriers designed to deliver drugs to the joint can be injected into the synovial fluid.
In another embodiment of the present invention, preferred targeting entities are molecules which specifically bind to receptors, antigens, or markers on cells that circulate within the vasculature, such as malignant B cells, or cells expressing antigens as a result of viral infection. Targeting entities attached to the liposomes 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 1000 daltons or less, which serve as ligands for a vascular target or vascular cell marker), such as the integrin-binding molecules described in copending United States Patent Application Serial No. 10/159,596, filed May 30, 2002, entitled Targeted Multivalent Macromolecules; proteins, such as antibodies and growth factors; peptides, such as RGD-containing peptides (e.g., those described in U.S. Patent No. 5,866,540), bombesin or gastrin-releasing peptide, peptides selected by phage-display techniques such as those described in U.S. Patent No. 5,403,484, and peptides designed de novo to be complementary to tumor-expressed receptors; antigenic determinants; or other receptor targeting groups. These head groups can be used to control the biodistribution, nonspecific adhesion, and blood pool half life of the liposomes. For example, β-D-lactose has been attached on the surface to target the asialoglycoprotein (ASG) found in liver cells which are in contact with the circulating blood pool. In some embodiments the targeting entity is an integrin-specific molecule, such as an
RGD peptide, see above, or an RGD peptidomimetic, such as 3-{4-[2-(3,4,5,6- tetrahydropyrimidin-2-ylamino)-ethyloxy]-benzoylamino}-2(S)-benzene-sulfonyl- aminopropionic acid.
In particular, 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. In some embodiments, the targeting entity is a tyrosine kinase specific molecule, such as the compounds AG1433 or SU1498.
In other embodiments, 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).
Glycolipids can be derivatized for use as targeting entities, for example, by converting the commercially available lipid (DAGPE) or the pentadicosanoic acid derivative _V-(8'- aminθ-3',6'-dioxaoctyl)-10,12-pentacosadiynamide (PEG-PDA amine) into its isocyanate followed by treatment with triethylene diamine spacer l,8-diamino-3,6-dioxaoctane to produce the amine terminated thiocarbamate lipid which by treatment with the para- isothiocyanophenyl glycoside of the carbohydrate ligand produces the desired targeting glycolipids. This synthesis provides a water soluble flexible spacer molecule spaced between the lipid that will form the internal structure or core of the liposome and the ligand that binds to cell surface receptors, allowing the ligand to be readily accessible to the protein receptors on the cell surfaces. The carbohydrate ligands can be derived from reducing sugars or glycosides, such as para-nitrophenyl glycosides, a wide range of which are commercially available or easily constructed using chemical or enzymatic methods. Liposomes coated with carbohydrate ligands can be produced by mixing appropriate amounts of individual lipids followed by sonication, extrusion, polymerization if polymerizable lipids are used, and filtration as described above. Suitable carbohydrate derivatized liposomes have about 1 to about 30 mole percent of the targeting glycolipid and filler lipid, such as PDA, DAPC, DAPE, or other phosphocholine based lipid, with the balance being metal-chelated lipid or metal- chelating lipid. Other lipids may be included in the liposomes to assure liposome formation and provide high contrast and recirculation.
In some embodiments, the targeting entity targets the liposomes to a cell surface. Delivery of the therapeutic or imaging agent can occur through endocytosis of the liposomes. Such deliveries are known in the art. See, for example Mastrobattista, et al., Immunoliposomes for the Targeted Delivery of Antitumor Drags, Adv. Drug Del. Rev. (1999)
40:103-27.
In a preferred embodiment, the targeting entity is attached to a carboxyl head group on the lipid. In another preferred embodiment, the targeting entity is attached to a maleimide or the alpha-methyl group of an acetamide. In one embodiment, the attachment is by covalent means. In another embodiment, the attachment is by non-covalent means. For example, antibody targeting entities may be attached by a biotin-avidin biotinylated antibody sandwich, to allow a variety of commercially available biotinylated antibodies to be used on the coated liposome. Other lipid head groups for the attachment of targeting agents include amines, cyano, carboxylic acids, isothiocyanates, thiols, disulfides, α-halocarbonyl compounds, ,β- 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 or radioactive isotopes. Other head groups may have an attached therapeutic entity, such as, for example, antibodies, peptidomimetics, and hormones and drugs for interaction with a biological site at or near the specific biological molecule to which the polymerized liposome particle attaches. Specific vasculature targeting agents of use in the invention include (but are not limited to) anti-VCAM-1 antibodies (VCAM = vascular cell adhesion molecule); anti-ICAM-1 antibodies (ICAM = intercellular adhesion molecule); anti-integrin antibodies (e.g., antibodies directed against αvβ3 integrin such as LM609, described in International Patent Application WO 89/05155 and Cheresh et al. J. Biol. Chem. 262:17703-11 (1987), and Vitaxin, described in International Patent Application WO 9833919 and in Wu et al., Proc. Natl. Acad. Sci. USA 95(11):6037-42 (1998); and antibodies directed against P- and E-selectins, pleiotropin and endosialin, endoglin, VEGF receptors, PDGF receptors, EGF receptors, matrix metalloproteases, G-protein coupled receptors, and prostate specific membrane antigen (PSMA).
In one embodiment of the invention, the 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. Alternatively, the vascular-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.
In a preferred embodiment, the invention provides a vascular targeted therapeutic agent that comprises an integrin targeting agent and a 90Y therapeutic entity.
Accordingly, in one embodiment, the vascular-targeted therapeutic agent has two targeting entities: a targeting entity directed towards a vascular marker, and a targeting entity directed towards a tumor cell marker. In another embodiment, an antitumor agent is administered with the vascular-targeted therapy agent. The antitumor agent can be administered simultaneously with the vascular-targeted therapy agent, or subsequent to administration of the vascular-targeted therapy agent. In particular, when the vascular- targeted therapy agent is relied upon to compromise vascular integrity in the area of the tumor, administration of the antitumor agent is preferably done at the point of maximum damage to the tumor vasculature.
The 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 therapy agent, but targeted against the tumor cell rather than the vasculature. A summary of monoclonal antibodies directed against various tumor markers is given in Table I of U.S. Patent No. 6,093,399, hereby incorporated by reference herein in its entirety. In general, when the vascular-targeted therapy agent compromises vascular integrity in the area of the tumor, the effectiveness of any drug which operates directly on the tumor cells can be enhanced. While the primary focus of the invention is on vascular-targeted agents, in principle, a targeted agent can be designed to focus on markers present in other fluids, body tissues, and body cavities, e.g., synovial fluid, ocular fluid, or spinal fluid. Thus, for example, an antibody-liposome-radioisotope agent can be administered to spinal fluid, where the antibody targets a site of pathology accessible from the spinal fluid. The agent may also be injected subcutaneously for administration to the lymphatic system.
Therapeutic Compositions
The present invention is also directed toward therapeutic compositions comprising the therapeutic agents of the present invention. Compositions of the present invention can also include other components such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier. For example, compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, mannitol, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer, Tris buffer, histidine, citrate, and glycine, or mixtures thereof, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration. In one embodiment of the present invention, the composition can also include an ' immunopotentiator, such as an adjuvant or a carrier. Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, Freund's adjuvant; other bacterial cell wall components; aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins; viral coat proteins; other bacterial-derived preparations; gamma interferon; block copolymer adjuvants, such as Hunter's Titermax adjuvant (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. One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).
Generally, the therapeutic agents used in the invention are administered to an animal in an effective amount. Generally, an effective amount is an amount effective to either (1) reduce the symptoms of the disease sought to be treated or (2) induce a pharmacological change relevant to treating the disease sought to be treated. For cancer, including but not limited to solid tumors, leukemias, lymphomas, and associated metastatic lesions, 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 anti-tumor 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 compostions of the present invention. The method comprises administering the therapeutic agent to a subject in need of such administration.
The therapeutic agents of the instant invention can be administered by any suitable means, including, for example, parenteral, topical, oral or local administration, such as intradermally, by injection, or by aerosol. In the preferred embodiment of the invention, the agent is administered by injection. Such injection can be locally administered to any affected area. A therapeutic composition can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration of an animal include powder, tablets, pills and capsules. Preferred delivery methods for a therapeutic composition of the present invention include intravenous administration and local administration by, for example, injection or topical administration. For particular modes of delivery, a therapeutic composition of the present invention can be formulated in an excipient of the present invention. A therapeutic reagent of the present invention can be administered to any animal, preferably to mammals, and more preferably to humans.
The particular mode of administration will depend on the condition to be treated. It is contemplated that administration of the agents of the present invention may be via any bodily fluid, or any target or any tissue accessible through a body fluid.
Preferred routes of administration of the cell-surface targeted therapeutic agents of the present invention are by intravenous, interperitoneal, or subcutaneous injection including administration to veins or the lymphatic system. While the primary focus of the invention is on vascular-targeted agents, in principle, a targeted agent can be designed to focus on markers present in other fluids, body tissues, and body cavities, e.g. synovial fluid, ocular fluid, or spinal fluid. Thus, for example, an agent can be administered to spinal fluid, where an antibody targets a site of pathology accessible from the spinal fluid. Intrathecal delivery, that is, administration into the cerebrospinal fluid bathing the spinal cord and brain, may be appropriate for example, in the case of a target residing in the choroid plexus endothelium of the cerebral spinal fluid (CSF)-blood barrier.
As an example of one treatment route of administration through a bodily fluid is one in which the disease to be treated is rheumatoid arthritis. In this embodiment of the invention, the invention provides therapeutic agents to treat inflamed synovia of people afflicted with rheumatoid arthritis. This type of therapeutic agent is a radiation synovectomy agent. Individuals with rheumatoid arthritis experience destruction of the diarthroidal or synovial joints, which causes substantial pain and physical disability. The disease will involve the hands (metacarpophalangeal joints), elbows, wrists, ankles and shoulders for most of these patients, and over half will have affected knee joints. Untreated, the joint linings become increasingly inflamed resulting in pain, loss of motion and destruction of articular cartilage.
Chemicals, surgery, and radiation have been used to attack and destroy or remove the inflamed synovium, all with drawbacks.
The concentration of the radiation synovectomy agent varies with the particular use, but a sufficient amount is present to provide satisfactory radiation synovectomy. For example, in radiation synovectomy of the hip, the concentration of the agent will generally be higher than when used for the radiation synovectomy of the wrist joints. The radiation synovectomy composition is administered so that preferably it remains substantially in the joint for 20 half-lifes of the isotope although shorter residence times are acceptable as long as the leakage of the radionuclide is small and the leaked radionuclide is rapidly cleared from the body.
The radiation synovectomy compositions may be used in the usual way for such procedures. For example, in the case of the treatment of a knee-joint, a sufficient amount of the radiation synovectomy composition to provide adequate radiation synovectomy is injected into the knee-joint. There are a number of different techniques which can be used and the appropriate technique varies on the joint being treated. An example for the knee joint can be found, for example, in Nuclear Medicine Therapy, J. C. Harbert, J. S. Robertson and K. D. Reid, 1987, Thieme Medical Publishers, pages 172-3.
The route of administration through the synovia may also be useful in the treatment of osteoarthritis. Osteoarthritis is a disease where cartilage degradation leads to severe pain and inability to use the affected joint. Although age is the single most powerful risk factor, major trauma and repetitive joint use are additional risk factors. Major features of the disease include thinning of the joint, softening of the cartilage, cartilage ulcers, and abraded bone. Delivery of agents by injection of targeted carriers to synovial fluid to reduce inflammation, inhibit degradative enzymes, and decrease pain are envisioned in this embodiment of the invention.
Another route of administration is through ocular fluid. In the eye, the retina is a thin layer of light-sensitive tissue that lines the inside wall of the back of the eye. When light enters the eye, it is focused by the cornea and the lens onto the retina. The retina then transforms the light images into electrical impulses that are sent to the brain through the optic nerve. The macula is a very small area of the retina responsible for central vision and color vision. The macula allows us to read, drive, and perform detailed work. Surrounding the macula is the peripheral retina which is responsible for side vision and night vision. Macular degeneration is damage or breakdown of the macula, underlying tissue, or adjacent tissue. Macular degeneration is the leading cause of decreased visual acuity and impairment of reading and fine "close-up" vision. Age-related macular degeneration (ARMD) is the most common cause of legal blindness in the elderly.
The most common form of macular degeneration is called "dry" or involutional macular degeneration and results from the thinning of vascular and other structural or nutritional tissues underlying the retina in the macular region. A more severe form is termed
"wet" or exudative macular degeneration. In this form, blood vessels in the choroidal layer (a layer underneath the retina and providing nourishment to the retina) break through a thin protective layer between the two tissues. These blood vessels may grow abnormally directly beneath the retina in a rapid uncontrolled fashion, resulting in oozing, bleeding, or eventually scar tissue formation in the macula which leads to severe loss of central vision. This process is termed choroidal neovascularization (CNV).
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. In histoplasmosis syndrome, a series of events occur in the choroidal layer of the inside lining of the back of the eye resulting in localized inflammation of the choroid and consequent scarring with loss of function of the involved retina and production of a blind spot (scotoma). In some cases, the choroid layer is provoked to produce new blood vessels that are much more fragile than normal blood vessels. They have a tendency to bleed with additional scarring, and loss of function of the overlying retina. Diabetic retinopathy involves retinal rather than choroidal blood vessels resulting in hemorrhages, vascular irregularities, and whitish exudates. Retinal neovascularization may occur in the most severe forms. When the vasculature of the eye is targeted, it should be appreciated that targets may be present on either side of the vasculature.
Delivery of the agents of the present invention to the tissues of the eye can be in many forms, including intravenous, ophthalmic, and topical. For ophthalmic topical administration, the agents of the present invention can be prepared in the form of aqueous eye drops such as aqueous suspended eye drops, viscous eye drops, gel, aqueous solution, emulsion, ointment, and the like. Additives suitable for the preparation of such formulations are known to those skilled in the art. In the case of a sustained-release delivery system for the eye, the sustained- release delivery system may be placed under the eyelid or injected into the conjunctiva, sclera, retina, optic nerve sheath, or in an intraocular or intraorbitol location. Intravitreal delivery of agents to the eye is also contemplated. Such intravitreal delivery methods are known to those of skill in the art. The delivery may include delivery via a device, such as that described in U.S. Patent No. 6,251,090 to Avery. In a further embodiment, the therapeutic agents of the present invention are useful for gene therapy. As used herein, the phrase "gene therapy" refers 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. For example, the genetic material of interest can encode a hormone, receptor, enzyme or (poly) peptide of therapeutic value. In a specific embodiment, 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; Olson, F. et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, F. et: al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, E. et al. Biochim. Biophys. Acta 775: 169, 1984; Kim, S. et al. Biochim.
Biophys. Acta 728:339, 1983; and Fukunaga, M. et al. Endocrinol. 115:757, 1984. In general 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. The 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. For example, 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. 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. In a further embodiment of the invention, the invention provides therapeutic agents to treat inflamed synovia of people afflicted with rheumatoid arthritis. This type of therapeutic agent is a radiation synovectomy agent. The concentration of the radiation synovectomy agent varies with the particular use, but a sufficient amount is present to provide satisfactory radiation synovectomy. For example, in radiation synovectomy of the hip, the concentration of the agent will generally be higher than when used for the radiation synovectomy of the wrist joints. The radiation synovectomy composition is administered so that preferably it remains substantially in the joint for 20 half- lifes of the isotope although shorter residence times are acceptable as long as the leakage of the radionuclide is small and the leaked radionuclide is rapidly cleared from the body. The radiation synovectomy compositions may be used in the usual way for such procedures. For example, in the case of the treatment of a knee-joint, a sufficient amount of the radiation synovectomy composition to provide adequate radiation synovectomy is injected into the knee-joint. There are a number of different techniques which can be used and the appropriate technique varies on the joint being treated. An example for the knee joint can be found, for example, in Nuclear Medicine Therapy, J. C. Harbert, J. S. Robertson and K. D. Reid, 1987, Thieme Medical Publishers, pages 172-3.
Imaging The present invention is directed to imaging agents displaying important properties in medical diagnosis. More particularly, the present invention is directed to magnetic resonance imaging contrast agents, such as gadolinium, ultrasound imaging agents, or nuclear imaging agents, such as Tc-99m, In-111, Ga-67, Rh-105, 1-123, Nd -147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, and Tl-201. The imaging agents of the present invention are useful in imaging a patient generally, and/or in specifically diagnosing the presence of diseased tissue in a patient. The imaging process may be carried out by administering an imaging agent of the invention to a patient, and then scanning the patient using ultrasound or magnetic resonance imaging to obtain visible images of an internal region of a patient and/or of any diseased tissue in that region. By region of a patient, it is meant the whole patient, or a particular area or portion of the patient. The imaging contrast agent may be employed to provide images of the vasculature, heart, liver, and spleen, and in imaging the gastrointestinal region or other body cavities, or in other ways as will be readily apparent to those skilled in the art, such as in tissue characterization, blood pool imaging, etc. Any of the various types of ultrasound or magnetic resonance imaging devices can be employed in the practice of the invention, the particular type or model of the device not being critical to the method of the invention.
Diagnostics
Targeting agent-conjugated lipid constructs of this invention achieve in vitro and in vivo targeting of specific molecules associated with specific body tissues and specific molecules associated with specific bodily functions and pathologies to provide sufficient signal enhancement for detection by imaging methods such as magnetic resonance imaging or nuclear scintigraphy. Such in vivo imaging of various disease or developmentally associated molecules permits following the relationship of these molecules to disease progression, their time course of progression, and their response to pharmacologic interventions. Characterization of these responses in individual animals simplifies assessment of the interventions, since expression and regression of the target can be confirmed as it relates to disease outcomes. As a diagnostic tool, this technique detects disease at early stages, thereby enabling more effective treatment. The lipid constructs of this invention are suitable for combination of imaging and delivery of drugs for therapeutic treatments. Various agents can be encapsulated or attached to the surface of liposomes for delivery to specific sites in vivo. By using target-specific drug/liposomes of this invention, the drug delivery can be simultaneously visualized by magnetic resonance imaging.
In one embodiment, the site-specific liposome having attached monoclonal antibodies for specific receptor targeting may be used to visualize abnormal pathology related to solid tumors, inflammation, rheumatoid arthritis, and osteoporosis using cell surface markers including the integrins, VEGF receptors, PDGF receptors, matrix metalloproteases, selectins, PSMA, endosialin, G-protein coupled receptors, and endoglin.
The present invention further provides methods and reagents for diagnostic purposes. Diagnostic assays contemplated by the present invention include, but are not limited to, receptor-binding assays, antibody assays, immunohistochemical assays, flow cytometry assays, genomics and nucleic acid detection assays. High-throughput screening arrays and assays are also contemplated.
This invention provides various methods for in vitro assays. For example, antibody- conjugated liposomes, according to this invention, provide an ultra-sensitive diagnostic assay for specific antigens in solution. Liposomes of this invention having a chelator head group chelated to spectroscopically distinct ions provide high sensitivity for assays involving protein-protein, ligand-protein , drug-protein, nucleic-acid protein, and nucleic acid-nucleic acid interactions. Liposomes of this invention having a fluorophore head group provide a method for detection of glycoproteins on cell surfaces.
In one embodiment, the invention provides an agent which may be used for both therapy and imaging. The dual function agent, in one embodiment comprises, a lipid construct, a therapeutic agent and an imaging agent. The dual function agent may also comprise a targeting agent. This invention further provides a method of assaying abnormal pathology in vitro comprising, introducing a plurality of liposomes of the present invention to a molecule involved in the abnormal pathology into a fluid contacting the abnormal pathology, the targeting liposome attaching to a molecule involved in the abnormal pathology, and detecting in vitro the liposome attached to molecules involved in the abnormal pathology. The following specific examples are set forth in detail to illustrate the invention and should not be considered to limit the invention in any way.
EXAMPLES
EXAMPLE 1.
A. Preparation of PC/Chol/BisM-PE-DTPA4 liposomes Phosphatidylcholine from egg (PC, Avanti, 232 mg), cholesterol (Choi, Avanti, 50 mg), and l,2-dimyristoyl-,s7z-glycero- 3-phosphoethanolamidotriamine tetraacetic acid (Avanti, 25 mg) were dissolved in chloroform and the solvent was removed by rotary evaporation. The mixture was hydrated with water (10 mL) and 0.5 M NaOH (87 microliters) was added followed by five freeze-thaw cycles where the mixture was repeatedly frozen in a dry ice/acetone bath and thawed at approximately 65°C. The solution was passed through a thermal barrel extruder heated at 95°C (Lipex Biomembranes Inc.) containing 30 nm polycarbonate filters to give a clear solution containing 60 nm liposomes as determined by dynamic light scattering with
Brookhaven Instruments ZetaPals particle sizer.
B. Attachment of antibodies to PC/Chol/BisM-PE-DTPA4 liposomes Ethyldimethylaminopropyl carbodiimide (EDC) was added to a solution of PC/Choi/ BisM- PE-DTPA4 liposomes (10 mg/mL) in 50 mM borate buffer at pH 8 containing humanized IgG (250 micrograms/mL). The final concentration of EDC was 5 mM. The solution was incubated for 18 h at room temperature and applied to a column containing Sepharose CL-4B resin (Pharmacia) equilibrated with 50 mM histidine buffer at pH 7.4 containing 5 M sodium citrate and 100-200 mM sodium chloride. Analysis of fractions by ELISA showed the presence of IgG on the liposome and the separation of the IgG-liposome conjugates from unbound IgG. The purity of the resulting antibody-liposome conjugates is shown in Figure 1.
In this figure the antibody-liposome conjugate is present in fractions 2-6 as determined by ELISA. On the same column, the unbound antibody, also detected by ELISA, elutes in fractions 7-12 (data not shown). For this preparation, unbound antibody was not detected.
C. Attachment of 90Y to antibody-liposome conjugates To 100 μL of antibody- liposome conjugates (0.1-50 mg/mL) in 50 mM histidine buffer containing 5 mM sodium citrate approximately 100-250 μCi of yttrium-90 chloride (DupontNEN orNordion) in 50 mM sodium citrate was added, mixed using a vortex mixer, and incubated at room temperature for 30 minutes. In duplicate, the percent 90Y bound to the liposome was determined by adding 100 μL of the 90Y-vesicle complex to a 100k MWCO NANOSEP™ (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 90Y" in the assembly is determined with the Capintec CRC-15R dosimeter. The filter portion of the assembly is removed and discarded. Using the dosimeter, the remaining part of the assembly containing the "unbound
90Y" that passed through the filter is counted. "Bound 90Y" is determined by subtracting the "unbound 90Y" from the "total 90Y". Percent 90Y bound is calculated by dividing the "bound 90Y" by the "total 90Y" and multiplying by 100. 90Y binding is greater than 95% for lipid based DTPA chelators. D. Preparation of antibody-liposome-yttrium-90 conjugates with diacetylene lipids Liposomes containing diacetylene lipids are prepared as described in Example 1 A, except l,2-bis(10,12-tricosadiynoyl)-i'7ϊ-glycero-3-phosphocholine and chelating lipid [PDA- PEG3]2-DTTA3 (Figure 3) or [TDA-PEG3]2-DTTA3 (Figure 5) or BisT-PE-DTTA4 (Figure 5) may be used instead of the lipids described in 1 A. Antibody or peptide or peptidomimetic may be attached as described in IB, and yttrium-90 or indium-111 may be attached as described in IC.
EXAMPLE 2.
An antibody specific to the human αvβ3 integrin was attached by the method in Example 1 B. and yttrium-90 was attached using the method in Example 1 C. Targeting was demonstrated in-vitro using a radioimmunoassay with the otvβ3 integrin that is specific for the three component complex. Briefly, 96 well plates coated with the αvβ3 integrin (Chemicon International, Inc.) were blocked with BSA. Samples of rabbit serum containing 0-100 micrograms/mL of the anti-θvβ3 integrin antibody-liposome-yttrium 90 complex were added and incubated for 1 hour at room temperature. The plate was washed three times with PBST buffer and the yttrium 90 was measured using a Microbeta scintillation counter (Wallac). The standard curve is shown in Figure 2.
EXAMPLE 3. Preparation of polymerized vesicles containing a succinylated lipid and the attachment of antibody and yttrium-90 l,2-bis(10,12-tricosadiynoyl)-5n-glycero-3-phosphocholine (Avanti 870016, 400 mg, 0.44 mmol) and l,2-dipalmitoyl-5n-glycero-3-phosphoethanolamine-N-succinate (Avanti 870225, 19 mg, 0.02 mmol) were dissolved in chloroform and the solvent was removed by rotary evaporation. The mixture was hydrated with water (8.3 mL) and 0.5 M NaOH (100 microliters) was added followed by five freeze-thaw cycles where the mixture was repeatedly frozen in a dry ice/acetone bath and thawed at approximately 65°C. The solution was passed through a thermal barrel extruder heated at 95°C (Lipex Biomembranes Inc.) containing 30 nm polycarbonate filters to give a clear solution containing 68 nm vesicles as determined by dynamic light scattering with Brookhaven Instruments ZetaPals particle sizer. A portion of the solution was heated at 90°C overnight. The solution was placed in a 1 x 15 cm round plastic dish and illuminated with UV light at 12.5°C for 3 h using a hand held lamp. The resulting vesicles where 65 nm as determined by dynamic light scattering analysis. Yttrium binding was determined as described in Example 1 by adding approximately 110 microcuries of yttrium-90 to 0.1 mg of lipid vesicle. The binding was found to be 80%. To assess the stability of the complex, the vesicle-90Y complex was incubated in the presence of 1 mM
DTPA for 30 minutes and the metal binding was 70%. For comparison, control vesicles containing only l,2-bis(10,12-tricosadiynoyl)->s'7ϊ-glycero-3-phosphocholine do not bind yttrium-90 under identical conditions.
EXAMPLE 4. Stability of Vitaxin-liposome-90Y conjugates in rabbit serum.
V2-liposomes from egg PC consist of the anti-αvβ3 integrin antibody Vitaxin covalently coupled to the carboxyl groups present on liposomes composed of egg phosphatidylcholine (PC), 1 ,2-dimyristoyl-5w-glycero-3 -phosphoethanolamidotriamine tetraacetic acid (BisM-PE-DTTA4), and cholesterol (65/5/30 mole percent) or Vitaxin coupled to the carboxyl groups on non-UV radiated liposomes prepared from l,2-bis(l 0, 12- tricosadiynoyl)-5«-glycero-3-phosphocholine (BisT-PC) and a diethylenetriaminetriacetic acid-diacetylene lipid derivative (compound 8a, Journal of the American Chemistry Society, 1995, pp 7301-7306). Briefly, V2-liposomes were labeled with yttrium-90 and incubated at 37°C in rabbit serum for 1, 30, 60, and 180 minutes. At each time point, samples were removed and added to 96-well plates coated with human αvβ3 integrin and blocked with BSA prior to the addition of the sample. After a 1 hour incubation at room temperature, the plate was washed three times with PBST buffer and the yttrium signal was determined using a Wallac MicroBeta reader. Results are shown in Figure 3. Liposomes lacking Vitaxin do not generate significant signal in this assay.
EXAMPLE 5. Administration of antibody-liposome conjugate
Rabbits that were selected for treatment were immobilized using a rabbit restrainer and the ear prepared with alcohol (70% isopropyl) for intravenous administration of test samples via the marginal ear vein. A 22-gauge catheter was used for ease of test article administration. Test samples containing antibody-liposome-conjugate or test samples containing this conjugate that are labeled with 90Y were properly drawn in sterile syringes and injected using a small needle (22-24 gauge). Intravenous injection was performed at a rate of no greater than 0.2 cc/sec. Upon delivery, gauze was applied with pressure to minimize bleeding. Biodistribution data is shown in Figure 6.
EXAMPLE 6. Preparation of integrin-targeting liposomes containing an integrin-targeting lipid and ammonium sulfate.
RGD peptidomimetic lipid is a lipid having three integrin-binding small molecules having the formula 3-{4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)-ethyloxy]- benzoylamino}-2(S)-benzene-sulfonyl-aminopropionic acid covalently attached. RGD peptidomimetic lipid has the following structure:
Figure imgf000040_0001
The preparation of RGD peptidomimetic is described in commonly owned, copending application serial number 10/158,777, filed May 30, 2002, and entitled Targeted Multivalent Macromolecules . l,2-bis(10,12-tricosadiynoyl)-OT-glycero-3-phosphocholine (BisT-PC) (500 mg, 546.9 μmole, 95 mole %) was weighed into a clean 100 ml round bottom flask. Chelator lipid [PDA-PEG3]2DTTA3 (3.15 ml, 31.5 mg, 23 μmole, 4 mole %), and RGD peptidomimetic lipid (1.54 ml, 15.4 mg, 5.74 μmole, 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 μl 0.5 N NaOH while rotating the flask in the 65°C water bath. Immediately prior to extrusion, 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. 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.
EXAMPLE 7. Attachment of 90Y to peptidomimetic-vesicle complexes The peptidomimetic-vesicle complexes containing chelator lipid [PDA-PEG3]2DTTA3 are labeled with 90Y in 50 mM histidine buffer containing 5 mM citrate at pH 7.4 by the following procedure. Yttrium-90 chloride in 50 mM HC1 (NEN Life Science Products) was diluted to a working solution containing approximately 20 mCi/mL. To 100 μL of the Integrin-targeted vesicles (0.1-50 mg/mL), approximately 100-250 μCi of yttrium-90 chloride (NEN Life Science Products) was added, mixed using a vortex mixer, and incubated at room temperature for 30 minutes. In duplicate, the percent 90Y bound to the therapeutic vesicle was determined by adding 100 μL of the 90Y-vesicle complex to a 100k MWCO Nanosep™ (Pall
Filtron) filter. The filter assembly was spun in a microfuge at 4000 φm for 1 hr or until all of the solution has passed through the filter. The "total 90Y" 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 90Y" that passed through the filter was counted. "Bound 90Y" was determined by subtracting the "unbound 90Y" from the "total 90γ". Percent90Y bound was determined by dividing the "bound 90Y" by the "total 90Y" and multiplying by 100.
EXAMPLE 8. Study of antitumor efficacy of 90Y-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. Invasion Metastasis 18(1) (1998) 1-14). The K1735 M2 tumor cells were grown in tissue culture flasks in Dubelco's medium with 10% fetal calf serum (FCS). Cells were harvested using Trypsin- EDTA solution (containing 0.05%> trypsin), resuspended in PBS at 10,000,000/ml, and kept on ice. Animals with tumors between 100 and 200 mm3 were selected for treatment as described in Table I.
Table I: Description of treatment groups
Figure imgf000042_0001
Figure 7 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 8 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, on the other hand, considers the order, or rank of the tumors in a given group compared to other treatments and therefore minimizes the impact of outliers. The Kruskal-Wallis test looks for significant differences in the medians of the treatment populations and is more reliable when the data contains significant outliers.
For normalized tumor volume seven days post treatment, the P- value for the ANOVA test was 0.052. The P-value for the Kruskal-Wallis test was 0.167. Neither of these tests is significant at the 95% confidence level. As Figure 7 and Figure 8 show, this study contains a number of control groups with small differences in efficacy. In order to determine if the number of treatment groups diluted the results of the statistical tests the tests were repeated after removing some of the less distinct control groups. When only buffer, IA-NP, IA-NP- Y90 (2.5) and IA-NP-Y90 (5) treatments are compared the P-values improve substantially
(0.009 for the ANOVA test and 0.034 for the Kruskal-Wallis test). This indicates that there is at least one significantly different treatment in this reduced comparison.
Pairwise, or one to one, comparisons of the different treatment groups were made with different statistical procedures. The results indicate that IA-NP and IA-NP-Y90 (2.5) treatments when compared to treatment with buffer may have significantly lower normalized tumor volumes depending on the how the data are analyzed. On the other hand, treatment with IA-NP-Y90 (5) compared to treatment with buffer yields significantly lower normalized tumor volume regardless of the statistical test employed.
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 7 summarizes the growth delay data for this study. Again ANOVA and Kruskal-Wallis tests were used to compare TVQT values from different treatment groups. The P-values associated with both tests were highly significant (0.001 for the Kruskal-Wallis test and <0.0005 for the ANOVA). Pairwise comparisons of the different treatment groups indicate that treatment with IA-NP-Y90 at higher radiation doses (5μCi./g) is significantly different from buffer, IA and both low and high IA-NP treatments. Table III on the following page shows the results of Tukey's W pairwise comparison procedure. These results were confirmed by non-parametric statistical tests as well.
Table III: Summary of P-values obtained using Tukey's pairwise comparisons with tumor volume quadrupling time data.
NP-Y90 NP-Y90 IA-NP-Y90
Buffer IA IA-NP (2.5uCi/g) (5uCi/g) (2.5uCi/g)
IA >0.05
IA-NP >0.05 >0.05
NP-Y90 (2.5uCi/g) >0.05 >0.05 >0.05
NP-Y90 (5uCi/g) >0.05 >0.05 >0.05 >0.05
IA-NP-Y90 (2.5uCi/g) >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
Eight days post treatment one tumor from each treatment group was selected at random for histological staining. The tumors were resceted and frozen in isopentane at liquid nitrogen temperatures.
Marin Biologic Laboratories, Inc. in Tiburon CA performed TUNEL assay, Von Willebrand's Factor and H&E staining on resceted tumors. TUNEL assay results indicate that Hist/Cit Buffer, IA, and NP-Y90 2.5 μCi/g treatment result in mostly healthy cells, while, IA-NP, IA-NP-Y90 2.5 μCi/g, and IA-NP- Y90 5μCi g, show inceasing amounts of apotosis and cell death. ° Treatment with IA-NP-Y90 at 5μCi/g significantly reduces tumor growth in this tumor model (significance was established at the 95% confidence level). On average the normalized tumor volume for tumors treated with IA-NP-Y90 at 5μCi/g were less than half the volume when compared to tumors treated with buffer. In addition the average TVQT for tumors treated with IA-NP-Y90 at 5μCi/g is 15.0 days compared to 6.4 days for tumors treated with buffer. Histological study of tumor samples confirms this result.
Interestingly, 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 9. Study of antitumor efficacy of peptidomimetic-dextran-vesicle Y complexes in a mouse melanoma model
Dextran coated vesicles were also tested in the mouse melanoma model as described in EXAMPLE 8. Results are shown in Figure 9. For these studies, dextran-coated vesicles containing BisT-PC and chelator lipid [PDA-PEG3]2DTTA3 were used. They were prepared as follows, and labeled with yttrium-90 as described in Example 7.
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 atpH 8. ED AC (Aldrich 16146-2, ethyldimethylaminodipropyl carbodimimide HC1 salt, 6 mg) in 200 μl of water was added dropwise to the coating mixture while stirring. The mixture was stirred at room temperature overnight. The clear reaction mixture was purified by size exclusion chromatography on a Sepharose CL 4B column (2.5 x 30 cm, Amersham Pharmacia Biotech AB product 17-0150- 01) equilibrated with 10 mM HEPES containing 200 mM NaCl at pH 7.4. When the coated vesicles began to elute, 4 ml fractions were collected. The peak fractions (2 thru 6) were pooled and filtered through a 0.45 μm filter (Nalgene 25 mm syringe filter, product 190-2545) followed by a 0.2 μm 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.
EXAMPLE 10. Study of antitumor efficacy of peptidomimetic-vesicle-90 Y complexes in a mouse colon cancer model.
In this study, 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 puφose of this study was to investigate the potential anti-tumor effects with a single intravenous administration of the IA-NP-Y90 complex.
Tumors 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.
Table I: Description of treatment groups
Figure imgf000045_0001
* 8 days post treatment one mouse was sacrificed for histological study from all but the IA groups.
** 0.1mg/g = 100 mg/kg
*** 6 μCi/g = 6 mCi kg (6 times rabbit dose)
Figure 10 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.
Figure imgf000045_0002
As Table III indicates there is a significant difference in normalized tumor volume eight days post treatment between the following therapies:
Buffer compared with IA-NP, NP-Y90 or IA-NP-Y90 IA compared with IA-NP or IA-NP-Y90
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
11 summarizes the growth delay data for this study.
P-values associated with the ANOVA and Kruskal-Wallis tests were both less than 0.0005 indicating that the differences between treatment groups shown in Figure 11 are not due to chance alone. Since there are no outliers in the tumor growth delay data, Tukey's Pairwise Comparison procedure was used to determine which treatments are significantly different from others. Table TV below shows the P-values obtained with Tukey's procedure.
Table FV.
Figure imgf000046_0001
As Table IV indicates there is a significant difference in tumor growth between the folio wingvtherapies :
Buffer compared with IA-NP, NP-Y90 and IA-NP-Y90 IA compared with IA-NP, NP-Y90 and IA-NP-Y90
IA-NP compared with IA-NP-Y90 (note, the significance value associated with this comparison is much lower than for the other significant comparisons). Interestingly, this tumor type is known to be resistant to radiation therapy. In vitro αvβ integrin binding assay Integrin binding of RGD peptidomimetic-liposome conjugates containing the chelator lipid [PDA-PEG3]2DTTA3 was demonstrated in vitro using a radiometric binding assay. Briefly, 96-well plates coated with the αvβ3 integrin were blocked with BSA. Samples of rabbit serum or buffer containing 0-100 micrograms/mL of the agonist-liposome-90Y complex were added and incubated for one hour at room temperature. The plate was washed 3X with PBST buffer and the 90Y was measured using a Wallac Microbeta scintillation counter.

Claims

CLAIMSWhat is claimed is:
1. A lipid construct comprising a chelator, a targeting entity, and a detectable entity or a therapeutic entity.
2. The lipid construct of Claim 1 , further comprising at least one lipid.
3. The lipid construct of Claim 1 , wherein said at least one lipid is a phosphatidylcholine derivative.
4. The lipid construct of Claim 1, further comprising cholesterol.
5. The lipid construct of Claim 1, further comprising a stabilization agent.
6. The lipid construct of Claim 5, wherein said stabilization agent is polyethylene glycol.
7. The lipid construct of Claim 1 , wherein said therapeutic entity is a radionuclide.
8. The lipid construct of Claim 7, wherein the radionuclide is selected from the group consisting of Y-90, 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.
9. The lipid construct of Claim 7, wherein the radionuclide is Y-90.
10. The lipid construct of Claim 7, wherein the Y-90 is associated with the surface of the lipid construct.
11. The lipid construct of Claim 7, wherein the Y-90 is encapsulated within the lipid construct.
12. The lipid construct of Claim 1 , wherein the chelator is part of a chelating lipid.
13. The lipid construct of Claim 12, wherein said chelating lipid is 1 ,2- dimyristoyl-5n-glycero-3-phosphoethanolamidotriamine tetraacetic acid (BisM-PE-DTPA4). Y-90.
14. The lipid construct Claim 13, wherein said therapeutic entity is Y-90.
15. The lipid construct of Claim 12, wherein said chelating lipid is N,N- bis[[[[(l 3 ', 15 '-pentacosadiynamido-3,6- doxaoctyl)carbamoyl]methyl](carboxymethyl)amino]ethyl]glycine ([PDA-PEG3]2-DTTA3).
16. The lipid construct of Claim 12, wherein said chelating lipid contains a diacetylene lipid.
17. The lipid construct of Claim 1, further comprising polymerizable lipids.
18. The lipid construct of Claim 12, further comprising polymerizable lipids .
19. The lipid construct of Claim 17 or 18, wherein the polymerizable lipid is 1,2- bis(l 0, 12-tricosadiynoyl)-sn-glycero-3 -phosphocholine.
20. The lipid construct of Claim 17 or 18, wherein the polymerizable lipid is [PDA-PEG3]2-DTTA3.
21. The lipid construct of Claim 12, wherein said chelating lipid is selected from the group consisting of a derivative of diethylenetriaminepentaacetic acid, a derivative of ethylaminediaminetetracetic acid, and a derivative of 1,4,7, 10-tetraazacyclododecane-
N,N',N",N" '-tetraacetic acid (DOTA).
22. The lipid construct of Claim 12, wherein said chelating lipid comprises an ionizable group selected from the group consisting of carboxyl, phosphate, phosphonate, sulfate, sulfonate, and sulfinate.
23. The lipid construct of Claim 12, wherein said chelating lipid comprises a single ionizable group, said single ionizable group generating a surface capable of binding an isotope or metal with a valency of +2 or greater.
24. The lipid construct of Claim 12, wherein said chelating lipid comprises a single ionizable group, said single ionizable group generating a surface capable of binding an isotope or metal with a valency of +3 or greater.
25. The lipid construct of Claim 1 , wherein said targeting entity is selected from the group consisting of a small molecule ligand and a protein.
26. The lipid constmct of Claim 1 , wherein said targeting entity targets the lipid construct to a cell surface.
27. The lipid constmct of Claim 1, wherein the detectable entity is a radionuclide.
28. The imaging agent of Claim 1, wherein the radionuclide is selected from the group consisting of Tc-99m, In-I ll, Ga-67, Rh-105, Nd -147, Pm-151, Sm-153, Gd-159, Tb-
161, Er-171, Re-186, Re-188, and Tl-201.
29. The lipid construct of Claim 2, wherem said targeting entity is associated with a carboxyl head group of said lipid.
30. The lipid constmct of Claim 2, wherein the targeting entity is associated with a maleimide group of said lipid or the alpha-methyl group of an acetamide of said lipid.
31. The lipid construct of Claim 2, wherein the targeting entity is associated with the lipid construct by covalent means.
32. The lipid construct of Claim 2, wherein the targeting entity is associated with the lipid construct through a group selected from the group consisting of amine, cyano, carboxylic acid, isothiocyanate, thiol, disulfide, α-halocarbonyl, α,β-unsaturated carbonyl and alkyl hydrazine.
33. The lipid construct of Claim 2, wherein the targeting entity is associated with the lipid construct by non-covalent means.
34. The lipid construct of Claim 33, wherein said non-covalent means is a biotin- avidin biotinylated antibody sandwich.
35. The lipid construct of claim 25, wherein said targeting entity is an antibody.
36. The lipid construct of claim 34, wherein said antibody has a target selected from the group consisting of P-selectin, E-selectin, pleiotropin, G-protein coupled receptors, endosialin, endoglin, VEGF receptor, PDGF receptor, EGF receptor, the matrix metalloproteases, and prostate specific membrane antigen (PSMA).
37. The lipid construct of claim 1, wherein said targeting entity has a vascular target.
38. The lipid construct of Claim 37, wherein said targeting entity is Vitaxin or LM609.
39. The lipid construct of claim 36, wherein said targeting entity is selected from the group consisting of an anti-VCAM-1 antibody, an anti-ICAM-1 antibody, an anti-integrin antibody.
40. The lipid construct of Claim 36, further comprising a targeting entity having a tumor cell target.
41. A therapeutic agent comprising a lipid construct, said lipid construct comprising a chelating lipid, a targeting entity, and a therapeutic entity, wherein said therapeutic entity is associated with said chelating lipid at the surface of said lipid construct.
42. The therapeutic agent of Claim 42, wherein the therapeutic entity is a metal ion.
43. The therapeutic agent of Claim 42, wherein the metal ion is a radioactive metal ion.
44. The therapeutic agent of Claim 43, wherein the metal ion is selected from the group consisting of Y-90, 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.
45. The therapeutic agent of Claim 43, wherein the therapeutic agent is a radiation synovectomy agent.
46. The therapeutic agent of Claim 42, wherem the therapeutic entity is Y-90 and the targeting entity is an integrin targeting agent.
47. An imaging agent comprising a lipid construct, said lipid construct comprising a chelating lipid, a targeting entity, and a detectable entity, wherein said detectable entity is associated with said chelating lipid at the surface of said lipid construct.
48. The imaging agent of Claim 47, wherein the detectable entity is a metal ion.
49. The imaging agent of Claim 48, wherein the metal ion is a radioactive metal ion.
50. The imaging agent of Claim 49, wherein the metal ion is selected from the group consisting of Tc-99m, In-Ill, Ga-67, Rh-105, Nd -147, Pm-151, Sm-153, Gd-159, Tb- 161, Er-171, Re-186, Re-188; and Tl-201.
51. A method for preparing a lipid construct comprising: a) preparing a liposome comprising a chelating lipid; and b) contacting the liposome with a metal ion.
52. The method of Claim 51 , wherein the metal ion is a radioactive metal ion.
53. The method of Claim 52, wherein the metal ion is selected from the group consisting of Y-90, 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.
54. The method of Claim 52, wherem the metal ion is selected from the group consisting of Tc-99m, In-Ill, Ga-67, Rh-105, 1-123, Nd -147, Pm-151, Sm-153, Gd-159, Tb- 161, Er-171, Re-186, Re-188, and Tl-201.
55. The method of Claim 52, further comprising contacting the liposome with a second metal ion.
56. The method of Claim 55, wherein the metal ion is selected from the group consisting of Y-90, 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, and the second metal ion is selected from the group consisting of Tc-99m, In-111, Ga-67, Rh-105, 1-123, Nd -147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, and Tl-201.
57. The method of Claim 56, wherem both metal ions reside in the same mixture.
58. The method of Claim 52, further comprising removing unbound metal ion.
59. A method of imaging a patient comprising a) administering an imaging agent to a patient in need thereof, said imaging agent comprising a lipid constract, said lipid constract comprising a chelating lipid and a detectable entity; and b) imaging the patient.
60. The method of Claim 59, wherein the imaging is magnetic resonance imaging or nuclear scintigraphy.
61. 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 lipid constract, said lipid construct comprising a chelating lipid and a therapeutic entity.
62. The method of Claim 61 , wherem the patient suffers from rheumatoid arthritis.
63. The method of Claim 61, wherein the patient suffers from a disorder selected from the group consisting of cancer, a solid tumor, a leukemia, a lymphoma, and metastatic lesions of any of the aforementioned disorders.
64. A lipid constract useful for therapy and imaging, comprising a chelating lipid, a targeting entity, a detectable entity, and a therapeutic entity, wherein said detectable entity and therapeutic entity are associated with said chelating lipid at the surface of said lipid constract.
65. The method of Claim 64, the therapeutic entity is selected from the group consisting of Y-90, 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, and the detectable entity is selected from the group consisting of Tc-99m, In-111, Ga-67, Rh-105, 1-123, Nd -
147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171, Re-186, Re-188, and Tl-201.
S:\ClientFolders\Targesome\02\PCT\Specification.doc
PCT/US2002/023947 2001-07-27 2002-07-26 Lipid constructs as therapeutic and imaging agents WO2003011345A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP02756736A EP1420831A1 (en) 2001-07-27 2002-07-26 Lipid constructs as therapeutic and imaging agents
JP2003516575A JP2005519861A (en) 2001-07-27 2002-07-26 Lipid constructs as therapeutic agents and imaging agents
CA002455598A CA2455598A1 (en) 2001-07-27 2002-07-26 Lipid constructs as therapeutic and imaging agents

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US30834701P 2001-07-27 2001-07-27
US60/308,347 2001-07-27
US09/976,254 2001-10-11
US09/976,254 US20020071843A1 (en) 2000-10-11 2001-10-11 Targeted therapeutic agents

Publications (1)

Publication Number Publication Date
WO2003011345A1 true WO2003011345A1 (en) 2003-02-13

Family

ID=26976209

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/023947 WO2003011345A1 (en) 2001-07-27 2002-07-26 Lipid constructs as therapeutic and imaging agents

Country Status (4)

Country Link
EP (1) EP1420831A1 (en)
JP (1) JP2005519861A (en)
CA (1) CA2455598A1 (en)
WO (1) WO2003011345A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1372739A2 (en) * 2001-03-08 2004-01-02 Targesome, Inc. Stabilized therapeutic and imaging agents
JP2008517904A (en) * 2004-10-21 2008-05-29 シィンボルックス イーペー ベー.ファウ. Vehicle for transporting selected molecules into cells
US8349360B2 (en) 2004-10-06 2013-01-08 Bc Cancer Agency Liposomes with improved drug retention for treatment of cancer
US8758724B2 (en) 2005-11-29 2014-06-24 Nihon Medi-Physics Co., Ltd. Unnatural amino acid radiolabeling precursor
RU2537175C2 (en) * 2013-03-26 2014-12-27 Российская Федерация, От Имени Которой Выступает Министерство Образования И Науки Российской Федерации Method for producing radioimmune preparation for diagnosing and treating oncological diseases
US9186377B2 (en) 2011-06-03 2015-11-17 Maguire Abbey, Llc Method, composition, and articles for improving joint lubrication
WO2016046793A3 (en) * 2014-09-26 2016-06-09 The South African Nuclear Energy Corporation Limited Radiopharmaceutical conjugate of a metabolite and an epr agent, for targeting tumour cells

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101272808B (en) * 2005-01-06 2012-03-21 通用电气医疗集团股份有限公司 Optical imaging
BR112013014735A2 (en) * 2010-12-14 2016-10-04 Rigshospitalet radionuclide capture in nanoparticle compositions
WO2013125232A1 (en) * 2012-02-23 2013-08-29 キヤノン株式会社 Dye-containing nanoparticle for photoacoustic contrast agent

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958371A (en) * 1995-06-08 1999-09-28 Barnes-Jewish Hospital Site specific binding system, nuclear imaging compositions and methods
US6045821A (en) * 1994-10-10 2000-04-04 Nycomed Salutar, Inc. Liposomal agents
US6132764A (en) * 1994-08-05 2000-10-17 Targesome, Inc. Targeted polymerized liposome diagnostic and treatment agents
US6139819A (en) * 1995-06-07 2000-10-31 Imarx Pharmaceutical Corp. Targeted contrast agents for diagnostic and therapeutic use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6132764A (en) * 1994-08-05 2000-10-17 Targesome, Inc. Targeted polymerized liposome diagnostic and treatment agents
US6045821A (en) * 1994-10-10 2000-04-04 Nycomed Salutar, Inc. Liposomal agents
US6139819A (en) * 1995-06-07 2000-10-31 Imarx Pharmaceutical Corp. Targeted contrast agents for diagnostic and therapeutic use
US5958371A (en) * 1995-06-08 1999-09-28 Barnes-Jewish Hospital Site specific binding system, nuclear imaging compositions and methods

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1372739A2 (en) * 2001-03-08 2004-01-02 Targesome, Inc. Stabilized therapeutic and imaging agents
EP1372739A4 (en) * 2001-03-08 2005-10-19 Targesome Inc Stabilized therapeutic and imaging agents
US8349360B2 (en) 2004-10-06 2013-01-08 Bc Cancer Agency Liposomes with improved drug retention for treatment of cancer
US8709474B2 (en) 2004-10-06 2014-04-29 Bc Cancer Agency Liposomes with improved drug retention for treatment of cancer
JP2008517904A (en) * 2004-10-21 2008-05-29 シィンボルックス イーペー ベー.ファウ. Vehicle for transporting selected molecules into cells
US9381259B2 (en) 2005-11-29 2016-07-05 Nihon Medi-Physics Co., Ltd. Precursor compound of radioactive halogen-labeled organic compound
US8758724B2 (en) 2005-11-29 2014-06-24 Nihon Medi-Physics Co., Ltd. Unnatural amino acid radiolabeling precursor
US10953112B2 (en) 2005-11-29 2021-03-23 Nihon Medi-Physics Co., Ltd. Precursor compound of radioactive halogen-labeled organic compound
US9387266B2 (en) 2005-11-29 2016-07-12 Nihon Medi-Physics Co., Ltd. Precursor compound of radioactive halogen-labeled organic compound
US10010632B2 (en) 2005-11-29 2018-07-03 Nihon Medi-Physics Co., Ltd. Precursor compound of radioactive halogen-labeled organic compound
US11083804B2 (en) 2005-11-29 2021-08-10 Nihon Medi-Physics Co., Ltd. Precursor compound of radioactive halogen-labeled organic compound
US9186377B2 (en) 2011-06-03 2015-11-17 Maguire Abbey, Llc Method, composition, and articles for improving joint lubrication
RU2537175C2 (en) * 2013-03-26 2014-12-27 Российская Федерация, От Имени Которой Выступает Министерство Образования И Науки Российской Федерации Method for producing radioimmune preparation for diagnosing and treating oncological diseases
WO2016046793A3 (en) * 2014-09-26 2016-06-09 The South African Nuclear Energy Corporation Limited Radiopharmaceutical conjugate of a metabolite and an epr agent, for targeting tumour cells
AU2015323328B2 (en) * 2014-09-26 2021-02-25 The South African Nuclear Energy Corporation Limited Radiopharmaceutical conjugate of a metabolite and an EPR agent, for targeting tumour cells
US10874753B2 (en) 2014-09-26 2020-12-29 The South African Nuclear Energy Corporation Limited Radiopharmaceutical conjugate of a metabolite and an EPR agent, for targeting tumour cells

Also Published As

Publication number Publication date
JP2005519861A (en) 2005-07-07
EP1420831A1 (en) 2004-05-26
CA2455598A1 (en) 2003-02-13

Similar Documents

Publication Publication Date Title
US20100111840A1 (en) Stabilized therapeutic and imaging agents
US20020071843A1 (en) Targeted therapeutic agents
US20030133972A1 (en) Targeted multivalent macromolecules
US20030082103A1 (en) Targeted therapeutic lipid constructs having cell surface targets
US20030129223A1 (en) Targeted multivalent macromolecules
Kim et al. Liposomes: biomedical applications
US20040067196A1 (en) Targeted therapeutic lipid constructs
JP5438317B2 (en) Novel liposome composition
JP5615483B2 (en) Cytotoxic preparations for combination therapy
JP6387400B2 (en) Pharmaceutical composition, its manufacture and use
CN103269722A (en) Aptamer conjugates for targeting of therapeutic and/or diagnostic nanocarriers
WO2002096367A2 (en) Targeted multivalent macromolecules
US20110190623A1 (en) Thermally-activatable liposome compositions and methods for imaging, diagnosis and therapy
AU2012253373A1 (en) Enhanced growth inhibition of osteosarcoma by cytotoxic polymerized liposomal nanoparticles targeting the alcam cell surface receptor
US20240066144A1 (en) Bone marrow-, reticuloendothelial system-, and/or lymph node-targeted radiolabeled liposomes and methods of their diagnostic and therapeutic use
Keshavarz et al. CAR, a homing peptide, prolongs pulmonary preferential vasodilation by increasing pulmonary retention and reducing systemic absorption of liposomal fasudil
WO2003028643A2 (en) Targeted therapeutic lipid constructs having cell surface targets
EP1420831A1 (en) Lipid constructs as therapeutic and imaging agents
Torchilin Surface-modified liposomes in gamma-and MR-imaging
CN101848703B (en) Improved liposomes and uses thereof
WO2004070009A2 (en) Targeted multivalent macromolecules
Harrington et al. Polyethylene glycol in the design of tumor-targetting radiolabelled macromolecules-lessons from liposomes and monoclonal antibodies
Bartsch et al. Oral Presentations—Abstracts
Amin et al. Liposomal Drug Delivery Systems for Cancer Therapy: The Rotterdam Experience. Pharmaceutics 2022, 14, 2165
Wang Tumor-specific targeting and intracellular delivery mediated by the folate endocytosis pathway

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VN YU ZA ZM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2455598

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2003516575

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2002756736

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2002756736

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 2002756736

Country of ref document: EP