Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2881—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/04—X-ray contrast preparations
  • A61K49/0433—X-ray contrast preparations containing an organic halogenated X-ray contrast-enhancing agent
  • A61K49/0447—Physical forms of mixtures of two different X-ray contrast-enhancing agents, containing at least one X-ray contrast-enhancing agent which is a halogenated organic compound
  • A61K49/0461—Dispersions, colloids, emulsions or suspensions
  • A61K49/0466—Liposomes, lipoprotein vesicles, e.g. HDL or LDL lipoproteins, phospholipidic or polymeric micelles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
  • A61K49/1806—Suspensions, emulsions, colloids, dispersions
  • A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
  • A61K51/1217—Dispersions, suspensions, colloids, emulsions, e.g. perfluorinated emulsion, sols
  • A61K51/1234—Liposomes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)

Definitions

• the present invention is in the fields of drag delivery, cancer treatment and diagnosis and pharmaceuticals.
• This invention provides a method of making antibody- or antibody fragment-targeted immunoliposomes for the systemic delivery of molecules to treat and image diseases, including cancerous tumors.
• the invention also provides immunoliposomes and compositions, as well as methods of imaging various tissues.
• the liposome complexes are useful for encapsulation of imaging agents, for example, for use in magnetic resonance imaging.
• the specificity of the delivery system is derived from the targeting antibodies or antibody fragments.
• Magnevist ® Gadopentetate Dimeglumine
• Mag Magnetic
• Magn Magnetic
• This metal chelate is metabolically inert.
• the meglumine ion dissociates from the hydhophobic gadopentetate, which is distributed only in the extracellular water. It cannot cross an intact blood-brain barrier, and therefore does not accumulate in normal brain tissue, cysts, post-operative scars, etc, and is rapidly excreted in the urine. It has a mean half-life of about 1.6 hours. Approximately 80% of the dose is excreted in the urine within 6 hours.
• contrast media are mainly based on perfusion and diffusion labels, and glucose uptake.
• these free (non-complexed) agents changes are seen in tumors, in inflammatory disease, and even with hormonal effects (in breast) (e.g. most gadolinium based and iodine based contrast agents document perfusion and diffusion into interstitial space, FDG-PET demonstrates glucose uptake).
• tumors are not specifically targeted by these contrast agents.
• active benign processes cannot always be separated from malignant, e.g. benign enhancing areas on breast MRI, chronic pancreatitis vs pancreatic carcinoma.
• the present invention provides, methods of preparing an antibody- or antibody fragment-targeted cationic immunoliposome complex comprising preparing an antibody or antibody fragment, mixing the antibody or antibody fragment with a cationic liposome to form a cationic immunoliposome, wherein the antibody or antibody fragment is not chemically conjugated to the cationic liposome, and mixing the cationic immunoliposome with an imaging agent to form the antibody- or antibody fragment-targeted-cationic immunoliposome complex.
• Exemplary antibody fragments for use in the practice of the present invention include, single chain Fv fragments, such as an anti-transferrin receptor single chain Fv (TfRscFv) and anti-HER-2 antibody or antibody fragment.
• the methods further comprise mixing the cationic imrnunoliposome with a peptide comprising the K[K(H)KKK] 5 -K(H)KKC (HoKC) (SEQ ID NO: 1) peptide.
• the antibody or antibody fragment is mixed with said cationic liposome at a ratio in the range of about 1 :20 to about 1 :40 (w:w).
• the cationic liposomes comprise a mixture of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and/or cholesterol; or a mixture of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and/or cholesterol.
• the cationic immunoliposomes are mixed with the imaging agent at a ratio in the range of about 0.1:10 to about 0.1:35 (mg imaging agent: ⁇ g liposome), suitably about 1:14 to about 1:28 (mg imaging agent: ⁇ g liposome), or about 1:21 (mg imaging agent: ⁇ g liposome).
• imaging agent for use in the practice of the present invention include, but are not limited to, magnetic resonance imaging (MRI) agents, such as gadolinium, gadopentetate dimeglurnine, iopamidol and iron oxide.
• MRI magnetic resonance imaging
• barium, iodine and saline imaging agents for CT, 18 F-2-deoxy-2-fluoro- D-glucose (FDG) and other imaging agents for PET can also be used.
• the present invention also provides cationic immunoliposome complexes prepared by the methods of the present invention and antibody- or antibody fragment- targeted cationic immunoliposome complexes comprising a cationic liposome, an antibody or antibody fragment, and an imaging agent, wherein the antibody or antibody fragment is not chemically conjugated to said cationic liposome.
• the present invention provides methods of imaging an organ or a tissue, and also for distinguishing between benign tissues/diseases and cancerous tissues/diseases in a patient comprising administering the cationic immunoliposome complexes of the present invention to the patient prior to performing the imaging.
• Administration can occur via any route, for example, intravenous administration, intramuscular administration, intradermal administration, intraocular administration, intraperitoneal administration, intratumoral administration, intranasal administration, intracereberal administration or subcutaneous administration.
• the tissue that is imaged using the methods and complexes of the present invention are cancerous tissues, including cancerous metastasis.
• the present invention also provides methods of imaging and treating a tumor tissue in a patient suffering from cancer comprising administering the cationic immunoliposome complexes of the present invention to the patient to image the tumor tissue and administering an anti-cancer agent to the patient to treat the tumor tissue.
• anti-cancer agents include nucleic acids, genes, proteins, peptides, small molecules, chemotherapeutic agents, such as docetaxel, mitoxantrone and gemcitabine, and antisense oligonucleotides or siRNA.
• Figure IA and IB show tumor-specific targeting of a CaPan-1 orthotopic metastasis model by the TfRscFv-Liposome-DNA nanocomplex.
• the same tumor nodule in the liver indicated by an arrow in IA exhibits intense ⁇ -galactosidase expression in IB.
• FIG. 2A -2C show In Vitro MR Imaging of K564 cells after transfection with the TfRscFv-Lip-Mag nanocomplex.
• IA time dependent transfection. The values given are relative intensity.
• IB shows variation in relative intensity with the amount of Magnevist® included in the complex (in ⁇ l).
• IC Comparison of relative intensity of the TfRscFv-Lip-Mag complex versus free Magnevist®. The small circles in all images are markers for sample orientation.
• Figure 3 A-I show improved MR imaging in two different models of cancer using the Ligand-Liposome-Mag nanocomplex.
• 3A, D, and G show the differences in MRI signal in a large pancreatic orthotopic tumor (arrow) (4 months after surgical implantation of the tumor) between the i.v. administered free contrast agent and the TfRscFv-Lip-Mag complex.
• 3B, E, and H show a similar effect in a second mouse with a subcutaneous pancreatic tumor and a much smaller abdominal pancreatic tumor (arrows).
• 3 C, F and I are the images of a third animal with a subcutaneous prostate tumor (arrow) in which the same effect is evident.
• Figure 4A-C show SPM phase images of liposomes without Magnevist®.
• the images appearing in 4A, 4B and 4C were obtained at setpoints of 1.68 V, 1.45 V, and 1.35 V, respectively.
• the corresponding phase differences between the noncompliant substrate and the mechanically compliant liposome are -3.5°, +8°, and +40°.
• the interaction of the SPM tip and liposome changes from attractive to repulsive as the setpoint is decreased.
• Figure 5A-C show SPM and SEM images of liposome-encapsulated Magnevist®
• 5 A is the Atomic Force Microscopy topographical image of the Liposome encapsulated Magnevist® particle.
• the SPM phase image (setpoint 1.6)
• 5B and 15 keV SEM (TE) [Transmission-mode electron detector] image (5C) possess similar contrast, although generated by entirely distinct complementary physical mechanisms.
• Figure 6A and 6B show SPM topographic and phase imaging of
• Figure 7 A and 7B show cross-sectional comparison of SPM topographic and magnetic phase image in lift mode using 25 -nm height displacement.
• 7 A is an SPM topographic/magnetic phase image of the full TfRscFv-Lip-Mag nanocomplex.
• the appearance of a double dipole-like signal in 7B consisting of attractive and repulsive in- plane magnetic interactions suggests that the cause of this interaction is the nonuniform toroidal distribution of Magnevist within the NDS, consistent with SEM and nonmagnetic SPM phase images.
• Figures 8A-8H show improved MR imaging in two different models of cancer using the Ligand-HK-Liposome-Mag nanocomplex.
• Human breast cancer MDA-MB- 435 Figure 8E-8H
• human prostate cancer cell line Figure 8A-8D
• Figure 9A-C shows tumor-specific targeting of a CaPan-1 subcutaneous tumor and orthotopic metastasis model by the TfRscFv-HK-Liposome-Mag nanocomplex.
• Figure 10 shows dynamic MRI showing the increase in intensity using Mag- delivered by the complexes of the present invention in a pancreatic carcinoma model, as compared to free Mag.
• Figure 1 IA-11C shows MR imaging of pancreatic cancer metastases by Mag- comprising complexes of the present invention.
• Figure 12A-12E shows a greater enhancement in MR imaging of lung metastases by Mag-comprising complexes of the present invention.
• Figure 13 A-13D shows a greater enhancement in MR imaging of renal cell carcinoma lung metastases by Mag-comprising complexes of the present invention.
• Figure 14A-14D shows greater sensitivity of detection by MR imaging of small renal cell carcinoma lung metastases by Mag-comprising complexes of the present invention.
• Figure 15A-15B shows MR imaging of very small metastases by Mag-comprising complexes of the present invention, demonstrating the sensitivity of the complexes of the present invention.
• Figure 16 shows sections of metastatic tissue confirming the detection/imaging seen by MRI using the Mag-comprising complexes of the present invention.
• Figure 17 shows higher magnification of Figure 16.
• Figure 18A- ISF shows MR imaging of metastases in the subpleura of the lung by
• Figure 19A-19B shows detection Of B 16 ZF 10 melanoma lung metastases by Mag- comprising complexes of the present invention.
• the present invention fulfills a critical need, that is, enhanced sensitivity and tumor-cell specificity for early detection and differential diagnosis of tumor versus benign tissue, by providing nanocomplexes for systemic delivery of imaging agents, such as magnetic resonance imaging (MRI) agents, such as gadolinium, gadopentetate dimeglumine (Magnevist ® ) and, iopamidol, iron oxide; barium, iodine and saline imaging agents for CT; and 18 F-2-deoxy-2-fluoro-D-glucose (FDG) and other imaging agents for PET to targeted tissues, for example tumors.
• imaging agents such as magnetic resonance imaging (MRI) agents, such as gadolinium, gadopentetate dimeglumine (Magnevist ® ) and, iopamidol, iron oxide; barium, iodine and saline imaging agents for CT; and 18 F-2-deoxy-2-fluoro-D-glucose (FDG) and other imaging agents for
• the present invention provides tumor-targeting delivery systems comprising contrast agents, for example magnetic resonance imaging (MRI) contrast agents.
• contrast agents for example magnetic resonance imaging (MRI) contrast agents.
• MRI magnetic resonance imaging
• U.S. Published Patent Application No. 2003/0044407 discloses these nan ⁇ -sized, cationic liposome encapsulating various agents. Decorating the surface of these liposomes are targeting molecules which can be a ligand, such as folate or transferrin, or an antibody or an antibody fragment directed against a cell surface receptor.
• the presence of the ligand/antibody on the liposomes facilitates the entry of the complexes into the cells through binding of the targeting molecule by its receptor followed by internalization of the bound complex via receptor mediated endocytosis, a highly efficient internalization pathway (Cristiano, RJ., and Curiel, D.T., Cancer Gene Therapy 3:49-57 (1996); Cheng, P.W., Human Gene Therapy 7:275-282 (1996)).
• This modification of the liposomes results in their being able not only to selectively deliver their payload to tumor cells, but also increases the transfection efficacy of the liposome.
• Transferrin receptor (TfR) levels are elevated in various types of cancer including oral, prostate, breast, and pancreas (Keer, H.N., et al, Journal of Urology 743:381-385 (1990); Rossi, M.C., and Zetter, B.R., Proc. Natl. Acad. ScL (USA) 89:6197-6201 (1992); Elliott, R.L., et al., Ann. KY. Acad. Sd. 698:159-166 (1993); Thorstensen, K., and Romslo, I., Scand. J. Clin. Lab. Investig.
• TfR recycles during internalization in rapidly developing cells such as cancer cells (Ponka, P. and Lok, C.N., Int'l. J. Biochem. Cell Biol. 37:1111- 1137 (1999)), thus contributing to the uptake of these transferrin targeted nanocomplexes even in cancer cells where TfR levels are not elevated.
• the nanocomplexes described herein employ an anti-transferrin receptor single chain antibody fragment (TfRscFv) as the targeting moiety (Haynes, B. F., et al, J. Immunol 727:347-351 (1981); Batra, J.K., et al, Molecular & Cellular Biology 77:2200-2205 (1991)).
• TfRscFv contains the complete antibody binding site for the epitope of the TfR recognized by the monoclonal antibody 5E9 (Batra, J.K., et al., Molecular & Cellular Biology 77:2200-2205 (1991)).
• TfRscFv has advantages over the Tf molecule itself, or an entire Mab, in targeting liposomes to cancer cells with elevated TfR levels: 1) the size of the scFv (28 kDa) is much smaller than the Tf molecule (80 kDa) or the parental Mab (155 kDa). The scFv-liposome-DNA complex may thus exhibit better penetration into small capillaries characteristic of solid tumors. 2) the smaller scFv has a practical advantage related to the scaled-up production necessary for the clinical trials. 3) the scFv is a recombinant molecule and not a blood product like Tf and thus presents no danger of a potential contamination by blood borne pathogens.
• Encapsulating Magnevist ® (Mag) within such a tumor-targeted nanocomplexes offers advantages for enhanced sensitivity and detection of tumor metastases and diagnosis of cancer.
• Antibody- or antibody fragment- targeted cationic liposome complexes in accordance with this invention are made by a simple and efficient non-chemical conjugation method in which the components of the desired complex are mixed together in a defined ratio and in a defined order (see, U.S. Published Patent Application No. 2003/0044407).
• the resultant complexes are as effective as, or more effective than, similar complexes in which the antibody or antibody fragment is chemically conjugated to the liposome or polymer.
• the terms "immunocomplex,” “immunoliposome,” “complex,” “nanocomplex,” “imniunonanocomplex,” “liposome complex” are used interchangeably throughout to refer to the cationic liposomes of the present invention.
• an antibody fragment is used.
• the antibody fragment is a single chain Fv fragment of an antibody.
• One preferred antibody is an anti-TfR monoclonal antibody and a preferred antibody fragment is an scFv based on an anti-TfR monoclonal antibody.
• a suitable anti-TfR monoclonal antibody is 5E9 ⁇ see, e.g., Hayes, B.F., et ah, "Characterization of a Monoclonal Antibody (5E9) that Defines a Human Cell Surface Antigen of Cell Activation," J. Immunol.
• scFv based on 5E9 antibody contains the complete antibody binding site for the epitope of the TfR recognized by this MAb as a single polypeptide chain of approximate molecular weight 26,000.
• An scFv is formed by connecting the component VH and VL variable domains from the heavy and light chains, respectively, with an appropriately designed linker peptide, which bridges the C-terminus of the first variable region and N-terminus of the second, ordered as either VH-linker-VL or VL-linker-VH.
• Another preferred antibody is an anti-HER-2 monoclonal antibody
• another preferred antibody fragment is an scFv based on an anti-HER-2 monoclonal antibody.
• a cysteine moiety is added to the C-terminus of the scFv.
• cysteine which provides a free sulfhydryl group, may enhance the formation of the complex between the antibody and the liposome, for example via a charge-charge interaction.
• the protein can be expressed in E.coli inclusion bodies and then refolded to produce the antibody fragment in active form.
• a first step in making the complex comprises mixing a cationic liposome or combination of liposomes or small polymer with the antibody or antibody fragment of choice (see Examples herein and in U.S. Published Patent Application No. 2003/0044407).
• a wide variety of cationic liposomes are useful in the preparation of the complexes of this invention.
• Published PCT application WO99/25320 describes the preparation of several cationic liposomes.
• desirable liposomes include those that comprise a mixture of dioleoyltrimethylammonium phosphate (DOTAP) and dioleoylphosphatidylethanolamine (DOPE) and/or cholesterol (chol), a mixture of dimethyldioctadecylammonium bromide (DDAB) and DOPE and/or chol.
• the ratio of the lipids can be varied to optimize the efficiency of uptake of the therapeutic molecule for the specific target cell type.
• the liposome can comprise a mixture of one or more cationic lipids and one or more neutral or helper lipids.
• a desirable ratio of cationic lipid(s) to neutral or helper lipid(s) is about l:(0.5-3), preferably l:(l-2) (molar ratio).
• the present invention also provides for targeted-cationic polymers for delivery of imaging agents.
• Suitable polymers are DNA binding cationic polymers that are capable of mediating DNA compaction and can also mediate endosome release.
• a preferred polymer is polyethyleneimine.
• Other useful polymers include polysine, protamine and polyamidoamine dendrimers.
• the antibody or antibody fragment is one which will bind to the surface of the target cell, and preferably to a receptor that is differentially expressed on the target cell.
• the antibody or antibody fragment is mixed with the cationic liposome or polymer at room temperature and at a proteinrlipid ratio in the range of about 1 :20 to about 1 :40 (w:w) or a protein polymer ratio in the range of about 0.1 : 1 to 10: 1 (molar ratio).
• the antibody or antibody fragment and the liposome or polymer are allowed to incubate at room temperature for a short period of time, typically for about 10-15 minutes, then the mixture is mixed with a therapeutic or diagnostic agent of choice.
• therapeutic molecules or agents which can be complexed to the antibody and liposome include genes, high molecular weight DNA (genomic DNA), plasmid DNA, antisense oligonucleotides, peptides, ribozymes, nucleic acids (including siRNA and antisense), viral particles, immunomodulating agents, proteins, small molecules and chemical agents.
• Preferred therapeutic molecules include genes encoding p53, Rb94 or Apoptin.
• RB94 is a variant of the retinoblastoma tumor suppressor gene.
• the agent is an antisense oligonucleotide or an siRNA molecule, such as a HER-2 antisense or siRNA molecule.
• a third type of preferred agent is a diagnostic imaging agent, such as an MRI imaging agent, such as a Gd-DTPA agent. Additional imaging agents include, but are not limited to, Gadolinium, gadopentetate dimeglumine (Magnevist®), iopamidol, iron oxide; barium, iodine and saline imaging agents for CT; and 18F-2-deoxy-2-fluoro- D-glucose (FDG) and other imaging agents for PET.
• the agent is DNA, such as the coding region of p53, it can be positioned under the control of a strong constitutive promoter, such as an RSV or a CMV promoter.
• the antibody or antibody fragment and liposome combination is mixed with the therapeutic or diagnostic agent at a ratio in the range of about 1:10 to 1:20 ( ⁇ g. of agentnmole of total lipid) or 1:10 to 1:40 (ug of agent:nmole of total polymer) and incubated at room temperature for a short period of time, typically about 10 to 15 minutes.
• the size of the liposome complex is typically within the range of about 50-400 nm as measured by dynamic light scattering using a Malvern Zetasizer 3000.
• the liposome used to form the complex is a sterically stabilized liposome.
• Sterically stabilized liposomes are liposomes into which a hydrophilic polymer, such as PEG, pofy(2-ethylacrylic acid), or poly(n- isopropylacrylamide (PNIPAM) have been integrated.
• a hydrophilic polymer such as PEG, pofy(2-ethylacrylic acid), or poly(n- isopropylacrylamide (PNIPAM)
• PEG pofy(2-ethylacrylic acid), or poly(n- isopropylacrylamide (PNIPAM)
• a cationic liposome is first mixed with a therapeutic or diagnostic agent as described above at a ratio in the range of about 1:10 to 1:20 ( ⁇ g of agentnmole of lipid).
• a solution of a PEG polymer in a physiologically acceptable buffer is added to this lipoplex and the resultant solution is incubated at room temperature for a time sufficient to allow the polymer to integrate into the liposome complex.
• the antibody or antibody fragment then is mixed with the stabilized liposome complex at room temperature and at a protein:lipid ratio in the range of about 1:5 to about 1:30 (w:w).
• the liposomal or polymer complexes prepared in accordance with the present invention can be formulated as a pharmacologically acceptable formulation for in vivo administration.
• the complexes can be combined with a pharmacologically compatible vehicle or carrier.
• the compositions can be formulated, for example, for intravenous administration to a human patient to be benefited by administration of the therapeutic or diagnostic molecule of the complex.
• the complexes are sized appropriately so that they are distributed throughout the body following i.v. administration.
• the complexes can be delivered via other routes of administration, such as intratumoral (IT), intralesional (IL), aerosal, percutaneous, endoscopic, topical, intramuscular (IM), intradermal (ID), intraocular (10), intraperitoneal (IP), intranasal (IN), intracereberal (IC) or subcutaneous administration.
• routes of administration such as intratumoral (IT), intralesional (IL), aerosal, percutaneous, endoscopic, topical, intramuscular (IM), intradermal (ID), intraocular (10), intraperitoneal (IP), intranasal (IN), intracereberal (IC) or subcutaneous administration.
• routes of administration such as intratumoral (IT), intralesional (IL), aerosal, percutaneous, endoscopic, topical, intramuscular (IM), intradermal (ID), intraocular (10), intraperitoneal (IP), intranasal (IN), intracereberal (IC) or subcutaneous administration.
• compositions comprising the antibody- or antibody fragment- targeted liposome (or polymer) and therapeutic agent complexes are administered to effect human gene therapy.
• the therapeutic agent component of the complex comprises a therapeutic gene under the control of an appropriate regulatory sequence.
• Gene therapy for various forms of human cancers can be accomplished by the systemic delivery of antibody or antibody fragment-targeted liposome or polymer complexes which contain a nucleic acid encoding wt p53 or RB94.
• the complexes can specifically target and sensitize tumor cells, both primary and metastatic tumors, to radiation and/or chemotherapy both in vitro and in vivo.
• the complexes can be optimized for target cell type through the choice and ratio of lipids, the ratio of antibody or antibody fragment to liposome, the ratio of antibody or antibody fragment and liposome to the therapeutic or diagnostic agent, and the choice of antibody or antibody fragment and therapeutic or diagnostic agent.
• the target cells are cancer cells.
• any tissue having malignant cell growth can be a target, head and neck, breast, prostate, pancreatic, brain, including glioblastoma, cervical, lung, liver, liposarcoma, rhabdomyosarcoma, choriocarcinoma, melanoma, retinoblastoma, ovarian, urogenital, gastric and colorectal cancers are suitable targets.
• the complexes made by the method of this invention also can be used to target non-tumor cells for delivery of a therapeutic molecule or any nucleic acid. While any normal cell can be a target, preferred cells are dendritic cells, endothelial cells of the blood vessels, lung cells, breast cells, bone marrow cells, thymus cells and liver cells.
• Undesirable, but benign, cells can be targeted, such as benign prostatic hyperplasia cells, over-active thyroid cells, lipoma cells, and cells relating to autoimmune diseases, such as B cells that produce antibodies involved in arthritis, lupus, myasthenia gravis, squamous metaplasia, macular degeneration, cardiovascular disease, neurologic disease such as Alzheimer's disease, dysplasia and the like.
• benign prostatic hyperplasia cells such as benign prostatic hyperplasia cells, over-active thyroid cells, lipoma cells, and cells relating to autoimmune diseases, such as B cells that produce antibodies involved in arthritis, lupus, myasthenia gravis, squamous metaplasia, macular degeneration, cardiovascular disease, neurologic disease such as Alzheimer's disease, dysplasia and the like.
• autoimmune diseases such as B cells that produce antibodies involved in arthritis, lupus, myasthenia gravis, squamous metaplasia, macular degeneration, cardiovascular
• the complexes can be administered in combination with another therapeutic treatment, such as either a radiation treatment or chemotherapeutic agent.
• the therapeutic treatments, or a combination of therapeutic treatments can be administered before or subsequent to the administration of the complex, for example within about 12 hours to about 7 days.
• Chemotherapeutic agents include, for example, doxorubicin, 5-fluorouracil (5FU), cisplatin (CDDP), docetaxel (TAXOTERE ® ), gemcitabine (GEMZAR ® ), pacletaxel, vinblastine, etoposide (VP- 16), camptothecia, actinomycin-D, mitoxantrone and mitomycin C.
• Radiation therapies/treatments include gamma radiation ( 137 Cs), X- rays, UV irradiation, microwaves, electronic emissions and the like. Additional therapeutic agents include small molecules, peptides, proteins and the like.
• Diagnostic or imaging agents also can be delivered to targeted cells via the liposome or polymer complexes.
• diagnostic agents and “imaging agents” are used interchangeably throughout to refer to agents which can be detected, visualized, imaged or observed in vivo following administration.
• Exemplary methods for detecting, visualizing, imaging or observing diagnostic and imaging agents are well known in the art and include, for example, optical imaging such as fluorescent imaging (fluorimeters) or bioluminescent imaging, positron emission tomography (PET) scanning, single photon emission computed tomography (SPECT) scanning, magnetic resonance imaging (MRI), x-ray, radionucleotide imaging (e.g., gamma camera, computed tomography (CT), quantitative autoradiography, etc.) and the like.
• Exemplary diagnostic agents include electron dense materials, iron, magnetic resonance imaging agents and radiopharmaceuticals.
• Radionuclides useful for imaging include radioisotopes of copper, gallium, indium, rhenium, and technetium, including isotopes 64 Cu, 67 Cu, 111 In, 99m Tc, 67 Ga or 68 Ga.
• MRI agents such as a Gd-DTPA agent, gadolinium, or Magnevist® (Gadopentetate Dimeglumine) (Mag) (Berlex Imaging, Montville, NJ). Imaging agents disclosed by Low et al. in U.S. Patent 5,688,488, incorporated herein by reference, are useful in the present invention.
• Additional imaging agents include, but are not limited to, iopamidol (e.g., ISOVUE ® , Regional Health Limited, Aukland, AU), iron oxide; barium, iodine and saline imaging agents for CT; and 18 F-2-deoxy-2-fluoro-D-glucose (FDG) and other imaging agents for PET.
• iopamidol e.g., ISOVUE ® , Regional Health Limited, Aukland, AU
• iron oxide e.g., iron oxide
• barium iodine and saline imaging agents for CT
• FDG F-2-deoxy-2-fluoro-D-glucose
• kits for use in the systemic delivery of a therapeutic or diagnostic molecule by the complex.
• Suitable kits can comprise, in separate, suitable containers (or in a single container), the liposome, the antibody or antibody fragment, and the therapeutic or diagnostic agent.
• the components can be mixed under sterile conditions in the appropriate order and administered to a patient within a reasonable period of time, generally from about 30 minutes to about 24 hours, after preparation.
• the kit components preferably are provided as solutions or as dried powders.
• Components provided in solution form preferably are formulated in sterile water-for-injection, along with appropriate buffers, osmolality control agents, etc.
• the present invention provides cationic liposomal complexes wherein one or more imaging agents are encapsulated within the interior of the liposome, contained within the hydrocarbon chain region of the bilayer, complexed/associated with the inner and/or outer monolayer (e.g., via static interaction or chemical/covalent interaction), or a combination of any or all of these possibilities;
• the imaging agents will be encapsulated within the interior of the liposome and/or associated with an inner and/or outer monolayer.
• imaging agents refer to agents which can be detected, visualized, imaged or observed in vivo following administration.
• imaging agents include electron dense materials, iron, magnetic resonance imaging agents and radiopharmaceuticals.
• Radionuclides useful for imaging include radioisotopes of copper, gallium, indium, rhenium, and technetium, including isotopes 64 Cu, 67 Cu, 111 In, 99m Tc, 67 Ga or 68 Ga.
• MRI agents such as a gadolinium, Gd-DTPA agent, or Magnevist® (Gadopentetate Dimeglumine) (Mag) (Berlex Imaging, Montville, NJ).
• Imaging agents disclosed by Low et al. in U.S. Patent 5,688,488, incorporated herein by reference, are also useful in the present invention. Additional imaging agents include, but are not limited to, iopaniidol (e.g., ISOVUE ® , Regional Health Limited, Aukland, AU), iron oxide; barium, iodine and saline imaging agents for CT; and 18 F-2- deoxy-2-fluoro-D-glucose (FDG) and other imaging agents for PET.
• iopaniidol e.g., ISOVUE ® , Regional Health Limited, Aukland, AU
• iron oxide e.g., iron oxide
• barium iodine and saline imaging agents for CT
• FDG F-2- deoxy-2-fluoro-D-glucose
• imaging agents are suitably encapsulated, contained or complexed/associated with the liposome complexes of the present invention by simply mixing the one or more imaging agents with the liposomes during processing.
• Suitable ratios of imaging agents:liposome complexes are readily determined by the ordinarily skilled artisan.
• the ratio of imaging agents to liposome complex is suitably in the range of about 0.1:10 to about 0.1:35 (mg imaging agent: ⁇ g liposome), more suitably about 1:14 to about 1:28 (mg imaging agent ⁇ g liposome), or about 1:21 (mg imaging agent ⁇ g liposome).
• examples of desirable cationic liposomes for delivery/encapsulation of imaging agents include those that comprise a mixture of dioleoyltrimethylammonium phosphate (DOTAP) and dioleoylphosphatidylethanolamine (DOPE) and/or cholesterol (chol); and a mixture of dimethyldioctadecylammonium bromide (DDAB) and DOPE and/or chol.
• DOTAP dioleoyltrimethylammonium phosphate
• DOPE dioleoylphosphatidylethanolamine
• chol cholesterol
• DDAB dimethyldioctadecylammonium bromide
• the liposome can comprise a mixture of one or more cationic lipids and one or more neutral or helper lipids.
• a desirable ratio of cationic lipid(s) to neutral or helper lipid(s) is about l:(0.5-3), preferably about l:(l-2) (molar ratio).
• ratios of various lipids useful in the practice of the present invention include, but are not limited to:
• the present invention provides methods of preparing imaging agent-comprising antibody- or antibody fragment-targeted cationic immunoliposome complexes comprising preparing an antibody or antibody fragment; mixing the antibody or antibody fragment with a cationic liposome to form a cationic immunoliposome, wherein the antibody or antibody fragment is not chemically conjugated to the cationic liposome; and mixing the cationic immunoliposome with one or more imaging agents to form the antibody- or antibody fragment-targeted-cationic immunoliposome complex.
• the antibody fragment is a single chain Fv fragment, for example, an anti-transferrin receptor single chain Fv (TfRscFv) or an anti-HER-2 antibody or antibody fragment.
• suitable lipids for use in preparing the imaging agent-comprising cationic immunoliposomes are described herein, and include, mixtures of dioleoyltrimethylammonium phosphate with dioleoylphosphatidylethanolamine and/or cholesterol; and mixtures of dimethyldioctadecylammonium bromide with dioleoylphosphatidylethanolamine and/or cholesterol.
• the antibody or antibody fragment is mixed with the cationic liposome at a ratio in the range of about 1 :20 to about 1 :40 (w:w) to form a cationic immunoliposome.
• the cationic immunoliposome is mixed with the imaging agent in the range of about 0.1:10 to about 0.1:35 (mg imaging agent: ⁇ g liposome), more suitably about 1:14 to about 1:28 (mg imaging agent ⁇ g liposome), or about 1:21 (mg imaging agent: ⁇ g liposome).
• Exemplary imaging agents include those described herein and known in the art.
• the imaging agent is an MRI imaging agent, such as gadolinium, gadopentetate dimeglumine, iopamidol (e.g., ISOVUE®, Regional Health Limited, Aukland, AU), or iron oxide; barium, iodine and saline imaging agents for CT; and ls F-2-deoxy-2-fluoro-D- glucose (FDG) and other imaging agents for PET.
• MRI imaging agent such as gadolinium, gadopentetate dimeglumine, iopamidol (e.g., ISOVUE®, Regional Health Limited, Aukland, AU), or iron oxide
• barium, iodine and saline imaging agents for CT and ls F-2-deoxy-2-fluoro-D- glucose (FDG) and other imaging agents for PET.
• FDG F-2-deoxy-2-fluoro-D- glucose
• the methods and immunoliposome complexes of the present invention further comprise mixing the cationic immunoliposome with a peptide comprising the K[K(H)KKK] 5-K(H)KKC (HoKC or HK) (SEQ ID NO: 1) peptide.
• the HoKC peptide carries a terminal cysteine to permit conjugation to a maleimide group.
• the liposome formulations also suitable include N- maleimide-phenylbutyrate-DOPE (MPB-DOPE) at 0.1 to 50 molar percent of total lipid, more preferably 1-10 molar percent of total lipid, most preferably 5 molar percent of total lipid.
• the HoKC liposomes are prepared as previously described (Yu, W. et al. Enhanced transfection efficiency of a systemically delivered tumor-targeting immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine peptide, Nucleic Acids Research 32, e48 (2004)).
• the present invention provides antibody- or antibody fragment-targeted cationic immunoliposome complexes comprising a cationic liposome, an antibody or antibody fragment, and one or more imaging agents, wherein the antibody or antibody fragment is not chemically conjugated to the cationic liposome.
• the antibody or antibody fragment is suitably associated with the liposome via an interaction (e.g., electrostatic, van der Walls, or other non-chemically conjugated interaction) between the antibody or antibody fragment and the liposome, suitably between a cystein residue on the antibody or antibody fragment and the liposome surface.
• a linker or spacer molecule e.g., a polymer or other molecule
• the imaging agent(s) can be encapsulated within the cationic liposome, contained with a hydrocarbon chain region of the cationic liposome, associated with an inner or outer monolayer of the cationic liposome, or any combination thereof.
• the cationic immunoliposomes of the present invention are unilamellar liposomes (i.e. a single bilayer), though multilamellar liposomes which comprise several concentric bilayers can also be used.
• Single bilayer cationic immunoliposomes of the present invention comprise an interior aqueous volume in which agents (e.g., imaging agents) can be encapsulated. They also comprise a single bilayer which has a hydrocarbon chain region (i.e., the lipid chain region of the lipids) in which agents (e.g., imaging agents) can be contained.
• agents (e.g., imaging agents) can be complexed or associated with either, or both, the inner monolayer and/or the outer monolayer of the liposome membrane (i.e., the headgroup region of the lipids), e.g., via a charge-charge interaction between the negatively charged imaging agents and the positively charged cationic liposomes.
• agents (e.g., imaging agents) can be encapsulated/associated/complexed in any or all of these regions of the cationic immunoliposome complexes of the present invention.
• the present invention provides methods of imaging an organ or a tissue in a patient comprising administering the imaging agent-comprising cationic immunoliposome complexes of the present invention to the patient prior to performing the imaging.
• the immunoliposome complexes can be administered via any desired route, including, but not limited to, intravenous (IV), oral, topical, via inhalation, intramuscular (BVl) injection, intratumoral (IT) injection, intradermal (ID) injection, intraperitoneal (D?) injection, intranasal (IN) injection, intraocular (10) injection, intracranial (IC) injection, or other routes.
• the term patient includes both animal patients (e.g., non-human mammals such as dogs, cats, pigs, sheep, etc,) as well as humans.
• Methods for imaging tissues of patients are well known in the art and include, but are not limited to, PET scanning, SPECT scanning, MRI imaging and the like. Any tissue or organ in a patient can be imaged using the methods and complexes of the present invention. Simply by modifying the targeting ligand on the liposomes, any over- expressed protein or molecule can be targeted.
• the methods of the present invention are used to image a cancerous tissue in a patient suffering from, or predisposed to, cancer.
• Cancerous tissues that can be imaged using the methods of the present invention include solid tumors, as well as metastasic lesions.
• the methods of the present invention can also distinguish cancerous tissues from non-cancerous (benign) tissues.
• the present invention provides methods of imaging and treating a tumor tissue in a patient suffering from, or predisposed to, cancer comprising administering the imaging-agent comprising immunoliposome complexes of the present invention to image the rumor tissue, and administering an anti-cancer agent to the patient to treat the tumor tissue.
• anti-cancer agents examples include, but are not limited to small molecules, proteins, peptides, and chemotherapeutic agents such as those described herein, genes, antisense oligonuclotides and siRNA.
• chemotherapeutic agents include, but are not limited to, doxorubicin, 5-fluorouracil (5FU), cisplatin (CDDP), docetaxel (TAXOTERE ® ), gemcitabine (GEMZAR ® ), pacletaxel, vinblastine, etoposide (VP- 16), camptothecin, actinomycin-D, mitoxantrone and mitomycin C, and an antibody therapy, such as a monoclonal antibody, e.g., HERCEPTIN ® (Genentech, San Francisco CA).
• antisense oligonucloetides and siRNA molecules for use in the practice of the present invention include, but are not limited to, those disclosed in U.S. Published Patent Application No. 2003/0044407 and U.S. Patent Application No. 11/520,796, filed September 14, 2006, the disclosures of each of which are incorporated herein by reference in their entireties.
• Additional anticancer agents include peptides, proteins and small molecules (see, e.g., U.S. Provisional Patent Application Nos. 60/800,163, filed May 15, 2006 and 60/844,352, filed September 14, 2006, the disclosures of each of which are incorporated herein by reference in their entireties).
• the anti-cancer agent e.g., the chemotherapeutic agent, small molecule, gene or the antisense or siRNA, etc.
• the anti-cancer agent can be associated with the cationic immunoliposome that also comprises the imaging agent, or it can be delivered separately, either in a different immunoliposome in accordance with the present invention, or via another carrier or delivery system (for example, IV injection of a chemotherapeutic per normal clinical standards).
• the methods of the present invention comprise administering an immunoliposome complex comprising an imaging agent (e.g., MRI imaging agent such as gadopentetate dimeglumine), and an anti-tumor agent at different times (i.e., the complex and the agent can be given at the same time or at different times).
• the anti-cancer agent is administered either before or after the imaging agent- comprising immunoliposome complex, (e.g., at least 1 hour before or after, at least 6 hours before or after, at least 12 hours before or after, at least 24 hours before or after, at least 48 hours before or after, etc., administration of the cationic immunoliposome complex).
• the methods of imaging and treating a tumor tissue in a patient suffering from cancer can further comprise administering radiation treatment to the patient.
• anti-cancer agents e.g., chemotherapy, genes, small molecules, proteins, peptides, antisense oligonucleotides or siRNA, etc.
• timing for administration in humans are easily determined by those of skill in the art, based on information contained herein and that is readily available in the art. Furthermore, such amounts can be estimated by extrapolating from experiments performed on animals, e.g., mouse, rat, dog or other studies.
• Exemplary benefits of utilizing the nanoimmunolipoosme complexes of the present invention include higher concentration in cancer tissues due to the tumor targeting nature of the complexes.
• the complexes and methods can be used to image tissues of interest at various depths.
• the methods and complexes of the present invention result not only in enhanced signal in the tumor, but also greater definition of the internal structure of the tumors. More significantly, smaller tumors can be detected leading to earlier detection and thus improved response/survival. These complexes can also be used to distinguish benign from malignant nodules. This helps to accelerate the decision on when to begin treatment. Currently, this is delayed to determine if the nodule increases since it is not certain if is malignant or not. However, since the complexes of this invention preferentially and specifically transfect tumor cells, this would also serve as a confirmation of malignancy, for example if the small nodules seen on lung CT are small malignancies or not. These last two points are of particular significance in lung and pancreatic cancer.
• Exemplary types of cancer imaging problems addressed by use of the imaging agent-comprising complexes of the present invention include, in pancreatic cancer, early detection and differentiation from chronic pancreatitis; early detection of metastatic disease to lungs; classification of solitary pulmonary nodules as benign or malignant; classification of small focal areas of increased MR enhancement in breast as benign or malignant.
• the complexes of the present invention can also be used to confirm that, using this delivery system, therapeutic genes will likely enter patient specific cancer cells. That is, the fact that the imaging agent-comprising complexes are able to enter cells provides an indication that delivery of therapeutic genes or other agents associated with the complexes of the present invention will also enter these specific cancer cells.
• Human lymphoblastic leukemia cell line K562 was obtained from the Lombardi
• Human prostate cancer cell line DUl 45 (ATCC, Manassas, VA) was originally derived from a lesion in the brain of a patient with widespread metastatic carcinoma of the prostate. It was maintained in Minimum Essential Medium with Earle's salts (EMEM) supplemented with 10% heat inactivated FBS plus L- glutamine and antibodies as above.
• EMEM Minimum Essential Medium with Earle's salts
• Cationic liposome (DOTAP:DOPE) was prepared by the ethanol injection method as previously described ⁇ see U.S. Published Patent Application No. 2003/0044407; Xu L 5 et ah, Molecular Cancer Therapeutics 1:337-346 (2002) the disclosures of each of which are incorporated herein by reference).
• the full complex was formed in a manner identical to that previously described (see U.S. Published Patent Application No. 2003/0044407).
• the TfRscFv was mixed with the liposome at a specific ratio and incubated at room temperature for 10 minutes.
• Magnevist® was added to this solution, mixed and again incubated at room temperature for 10 minutes.
• the complex is stable fqr at least 8 days, as determined by size measurements using a Malvern Zetasizer 3000H.
• the cumulants (Z average) average of measurements over this time frame is 112.3 ⁇ 4.67 (S.E.) while the polydispersity (representing the reproducibility of the values during repeat scans) is 0.445 ⁇ 0.03.
• a range of acceptable sizes for the nanocomplexes is from about 20 to 1000 nm, suitably about 50 to 700 nm and more suitably about 100 to 500 nm.
• 2 ml of serum-free media was added to the complex prior to transfection.
• the complex is formed at a ratio of 1 mg imaging agent to 0.33-1.17 ⁇ g TfRscFv to 10-35 ⁇ g Liposome (suitably 1 mg imaging agent to 0.5 to 1.0 ⁇ g TfRScFv to 14-28 ⁇ g Liposome, most suitably 1 mg imaging agent to 0.71 ⁇ g TfRscFv to 21 ⁇ g Liposome) using the above procedure.
• dextrose was added to a final concentration of 5%.
• transfect suspension cells K562 15x10 6 cells in a total volume of 4.0 ml of medium with all supplements except serum (serum free medium) were placed into a 100 mm 2 tissue culture dish. Two ml of the transfection solution from above, containing varying amounts of Magnevist®, was added to the cell suspension. The plate was incubated at 37 0 C with gentle rocking for the length of time given in the Results section (up to 90 min), after which the cells were gently pelleted (600xg for 7 minutes) at 4°C in 0.5 ml microcentrifuge tubes and washed three times with 10 ml of serum free medium to remove any excess transfection solution and placed on wet ice until imaged.
• serum serum free medium
• the cell pellets in microcentrifuge tubes were positioned at the center of the magnet.
• the MR imaging was performed at Howard University using a 4.7T horizontal bore NMR machine (Varian Inc, Palo Alto, CA).
• the imaging protocols consist of a multi-slice Tl -weighted spin echo imaging sequence and a saturation-recovery sequence.
• the repetition time (TR) was 1000 ms
• the echo time (TE) was 13 ms.
• the Tl- weighted spin-echo imaging technique was applied to verify the positive image enhancement.
• the saturation- recovery MR sequence with variable echo times was used for the Tl measurement.
• the slice thickness of images was 0.5mm.
• the RF coil employed was a 30 mm single loop coil.
• the RF coil serves as an RF transmitter and receiver.
• the RF pulse was a selective 5 ms sine pulse.
• the number of phase encoding steps was 256.
• the fleld-of-view was 15 mm x 15 mm.
• the image area chosen in the study was located at the center of the RF coil for RF homogeneity.
• the MR images were taken in the cross-section direction of the microcentrifuge tube.
• the height of the cell pellet was 12 mm.
• the range of the multi- slice images covers the whole pellet.
• the center slice images, which were not influenced by the image distortion due to the susceptibility effect from the air-pellet boundary, were utilized for the studies.
• the image intensity was measured using the Varian Image Browser software.
• the signal is taken from a region-of-interest, which is big enough to cover two thirds of the image from each microcentrifuge tube.
• mice bearing CaPan-1 orthotopic tumors or DUl 45 subcutaneous xenograft tumors were employed.
• the CaPan-1 tumors were induced as described above.
• DU145 tumors were induced by the subcutaneous inoculation of 7x10 6 cells in Matrigel. These studies were performed at Georgetown University. Animals to be imaged were anesthetized and placed in a proprietary, in-house designed, animal management system. This system incorporates a warm water heating system that maintains the temperature at 37°C, as well as a four channel thermal optical monitoring system used to monitor animal's skin temperature, ambient temperature and wall temperature of the device.
• anesthesia was induced using isoflurane at 4%, with the remaining gas comprised of a 66% oxygen and 30% nitrous oxide mixture. Maintenance of anesthesia was achieved with 1.5% isoflurane under similar gaseous conditions of oxygen and nitrous oxide as noted.
• the anesthetized animal was positioned inside of a cylindrical variable radiofrequency resonant antenna (bird cage resonator volume coil) and tuned to a center frequency of approximately 300 MHz (the resonant frequency of water molecules when subject to a field strength of 7 Tesla).
• the imaging protocol used was Tl-weighted Turbo RARE (rapid acquisition with rapid enhancement) three-dimensional imaging sequences performed on a 7T Bruker BioSpin (Germany/USA) imaging console.
• the imaging parameters used were: Tl-weighted Turbo-RARE 3D (3 -dimensional), TE 13.3 ms, TR 229.5, Flipback on, 4 echoes with a field of view of 8.0/3.5/3.5 cm and a 256 x 256 x 256 matrix.
• Topography by tapping mode with Z control (Veeco RTESP cantilevers for -320-360 kHz and k ⁇ 20-60 N/m), phase imaging, and magnetic force microscopy using magnetic coated tips (Veeco MESP 68 kHz) were performed in life mode.
• mice TfRscFv-LipA complex carrying pSVb (LacZ) plasmid DNA for ⁇ -galactosidase expression was i.v. injected into the mice three times over a 24 hour period (40 ⁇ g of plasmid DNA per injection). All five mice were sacrificed 60 hours post-injection and various organs including the liver, lung, spleen, pancreas and diaphragm were harvested and examined for the presence of metastasis and tumor specific staining. Fresh samples, sliced at 1 mm thickness, were stained with X-gal to produce a blue color where the gene is expressed.
• LacZ pSVb
• Magnevist ® is one of the most frequently employed contrast agent in the clinic, it was chosen as for use in these studies. In these initial experiments, it was examined whether the complex could be prepared with Magnevist ® and if doing so would enhance the MRI signal. Since trypsinization could lead to membrane damage and leakage of contrast agent from the cells, adherent cells were not employed in these studies. Instead, a human lymphoblastic leukemia cell line, K562, which grows as a suspension culture was used. Moreover, gentle pelleting and washing of the cells would remove any excess Magnevist ® or complex prior to imaging, allowing only cell associated signal to be detected.
• the intensity of the untreated cells (202 ⁇ 48) was not significantly different than that of an empty marker tube (194 ⁇ 43) indicating that the cells themselves do not contribute to the signal detected. More importantly, the transfection efficiency plateaus at approximately 60 minutes since the relative intensity of the cells transfected for 60 and 90 minutes were identical (317 ⁇ 46 and 317 ⁇ 47, respectively).
• Magnevist ® on the TfRscFv-Lip-Mag complex image enhancement was then assessed.
• the doses tested were 0.05, 0.3 and 0.9 mMole/kg. Corrected for size and blood volume of the mouse, the volumes of Magnevist ® used in the complex per 250ul of transfection solution were 0.25 ⁇ l, 1.5 ⁇ l and 4.5 ⁇ l.
• Figure 2B and Table 1 the image intensity increases and the Tl relaxation time shortens as a function of the amount of contrast agent included in the complex.
• a baseline image was obtained using a TI-weighted Turbo RARE (rapid acquisition with rapid enhancement) three-dimensional imaging sequence.
• Magnevist ® administered to the mouse, either free (as is performed in the clinic) or included in the complex was 10 ⁇ l. This amount is equivalent to 0.2 mM/Kg or twice what is used in humans. This amount was selected since the standard human dose of 0.1 mM/Kg Magnevist ® alone gave a very poor signal in the mice.
• the imaging with free Magnevist ® and the TfRscFv-Lip-Mag complex were performed on two consecutive days.
• a baseline scan was also performed prior to administration of nanocomplex to confirm that all of the Magnevist® from the previous day had been washed out.
• MR technique and windows were consistent between the two sets of images with the windows adjusted to correct for an automatic windowing feature of the scanner.
• the SPM images surface topography in tapping mode by oscillating the tip and cantilever to which it is attached close to the cantilever resonance frequency.
• a feedback circuit maintains the oscillation of the cantilever at a constant amplitude. This constant amplitude is given a by a set point which is somewhat smaller than that of the freely oscillating cantilever. Since the SPM tip interacts with the surface through various small forces, there is a phase shift between the cantilever excitation and its response at a given point on the surface. For an inhomogeneous surface, the tip-surface interactions will vary according to surface charge, steep topographical changes, and mechanical stiffness variations, for example.
• FIG. 4A-C A sequence of SPM phase images of a pair of isolated liposomes without payload is shown in Figure 4A-C.
• Figure 4A was imaged at a set point of 1.68 V and the corresponding negative phase difference between the substrate and liposome indicates that the tip-sample interaction is attractive for the liposome, given by a phase value of -3.5 degrees.
• the phase image of the liposome appears dark, except for a topographically keyed ring at the liposome edge.
• Figure 4B demonstrates the effect of reducing the set point to 1.45 V: The liposome now appears bright since the tip-sample interaction becomes repulsive, and in this case the phase difference between the liposome and substrate is +8 degrees. Finally, Figure 4C shows that the phase difference recorded at a set point of 1.35 V increases. further, becoming +35 degrees.
• Figure 5A-C presents SPM and SEM images of isolated liposome-encapsulated
• Lip+Mag Magnevist nanoparticles.
• the size distribution of single Lip+Mag particles is in the range of 100-200 nm diameter and scales according to optical measurements that indicate that payload-encapsulating liposomes are approximately 50% larger than liposomes alone in their spherical state.
• Magnevist ® have a bimodal surface shape after drying that is more complex than that of the simple elliptical surface of a liposome containing no payload (not shown).
• the SPM phase behavior differs markedly from that of payloadless liposomes, the outer ring is repulsive relative to the center, and a corresponding SPM phase image is shown in Figure 5B. Regions of both attractive and repulsive tip-sample interaction appear at moderate set point values.
• a correlation between the SPM phase image obtained at a set point of 1.6 and the SEM image in TE mode is evident in Figures 5B and 5C. Liposomes appear uniformly bright across the entire particle in SEM images (not shown), similar to the uniform phase images we obtain by SPM.
• Tips and cantilevers change with time and usage. Moreover, it is important to verify that the images produced are not affected by tip instabilities due to foreign material on the tip. Thus, they are changed frequently. Since each cantilever is somewhat different with respect to its resonance properties, the set points used in Figures 4 and 5 are different.
• SEM TE images indicate that the well-defined boundary between the outer ring and center of the liposome seen with the (Lip+Mag) particles is less apparent and the shape much more variable. This is consistent with the view that the presence of protein within the liposome has altered the osmotic outflow across the liposome during film drying.
• FIG. 7A is an SPM topographic/magnetic phase image of the full TfRscFv- Lip-Mag nanocomplex.
• Figure 7B consisting of attractive and repulsive in-plane magnetic interactions suggests that the cause of this interaction is the nonuniform toroidal distribution of Magnevist ® within the NDS, consistent with SEM and nonmagnetic SPM phase images.
• the nanocomplexes of the present invention can target metastatic disease, thereby enhancing detection sensitivity for metastases.
• SEM and SPM it has been demonstrated that the TfRscFv-liposome complex maintains its nanometer size when Magnevist ® is encapsulated (particles of approximately 100-200 nm are shown in Figure 6 and 7). It has also been demonstrated that the structural and mechanical properties of liposomes containing a payload are sufficiently different from those without one, thereby confirming that Magnevist is indeed encapsulated with the liposome. This was further confirmed by MFM imaging of the complex.
• Figures 8A-8H show improved imaging in two different models of cancer using the Ligand-HK-Liposome-Mag nanocomplex.
• Nanocomplexes for use in this Example were prepared using the same ratios and procedures as set forth in Example 1.
• Human breast cancer MDA-MB-435 Figure 8E-8H
• human prostate cancer cell line Figure 8A-8D
• Free Magnevist® or the TfRscFv-liposome nanocomplex (scLip- Mag), or the TfRscFv-HK-liposome nanocomplex (scLip-HK-Mag) comprising the HoKc peptide, containing the same dose of Magnevist® were i.v. injected (via the tail vein) into each of the three mice on three consecutive days. This amount of Magnevist ® is equivalent to twice the dose that would be administered to a human patient. The total volume of solution administered in all cases was 400 ⁇ l. A baseline scan was performed just prior to administration of both nanocomplexes to, confirm that all of the Magnevist ® from the previous day had been washed out.
• the panel shows the difference in MRI signal in a mouse with a subcutaneous tumor in which the increased definition and contrast are evident in both the prostate tumor (DU145) ( Figure 8A-8D) and the breast tumor (435) ( Figure 8E-8H) after injection with the scLip-Mag and even more so after injection with the scLip-HK-Mag.
• Figure 9A-9C shows tumor-specific targeting of a CaPan-1 subcutaneous tumor and orthotopic metastasis model by the TfRscFv-HK-Liposome-Mag nanocomplex.
• Subcutaneous CaPan-1 xenograft tumors were induced in female athymic nude mice as described in Methods in Example 1. The tumors were harvested and a single cell suspension in MATRIGEL ® was injected into the surgically exposed pancreas. Eight weeks post injection the TfRscFv-Liposome complex with or without HoKC (HK) peptide carrying Magnevist ® was injected into the mouse on two consecutive days. The total volume of solution administered in all cases was 400 ⁇ l.
• TfRscFv anti-transferrin receptor single chain antibody fragment
• the TfRscFv was mixed with the liposome at a specific ratio and incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most suitably 10-12 minutes.
• Magnevist ® was added to this solution, mixed and again incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most suitably 10- 12 minutes.
• sucrose or dextrose was added to a final concentration of 0.5-50%, suitably 1-20%, most suitably 10% for sucrose and 5% for dextrose, and incubated at room temperature for 1- 30 minutes, suitably 5-25 minutes, most suitably 15-20 minutes.
• the complex is formed at a ratio of lmg imaging agent to 0.33-1.17 ug TfRscFv to 10-35 ug Liposome (suitably lmg imaging agent to 0.5 to l.Oug TfRScFv to 14-28 ug Liposome, most suitably lmg imaging agent to 0.71 ug TfRscFv to 21ug Liposome) using the above procedure.
• a range of acceptable sizes of the complex is from about 20 to 1000 nm, suitably about 50 to 700 nm and most suitably about 100 to 500 nm.
• the complex was formed using 4.7 mg Magnevist, 99 ug Liposome and 3.3 ug TfRscFv with dextrose to a final concentration of 5%.
• a mouse bearing PANC-I subcutaneous tumors induced as above was anesthetized and placed in an animal holder system.
• Anesthesia was induced using isofiurane at 4%, with the remaining gas comprising a 66% oxygen and 30% nitrous oxide mixture. Maintenance of anesthesia was achieved with 1.0 to 2.0% isofiurane (preferably 1.5%) under similar gaseous conditions of oxygen and nitrous oxide as noted.
• the anesthetized animal was positioned inside of a cylindrical variable radiofrequency resonant antenna (bird cage resonator volume coil) and tuned to a center frequency of approximately 300 MHz (the resonant frequency of water molecules when subject to a field strength of 7 Tesla).
• the imaging protocol used was Tl -weighted two dimensional Turbo RARE (rapid acquisition with rapid enhancement) imaging sequences performed on a 7T Bruker BioSpin (Germany/USA) imaging console.
• the imaging parameters used were: Tl-weighted 2D (2-dimensional), TE 10.21 ms, TR 420.3, Flipback off, 8 echoes with a field of view of 5.12/5.12 cm.
• Lip-HoKC-Mag complex of this invention to detect and enhance imaging of metastatic tumors.
• metastases from a pancreatic cancer was examined, however, imaging of metastases from any type of cancer can be achieved using the complexes and methods of the present invention (e.g. prostate, melanoma, renal, breast, gastric, liver, ovarian, bladder, head and neck, brain, bone and any other type of solid tumor).
• Subcutaneous xenograft tumors of CaPan-1 were induced in female athymic nude mice by injection of 0.5 to IxIO 7 CaPan-1 cells suspended in MatrigelTM collagen basement membrane matrix (BD Biosciences). Approximately eight weeks later the tumors were harvested and a single cell suspension of the tumor was prepared.
• Cationic liposome (DOTAP:DOPE) was prepared by the ethanol injection method as previously described (see U.S. Published Patent Application No. 2003/0044407; Xu L, et al, Molecular Cancer Therapeutics 7:337-346 (2002) the disclosures of each of which are incorporated herein by reference).
• the HoKC peptide (K[K(H)KKK] 5 -K(H)KKC) (SEQ ID NO:1) carries a terminal cysteine to permit conjugation to a maleimide group.
• the liposome formulation also included N- maleimide-phenylbutyrate-DOPE (MPB-DOPE) at 0.1 to 50 molar percent of total lipid, more preferably 1-10 molar percent of total lipid, most preferably 5 molar percent of total lipid.
• MPB-DOPE N- maleimide-phenylbutyrate-DOPE
• the HoKC liposomes were prepared as previously described (Yu, W. et al. Enhanced transfection efficiency of a systemically delivered tumor-targeting immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine peptide, Nucleic Acids Research 32, e48 (2004)).
• the targeting moiety used in these studies is the anti- transferrin receptor single chain antibody fragment (TfRscFv).
• the TfRscFv was mixed with the liposome at a specific ratio and incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most suitably 10-12 minutes.
• Magnevist ® was added to this solution, mixed and again incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most suitably 10- 12 minutes.
• sucrose or dextrose was added to a final concentration of 0.5-50%, suitably 1-20%, most suitably 10% for sucrose and 5% for dextrose, and incubated at room temperature for 1- 30 minutes, suitably 5-25 minutes, most suitably 15-20 minutes.
• the complex is formed at a ratio of lmg imaging agent to 0.33-1.17 ug TfRscFv to 10-35 ug Liposome-HoKC (suitably lmg imaging agent to 0.5 to l.Oug TfRScFv to 14-28 ug Liposome-HoKC, most suitably lmg imaging agent to 0.71 ug TfRscFv to 21ug Liposome-HoKC) using the above procedure.
• a range of acceptable sizes of the complex is from about 20 to 1000 nm, suitably about 50 to 700nm and most preferable 100 to 500 nm.
• the complex was formed using 4.7 mg Magnevist ® , 99 ug Liposome-HoKC and 3.3ug TfRscFv with dextrose to a final concentration of 5%.
• the anesthetized animal was positioned inside of a cylindrical variable radiofrequency resonant antenna (bird cage resonator volume coil) and tuned to a center frequency of approximately 300 MHz (the resonant frequency of water molecules when subject to a field strength of 7 Tesla).
• a cylindrical variable radiofrequency resonant antenna bird cage resonator volume coil
• the imaging protocol used was Tl -weighted Turbo RARE (rapid acquisition with rapid enhancement) three-dimensional imaging sequences performed on a 7T Bruker BioSpin (Germany/USA) imaging console.
• the imaging parameters used were: Tl -weighted Turbo-RARE 3D (3 -dimensional), TE 13.3 ms, TR 229.5 ms, Flipback on, 4 echoes with a field of view of 8.0/3.5/3.5 cm and a 256 x 256 x 256 matrix.
• the animal was kept immobilized in the animal holder and the TfRscFv- Lip-HoKC-Mag complex (total volume 50-1000 ul, more preferably 100-500ul, most preferably 200-40OuI) was systemically administered using a 27G needle by intravenous injection into the tail vein of the animal and the 3D imaging sequence was immediately initiated.
• TfRscFv- Lip-HoKC-Mag complex total volume 50-1000 ul, more preferably 100-500ul, most preferably 200-40OuI
• Figure 1 IA-I IA Figure 11A: pre-contrast.
• Figure HB TfRcFv-Lip-HoKC-Mag injection
• Figure HC histology.
• the orthotopic pancreatic cancer shows enhancement with TfRcFv-Lip-HoKC-Mag (short white arrows). The two areas identified with the short white arrows are connected on more posterior slices and represent the primary orthotopic placed tumor. A small metastasis (thick white arrows) enhances in the same pattern seen with the primary tumor. The thin extension of liver (long thin arrow) lies adjacent to the metastasis. Necropsy (not shown) and histology (right image) confirm presence of metastasis (black arrows) directly adjacent to long thin extension of liver. Note the similarity of shape of one of the pieces of metastatic tumor to the appearance on the MRI.
• Cationic liposome (DOTAP:DOPE) was prepared by the ethanol injection method as previously described ⁇ see U.S. Published Patent Application No. 2003/0044407; Xu L, et al, Molecular Cancer Therapeutics 7:337-346 (2002) the disclosures of each of which are incorporated herein by reference).
• the targeting moiety used in these studies is the anti-transferrin receptor single chain antibody fragment (TfRscFv).
• the TfRscFv was mixed with the liposome at a specific ratio and incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most suitably 10-12 minutes.
• Magnevist ® was added to this solution, mixed and again incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most suitably 10-12 minutes.
• sucrose or dextrose was added to a final concentration of 0.5-50%, suitably 1-20%, most suitably 10% for sucrose and 5% for dextrose, and incubated at room temperature for 1- 30 minutes, more suitably 5-25 minutes, most suitably 15-20 minutes.
• the complex is formed at a ratio of lmg imaging agent to 0.33-1.17 ug TfRscFv to 10-35 ug Liposome (suitably lmg imaging agent to 0.5 to l.Oug TfRScFv to 14-28 ug Liposome, most suitably lmg imaging agent to 0.71 ug TfRscFv to 2 lug Liposome) using the above procedure.
• a range of acceptable sizes of the complex is from about 20 to 1000 nm, suitably about 50 to 700nm and most suitably about 100 to 500 nm.
• the complex was formed using 4.7 mg Magnevist, 99ug Liposome and 3.3ug TfRscFv with dextrose to a final concentration of 5%.
• a mouse bearing lung tumors induced above was anesthetized and placed in an animal holder system.
• Anesthesia was induced using isoflurane at 4%, with the remaining gas comprising a 66% oxygen and 30% nitrous oxide mixture. Maintenance of anesthesia was achieved with 1.0 to 2.0% isoflurane (preferably 1.5%) under similar gaseous conditions of oxygen and nitrous oxide as noted.
• the anesthetized animal was positioned inside of a cylindrical variable radiofrequency resonant antenna (bird cage resonator volume coil) and tuned to a center frequency of approximately 300 MHz (the resonant frequency of water molecules when subject to a field strength of 7 Tesla).
• the imaging protocol used was Tl -weighted two dimensional Turbo Multislice-Multiecho imaging sequence performed on a 7T Bruker BioSpin (Germany/USA) imaging console.
• the imaging parameters used were: Tl -weighted 2D (2-dimensional), TE 10.21 ms, TR 400 ms, Flipback off, 8 averages with a field of view of 3.84 x 3.84 cm and a 256 x 256 matrix.
• the animal was kept immobilized in the animal holder and either the free (uncomplexed) Magnevist ® (gad-d) or the TfRscFv-Lip- Mag complex containing the identical amount of Mag (total volume 50-1000 ul, more suitably 100-500ul, most suitably 200-40OuI) was systemically administered using a 27G needle by intravenous injection into the tail vein of the animal and the imaging sequence was immediately initiated. The pixel intensity of the images was measured and plotted. The same mouse was used for imaging with both the free and the complex. The imaging was performed on sequential days.
• the complex of this invention also enhances detection of relatively large metastases in the lung as compared to the currently used method of administering free imaging agent.
• Cationic liposome (DOTAP:DOPE) was prepared by the ethanol injection method as previously described (see U.S. Published Patent Application No. 2003/0044407; Xu L, et ah, Molecular Cancer Therapeutics i:337-346 (2002) the disclosures of each of which are incorporated herein by reference).
• the targeting moiety used in these studies is the anti-transferrin receptor single chain antibody fragment (TfRscFv).
• the TfRscFv was mixed with the liposome at a specific ratio and incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most suitably 10-12 minutes.
• Magnevist ® was added to this solution, mixed and again incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most suitably 10- 12 minutes.
• sucrose or dextrose was added to a final concentration of 0.5-50%, suitably 1-20%, most suitably 10% for sucrose and 5% for dextrose, and incubated at room temperature for 1- 30 minutes, suitably 5-25 minutes, most suitably 15-20 minutes.
• the complex is formed at a ratio of lmg imaging agent to 0.33-1.17 ug TfRscFv to 10-35 ug Liposome (suitably lmg imaging agent to 0.5 to l.Oug TfRScFv to 14-28 ug Liposome, most suitably lmg imaging agent to 0.71 ug TfRscFv to 21ug Liposome) using the above procedure.
• a range of acceptable sizes of the complex is from about 20 to 1000 nm, suitably about 50 to 700nm and most suitably about 100 to 500 nm.
• the complex was formed using 4.7 mg Magnevist, 99ug Liposome and 3.3ug TfRscFv with dextrose to a final concentration of 5%.
• a mouse bearing lung tumors induced above was anesthetized and placed in an animal holder system.
• Anesthesia was induced using isoflurane at 4%, with the remaining gas comprising a 66% oxygen and 30% nitrous oxide mixture. Maintenance of anesthesia was achieved with 1.0 to 2.0% isoflurane (preferably 1.5%) under similar gaseous conditions of oxygen and nitrous oxide as noted.
• the anesthetized animal was positioned inside of a cylindrical variable radiofrequency resonant antenna (bird cage resonator volume coil) and tuned to a center frequency of approximately 300 MHz (the resonant frequency of water molecules when subject to a field strength of 7 Tesla).
• the imaging protocol used was Tl -weighted two dimensional Turbo Multislice-Multiecho imaging sequence performed on a 7T Bruker BioSpin (Germany/USA) imaging console.
• the imaging parameters used were: Tl -weighted 2D (2-dimensional) imaging sequence, TE 10.21 ms, TR 572.99ms, Flipback off, 8 averages with a field of view of 2.56 x 2.56 cm and a 256 x 256 matrix.
• the animal was kept immobilized in the animal holder and either the free (uncomplexed) Magnevist ® (gad-d) or the TfRscFv-Lip-Mag complex containing the identical amount of Mag (total volume 50-1000 ul, suitably 100-500ul, most suitably 200-40OuI) was systemically administered using a 27G needle by intravenous injection into the tail vein of the animal and the imaging sequence was immediately initiated. The pixel intensity of the images was measured. The same mouse was used for imaging with both the free and the complex. The imaging was performed on sequential days. At this field of view 5 pixels is equivalent to approximately a 3 mm human tumor detected by CT.
• tumors of even smaller size can be detected after intravenous injection of the complex of the invention.
• Figure 15A- 15B nodules of 1-2 pixels were detectable by the complex. Nodules Nl and N2 were visualized on the MRI scan.
• Cationic liposome (DOTAP:DOPE) was prepared by the ethanol injection method as as previously described ⁇ see U.S. Published Patent Application No. 2003/0044407; Xu L, et ah, Molecular Cancer Therapeutics. 7:337-346 (2002) the disclosures of each of which are incorporated herein by reference).
• the targeting moiety used in these studies is the anti-transferrin receptor single chain antibody fragment (TfRscFv).
• the TfRscFv was mixed with the liposome at a specific ratio and incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most suitably 10-12 minutes.
• Magnevist ® was added to this solution, mixed and again incubated at room temperature for 1- 30 minutes, suitably 5-20 minutes, most suitably 10- 12 minutes.
• sucrose or dextrose was added to a final concentration of 0.5-50%, suitably 1-20%, most suitably 10% for sucrose and 5% for dextrose, and incubated at room temperature for 1- 30 minutes, suitably 5-25 minutes, most suitably 15-20 minutes.
• the complex is formed at a ratio of lmg imaging agent to 0.33-1.17 ug TfRscFv to 10-35 ug Liposome (suitably lmg imaging agent to 0.5 to l.Oug TfRScFv to 14-28 ug Liposome, most suitably lmg imaging agent to 0.71 ug TfRscFv to 2 lug Liposome) using the above procedure.
• a range of acceptable sizes of the complex is from about 20 to 1000 nm, suitably 50 to 7Q nm and most suitably 100 to 500 nm.
• the complex was formed using 4.7 mg Magnevist, 99ug Liposome and 3.3ug TfRscFv with dextrose to a final concentration of 5%.
• a mouse bearing lung tumors induced above was anesthetized and placed in an animal holder system.
• Anesthesia was induced using isoflurane at 4%, with the remaining gas comprising a 66% oxygen and 30% nitrous oxide mixture. Maintenance of anesthesia was achieved with 1.0 to 2.0% isoflurane (preferably 1.5%) under similar gaseous conditions of oxygen and nitrous oxide as noted.
• the anesthetized animal was positioned inside of a cylindrical variable radiofrequency resonant antenna (bird cage resonator volume coil) and tuned to a center frequency of approximately 300 MHz (the resonant frequency of water molecules when subject to a field strength of 7 Tesla).
• the imaging protocol used was Tl -weighted two dimensional Turbo Multislice-Multiecho imaging sequence performed on a 7T Bruker BioSpin (Germany/USA) imaging console.
• the imaging parameters used were: Tl-weighted 2D (2-dimensional) imaging sequence, TE 10.21 ms, TR 1418.13ms, Flipback off, 8 averages with a field of view of 3.84 x 3.84 cm and a 256 x 256 matrix.
• the animal was kept immobilized in the animal holder and either the free (uncomplexed) Magnevist ® (gad-d) or the TfRscFv-Lip-Mag complex containing the identical amount of Mag (total volume 50-1000 ul, suitably 100-500ul, most suitably 200-40OuI) was systemically administered using a 27G needle by intravenous injection into the tail vein of the animal and the imaging sequence was immediately initiated. The pixel intensity of the images was measured. The same mouse was used for imaging with both the free and the complex. The imaging was performed on sequential days.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Immunology (AREA)
• Dispersion Chemistry (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Microbiology (AREA)
• General Chemical & Material Sciences (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Mycology (AREA)
• Oncology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Physics & Mathematics (AREA)
• Biomedical Technology (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Optics & Photonics (AREA)
• Radiology & Medical Imaging (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicinal Preparation (AREA)
• Magnetic Resonance Imaging Apparatus (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Priority Applications (6)

Applications Claiming Priority (2)

Publications (2)

Family

ID=37963345

Family Applications (1)

Country Status (7)

Cited By (6)

Families Citing this family (6)

Family Cites Families (9)

• 2006
  • 2006-10-20 JP JP2008536843A patent/JP5478886B2/ja not_active Expired - Fee Related
  • 2006-10-20 CN CN2006800465731A patent/CN101351225B/zh not_active Expired - Fee Related
  • 2006-10-20 AU AU2006304790A patent/AU2006304790B2/en not_active Ceased
  • 2006-10-20 CA CA2638899A patent/CA2638899C/en not_active Expired - Fee Related
  • 2006-10-20 KR KR1020087011910A patent/KR20080064169A/ko not_active Ceased
  • 2006-10-20 WO PCT/US2006/041139 patent/WO2007047981A2/en not_active Ceased
• 2008
  • 2008-04-10 IL IL190773A patent/IL190773A/en active IP Right Grant

Also Published As

Similar Documents

Legal Events