WO2010032248A2 - Multifunctional albumin conjugates - Google Patents

Multifunctional albumin conjugates Download PDF

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Publication number
WO2010032248A2
WO2010032248A2 PCT/IL2009/000911 IL2009000911W WO2010032248A2 WO 2010032248 A2 WO2010032248 A2 WO 2010032248A2 IL 2009000911 W IL2009000911 W IL 2009000911W WO 2010032248 A2 WO2010032248 A2 WO 2010032248A2
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WIPO (PCT)
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group
conjugate
cancer
daidzein
isoflavone
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PCT/IL2009/000911
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French (fr)
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WO2010032248A3 (en
WO2010032248A8 (en
Inventor
Michal Neeman
Fortune Kohen
Helena Sheikhet Migalovich
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Yeda Research And Development Co. Ltd.
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Publication of WO2010032248A2 publication Critical patent/WO2010032248A2/en
Publication of WO2010032248A8 publication Critical patent/WO2010032248A8/en
Publication of WO2010032248A3 publication Critical patent/WO2010032248A3/en

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    • 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
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • 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
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A61K49/143—Peptides, e.g. proteins the protein being an albumin, e.g. HSA, BSA, ovalbumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to conjugates comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety including a diagnostic agent or a therapeutic agent, the conjugate being capable of delivering the bioactive moiety into a tumor cell.
  • the invention further relates to the use of said conjugate for the diagnosis and treatment of cancer.
  • the detection of a target site benefits from a high signal-to-background ratio of detection agent. Therapy benefits from higher percentages of the administered therapeutic agent reaching the target site, as well as a reasonably long duration of uptake and binding. Furthermore, drug targeting spares normal cells and significantly diminishes drug-toxicity.
  • Chemotherapy constitutes one of the major therapeutic approaches for the treatment of cancer, along with surgery and radiotherapy.
  • anti-cancer drugs such as daunomycin and adriamycin
  • these drugs are affected by the mechanisms of multi-drug resistance.
  • Affinity targeting of these cytotoxic drugs to tumor cells offers an approach that might overcome some of these drawbacks.
  • the targeting ratio and amount of agent delivered to a target site can be improved using targeting agents conjugated to diagnostic or therapeutic agents for preferential localization.
  • a variety of materials have been developed over the years for targeted delivery of diagnostic agents and drugs. Examples include specific targeting of tumor cells with monoclonal antibody-drug conjugates (U.S. Patents No.
  • albumin spherules as medicament carriers include their ease of preparation, complete removal from the body by metabolism, nonantigenicity, proven safety for intravascular administration and the capability of accommodating a wide variety of drug molecules in a relatively non-specific fashion.
  • the use of albumin as a carrier of imaging probes has been described (U.S. Pat. No. 4,337,240; Kobayashi H and Brechbiel M.W. 2005 Adv. Drug Deliv. Rev. 57 2271- 2286).
  • biotin-BS A-GdDTPA was used for tracking tumor stroma fibroblasts by MRI.
  • histological staining by the biotinylated albumin based marker showed high specificity to myofibroblast stroma tracks.
  • albumin is used as an immunogenic carrier for small molecules which are usually non immunogenic. Wang et al. J. Agric. Food. Chem., 1994, 42:1584-7, disclosed the preparation of a BSA-formononetin conjugate for the preparation of anti-formononetin antibodies.
  • the present invention relates to conjugates comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety including but not limited to a diagnostic agent or a therapeutic agent, said conjugates being capable of delivering the bioactive moiety into a cell and their use for the diagnosis and treatment of cancer.
  • the present invention also provides improved targeting methods where internalization and cellular retention of a bioactive moiety (e.g. a diagnostic or a therapeutic agent) are enhanced by binding of the bioactive moiety to an isoflavone- albumin conjugate. It is now disclosed that is the conjugates of the present invention are unexpectedly internalized in target cells to a greater extent and/or faster than without such internalization agent (e.g. isoflavone-albumin).
  • the present invention is based in part on the unexpected discovery that the conjugation of carboxy isoflavone derivatives to albumin significantly increase the uptake of the conjugate by cancerous cell lines sensitive to isoflavone. Specifically it was shown, that an imaging agent attached to a daidzein-BSA conjugate was specifically localized in ovarian carcinoma tumor cells, and displayed prolonged retention in the tumor of up to 7 days after administration as compared to the same imaging agent attached to BSA alone. Furthermore, unexpectedly, addition of nystatin, which usually inhibits BSA cellular uptake, resulted in significant augmentation of the cellular uptake of the daidzein-BSA conjugate.
  • the conjugate is regulating endocytosis by cancerous cell lines sensitive to isoflavone, involving competition between receptor mediated internalization through the binding of the carboxy isoflavone derivative and caveolae mediated internalization via binding of albumin.
  • Cells sensitive to isoflavone refers to cells whose growth may be affected by isoflavones or by carboxy derivatives of isoflavones. At certain concentrations the isoflavones may enhance growth, while at other concentrations the isoflavones may result in inhibition of cancer cell growth.
  • cells sensitive to isoflavone are cancer cells.
  • Non limiting examples to cancer cells which are sensitive to isoflavones include ovarian carcinoma cells, colon cancer cells, breast cancer cells, melanoma, non-small lung cancer, leukemia, endometrial cancer and prostate cancer cells.
  • cells sensitive to isoflavone may also be estrogen sensitive cells.
  • cells sensitive to isoflavone are cells expressing an estrogen receptor.
  • the cells sensitive to isoflavone are cells expressing a nuclear or putative membranal estrogen receptor subtype ⁇ .
  • cell sensitive to isoflavone are cells susceptible to isoflavone.
  • the present invention provides a conjugate comprising at. least one isoflavone derivative bound to albumin and at least one compound D, the conjugate represented by the structure of general formula (I): Albumin — Dn 1
  • D is a active agent selected from the group consisting of a diagnostic agent (e.g. an imaging agent) and a therapeutic agent;
  • R 7 is selected from the group consisting Of Cj-C 6 alkylene, (C 2 -C 2 o) alkenylene; and (C 2 -C 20 ) heteroalkylene;
  • the conjugate of the invention is capable of delivering the bioactive moiety into a cell sensitive to isoflavone.
  • the conjugate displays improved properties having at least one of increased partition into a cancer cell (e.g.
  • conjugates of the invention comprising an isoflavone bound to albumin and a bioactive moiety bound to the isoflavone, albumin or both, have improved qualities selected from partition to the tumor, tumor retention and circulation time compared to the bioactive moiety alone, the bioactive moiety bound to albumin, or the bioactive moiety bound to isoflavone.
  • the isoflavone derivative is selected from a derivative of diadzein, genistein, formononetin and biochanin A.
  • the isoflavone derivative is daidzein derivative.
  • the carboxy-isoflavone derivative is selected from the group consisting of 7-(O)-carboxymethyl daidzein, 6-carboxymethyl biochanin A, 2-carboxyalkyl biochanin A, 8-carboxymethyl biochanin A, 7-(O)- carboxymethyl formononetin, 2-carboxyalkyl genistein and 6-carboxymethyl genistein.
  • the carboxy-isoflavone derivative is 2- carboxyalkyl biochanin A.
  • the carboxy-isoflavone derivative is 6-carboxymethyl biochanin A.
  • the carboxy-isoflavone derivative is 8-carboxymethyl biochanin A.
  • the carboxy-isoflavone derivative is 7-(O)-carboxymethyl daidzein (i.e., R 1 is -0-CH 2 -C(O)- and R 2 - R 6 are all -H).
  • D is an imaging agent selected from, yet not restricted to paramagnetic particles (such as gadolinium, yttrium, lutetium, europium and gallinium); radioactive moieties (such as radioactive indium, rhenium and technetium); fluorescent dyes (such as fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), Cyan fluorescent protein
  • paramagnetic particles such as gadolinium, yttrium, lutetium, europium and gallinium
  • radioactive moieties such as radioactive indium, rhenium and technetium
  • fluorescent dyes such as fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), Cyan fluorescent protein
  • the paramagnetic particles as well as the radioactive moieties can be provided within a chelator.
  • chelators include: tetraazacyclododecanetetraacetic acid (DOTA), mercaptoacetylglycylglycyl-glycine (MAG3), diethylenetriamine pentaacetic acid (DTPA) and N-l- ⁇ -isothiocyanatopheny ⁇ diethylenetriamine-N ⁇ N ⁇ N 3 - tetraacetate(DTTA).
  • D is a therapeutic agent selected from the group consisting of a cytotoxic compound, a cytostatic compound, an antisense compound and an anti-viral agent. According to some embodiments, D is a cytotoxic compound.
  • the cytotoxic compound D is selected from, but not restricted to DNA synthesis and function inhibitory agents (such as adriamycin, bleomycin, chlorambucil, cisplatin, carboplatin, oxaloplatin, daunomycin, ifosfamide and melphalan); microtubule (mitotic spindle) formation and function inhibitory agents (such as vinblastine, vincristine, vinorelbine, paclitaxel (taxol) and docetaxel); anti metabolites (such as cytarabine, fluorouracil, fluroximidine, mercaptopurine, methotrexate, gemcitabin and thioquanine); alkylating agents (such as mechlorethamine, chlorambucil, cyclophosphamide, melphalan and methotrexate); antibiotics (such as bleomycin and mitomycin); nitrosoureas (such as carmustine (BC).
  • the cytotoxic substance D is an anti-tumor agent.
  • the anti-tumor agent D is selected from the group consisting of adriamycin, bleomycin, chlorambucil, cisplatin, daunomycin and melphalan.
  • the anti-tumor agent is daunomycin.
  • the conjugate of the present invention may further comprise an affinity molecule.
  • the affinity molecule is attached to the albumin.
  • the affinity molecule is selected from the group consisting of at least the antigen-binding portion of a specific antibody and biotin.
  • n t is greater than 1 and D may be the same or different at each occurrence.
  • the present invention provides a diagnostic composition comprising an effective amount (e.g. an amount effective for enhancing image contrast in an imaging procedure) of a conjugate comprising at least one isoflavone derivative which is bound to albumin and at least one compound D which is an imaging agent.
  • a pharmaceutical composition comprising an effective amount of a conjugate comprising at least one isoflavone derivative which is bound to albumin and at least one compound D which is a therapeutic agent.
  • the present invention provides a method of targeting cells sensitive to isoflavone comprising administering to a subject in need thereof an effective amount of a conjugate comprising at least one isoflavone derivative which is bound to albumin and at least one bioactive moiety.
  • the invention provides a method for generating enhanced images of a human or animal body and generation of an image of at least part of said body.
  • the present invention provides a method for diagnosing cancer in a subject comprising administering to the subject a conjugate comprising at least one isoflavone derivative bound to albumin and at least one diagnostic compound for affinity targeting of the diagnostic compound to cells sensitive to isoflavone.
  • the cells are cancer cells.
  • diagnosis of tumors may be performed in vitro or in vivo.
  • diagnosis may be performed either directly or indirectly in an in vivo diagnostic imaging procedure e.g. by magnetic resonance imaging (MRI), near infrared (NIR), optical imaging, scintigraphy, SPECT, PET, X-ray, ultrasound imaging, electrical impedance or magnetometric procedures.
  • MRI magnetic resonance imaging
  • NIR near infrared
  • optical imaging scintigraphy
  • SPECT single photontigraphy
  • PET PET
  • X-ray X-ray
  • ultrasound imaging electrical impedance or magnetometric procedures.
  • the diagnostic imaging procedure is MRI.
  • the diagnostic imaging procedure is NIR.
  • in vivo diagnosis may be used to detect the presence of a disease state and/or monitor the progression of the disease.
  • the conjugates (compositions of matter) of the invention are useful diagnostic contrast agents for in vivo imaging of a mammalian body.
  • the present invention provides a method for treating cancer comprising administering to a subject in need thereof a therapeutical composition comprising a therapeutically effective amount of a conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound D being a therapeutic agent, thereby targeting and internalizing the conjugate to cells susceptible to isoflavone.
  • cancer treated by methods of the invention is selected from the group consisting of ovarian cancer, colon cancer, breast cancer, melanoma, non-small lung cancer, leukemia, endometrial cancer and prostate cancer.
  • the present invention provides a method for treating ovarian cancer.
  • the methods of the invention may be used to treat diseases other than cancer.
  • the disease other than cancer is an estrogen related condition such as osteoporosis, endometriosis, atherosclerosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy, obesity, menopausal syndromes, type-II diabetes, and Alzheimer's disease.
  • an estrogen related condition such as osteoporosis, endometriosis, atherosclerosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy, obesity, menopausal syndromes, type-II diabetes, and Alzheimer's disease.
  • FIG. IA shows the specific binding and endocytosis of daidzein-BSA-FAM by MLS ovarian carcinoma cells.
  • panel c overlay of panels a and b.
  • Fig. IB shows the specific binding and endocytosis of BSA-ROX and Daidzein-BSA-FAM by MLS ovarian carcinoma cells.
  • FIG. 2 shows the binding and endocytosis of BSA-ROX and Daidzein-B SA-FAM by
  • BSA-ROX uptake grey-control, black- BSA-ROX uptake, dark gray-
  • FIG. 3 Competition of Daidzein-BSA-FAM (light gray) by BSA-ROX (gray) in the absence (A) and presence of nystatin (B) (FLl filter for the collection of FAM fluorescence, FL2 filter for the collection of ROX fluorescence), C and D display control samples without and with nystatin respectively.
  • B FLl filter for the collection of FAM fluorescence
  • C and D display control samples without and with nystatin respectively.
  • E Statistical analysis of the competition assay. Change in percent of positively stained fluorescent cells in the absence and presence of nystatin (n) (light gray-Daidzein-B SA-FAM (dbf), dark gray- BSA-ROX (br)).
  • FIG. 4 shows the in vivo targeted delivery of daidzein-BSA-CyTE-777 to ovarian carcinoma tumors followed by NIR Imaging.
  • daidzein-BSA-CyTE-777 A
  • Control BSA-CyTE-777 B
  • daidzein-BSA-CyTE-777 + BSA-FAM 48 hours (C) and 72 hours (D) respectively
  • In Vivo Imaging of daidzein-BSA-CyTE-777 localization in tumor-free CD-I nude mice.
  • CD-I nude mice were injected intravenously with daidzein-BSA-CyTE-777 (left) or non injected (right). The NIR signal after 24 hours is shown.
  • FIG. 5 shows the biodistribution of daidzein-BS A-EuDTTA versus BSA-EuDTTA in MLS tumor bearing mice.
  • A. Europium signal 24h after injection (n 3-5).
  • FIG. 6 demonstrates that while Daidzein-BSA-FAM accumulates in tumor cells, BSA-FAM is localized in tumor blood vessels.
  • FIG. 7 demonstrates the advantages of using daidzein-B SA-GdDTPA as a contrast material to MRI.
  • Tl weighted MRI images and R 1 maps were obtained 24, 48 and 72h after injection, and used for derivation of mean R 1 values of the tumor;
  • the present invention relates to conjugates comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety including but not limited to a diagnostic agent (e.g. imaging agent) or a therapeutic agent and their use for the diagnosis and treatment of cancer.
  • a diagnostic agent e.g. imaging agent
  • a therapeutic agent e.g. a therapeutic agent
  • the present invention relates to the use of the conjugate comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety selected from an imaging agent for targeting of the imaging agent to cancer cells for diagnostic purposes.
  • the cancer cells are sensitive to isoflavone.
  • the present invention relates to the use of the conjugate comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety selected from a therapeutic agent for targeting of drugs to cancer cells for cancer treatment purposes.
  • the cancer cells are sensitive to isoflavone.
  • targeting means that the conjugate is able to induce the formation of a functional response (preferably the accumulation of a diagnostic agent or an anti-cancer drug in cells sensitive to isoflavone) in cancer cells preferentially over normal cells as compared with the diagnostic agents or drugs commonly used which are not given as part of the conjugate of the invention..
  • a "diagnostic agent” is a molecule or atom which is conjugated to a targeting moiety (e.g. isoflavone or isoflavone-albumin), to produce a conjugate, which is useful for diagnosis.
  • a targeting moiety e.g. isoflavone or isoflavone-albumin
  • diagnostic agents include paramagnetic particles; radioactive moieties; fluorescent dyes and infrared dyes.
  • the diagnostic agent is a contrast agent.
  • the diagnostic agent may be provided within a chelator.
  • the conjugate of the invention comprising isoflavone derivative bound to albumin and a diagnostic agent may be used as a contrast agent.
  • therapeutic agent refers to a molecule or atom which is conjugated to a targeting moiety (e.g. isoflavone or isoflavone-albumin conjugate) to produce a conjugate which is useful for therapy.
  • a targeting moiety e.g. isoflavone or isoflavone-albumin conjugate
  • therapeutic agents include cytotoxic compounds, cytostatic compounds, antisense compounds or anti-viral agents. Delivery of bioactive moieties to tumor cells:
  • the present invention is directed to a bifunctional targeted conjugate comprising isoflavone, albumin and an active agent (diagnostic and/or therapeutic agents) which effectively overcomes the tumor-blood barrier, thus increasing the delivery of the conjugate to the rumor cells.
  • tumor cells escape from delivery of extravasated albumin based compounds, through caveolae mediated sequestration of the albumin based compound by perivascular myofibroblasts, thus generating an effective tumor-blood barrier.
  • Evoking an alternate competing endocytic pathway using isoflavone resulted in significant partition of the isoflavone-albumin based compound (e.g. the conjugate of the invention) into the tumor.
  • the partition of the conjugate of the invention into the tumor could be further enhanced in the presence of free albumin (non conjugated albumin).
  • the partition of the conjugate of the present invention into the tumor could be further enhanced by the addition or co-administration of nystatin.
  • nystatin a substance that influences the partition of the conjugate through suppression of caveolae mediated endocytosis by competition with the conjugate, in the absence of direct modulation of the tumor interstitial pressure.
  • partition into a cell or “partition into a cancer cell” or “partition into a tumor cell” as used herein refers to the amount of bioactive moiety (diagnostic agent and/or therapeutic agent) found in the interior part of a cell versus the amount of the bioactive moiety found at the exterior part of the cell, specifically, within or on the cell's membrane.
  • the partition into a tumor cell of the bioactive moiety is at least 10% higher than the partition into a tumor cell of the bioactive moiety when administered alone and/or bound to albumin alone and/or bound to isoflavone alone; alternatively, at least 20% higher; alternatively at least 30% higher; alternatively, at least 40% higher; alternatively, at least 50% higher; alternatively, at least 60% higher; alternatively, at least 70% higher; alternatively, at least 80% higher; alternatively, at least 90% higher; alternatively, at least 2 times higher; alternatively at least 3 times higher.
  • the conjugate of the present invention displays prolonged tumor retention and/or increased circulation time as compared to tumor retention and circulation time of the bioactive agent alone, the bioactive agent bound to albumin alone, or the bioactive agent bound to isoflavone alone.
  • tumor retention refers to the duration of the presence of a sufficient amount of a bioactive moiety inside a tumor cell since the time it was administered to a subject, or introduced to the cell, or internalized or up taken by the cell.
  • the term "sufficient amount” implies a sufficient amount of a diagnostic agent that will enable detection (e.g. tumor detection) using various diagnostic methods, for example, the diagnostic imaging procedures described.
  • "sufficient amount” implies an amount of a therapeutic agent which is sufficient to cause a desired therapeutic effect.
  • the bioactive moiety of the conjugate is retained inside the tumor cell for at least 7 days; alternatively, for at least 5 days, alternatively, for at least 3 days, alternatively for at least 48 hours, alternatively, for at least 24 hours.
  • Cirulation time refers to the duration of the presence of the conjugate of the invention in the blood or plasma; specifically, to the duration of the presence of the bioactive moiety, which is part of the conjugate, in the blood or plasma.
  • the circulation time of the bioactive moiety is determined by its distribution, metabolism and its removal from the body via excretion.
  • circulation time may be referred to as the half-life of the bioactive moiety in the plasma.
  • Half- life refers to the period of time required for the concentration or amount of the bioactive moiety in the body to be reduced by one-half. A plasma half-life of a bioactive moiety depends on how quickly the bioactive moiety is eliminated from the plasma.
  • a bioactive moiety that leaves plasma may have any of several fates. It can be eliminated from the body, or it can be translocated to another body fluid compartment such as the intracellular fluid or it can be destroyed in the blood.
  • the removal of a the bioactive moiety from the plasma is known as clearance and the distribution of the bioactive moiety in the various body tissues is known as the volume of distribution. Both of these pharmacokinetic parameters are important in determining the circulation time or half life of a bioactive moiety.
  • the half life can be determined by giving a single dose, usually intravenously, and then measure the concentration of the bioactive moiety in the plasma at regular intervals. The concentration of the drug will reach a peak value in the plasma and will then fall as the bioactive moiety is hydrolyzed or broken down and cleared from the blood.
  • the circulation time or the half-life of a bioactive moiety is increased upon introduction or binding of the bioactive moiety to an isoflavone-albumin conjugate.
  • the circulation time or half-life of the bioactive moiety is at least 10% higher than the circulation time or half-life of the bioactive moiety when administered alone, bound to albumin, or bound to isoflavone; alternatively, at least 20% higher; alternatively at least 30% higher; alternatively, at least 40% higher; alternatively, at least 50% higher; alternatively, at least 60% higher; alternatively, at least 70% higher; alternatively, at least 80% higher; alternatively, at least 90% higher; alternatively, at least 2 times higher; alternatively at least 3 times higher.
  • Cl to C6 alkylene refers to a saturated, linear or branched hydrocarbon moiety, such as -CH 2 -, -CH 2 -CH 2 -; -CH 2 -CH 2 -CH 2 -; -CH 2 -(CH 2 ) 2 -CH 2 ; -CH 2 -(CH 2 ) 3 -CH 2 ; -CH 2 -(CH 2 ) 4 -CH 2 ;.
  • C2 to C20 alkenylene denotes a divalent group derived from a straight chain or branch hydrocarbon moiety containing from two to twenty carbon atoms having at least one carbon-carbon double bond.
  • Alkenylene groups include, but are not limited to, for example, ethenylene, 2-propenylene, 2- butenylene, l-methyl-2-buten-l-ylene, and the like.
  • heteroalkylene refers to an alkylene or alkenylene moiety having at least one heteroatom (e.g., N, O, or S). Preferred are heteroalkylenes having at least one O.
  • GIc denotes glucosyl or glucoside.
  • Isoflavone compounds Epidemiological in vitro as well as in vivo animal studies indicate that isoflavones present in large quantities in soybeans and soy products (e.g., genistein, daidzein) and red clover (biochanin A) are promising agents for cancer therapy, chemoprevention and inhibition of tumor progression (Aggarwal, B. B. at al, Biochem. Pharmacol. 2006, 71, 1397).
  • estrogen-sensitive cancer cell lines e.g., breast, colon, etc.
  • these isoflavone compounds can act as weak estrogens and stimulate cell growth at concentrations ranging from 0.1 to 20 ⁇ M.
  • the affinity of most isoflavones to the two subtypes of ER is low (Kuiper, G. G. at al, Endocrinology 1998, 139, 4252) with the exception of genistein, which shows a strong selectivity for Er ⁇ over Era.
  • Both genistein and daidzein display 100-fold greater sensitivity for activating transcription in transfected cells via Er ⁇ compared to Era (Harris, D. M. at al., Exp. Biol. Med.
  • isoflavones inhibit cell growth at concentrations greater than 20 ⁇ M.
  • isoflavone compounds display estrogenic effects (e.g. stimulate cell growth).
  • isoflavone compounds display anti-estrogenic effects (e.g. inhibit cell growth).
  • isoflavone compounds may be referred to as modulators of cell growth.
  • isoflavones show a wide spectrum of biological activities not ascribed to activation of the ER such as regulation of cell-signaling pathways, and can inhibit proliferation and induce apoptosis in ER-negative breast cancer cell lines as well as in ER-positive cell lines.
  • Examples include genistein which was shown to inhibit the protein-tyrosine kinase pathway and was further used in a treatment of choroidal neovascularization (U.S. Pat. No. 6,028,099). Genistein was also shown to display topoisomerase II activity, and to induce apoptosis and cell differentiation. Moreover, genistein has been shown to inhibit the proliferation of both cancer and normal cells, and was used for prophylactic treatment of cataract (WO 00/37066).
  • the 4-methoxy derivative of genistein, biochanin A, is equally potent to genistein as a growth inhibitor in breast cancer lines due to its conversion to genistein
  • Daidzein exhibits unique properties. Daidzein is known to interact with the lipid interface on the cell surface (Lehtonen J.Y et al. Biochim Biophys Acta, 1996 1285: 91-100) thus facilitating endocytosis. Furthermore, acting as a weak estrogen, daidzein may recognize a putative plasma membrane estrogen receptor (Somjen D. et al. J Steroid Biochem MoI Biol, 2005 93: 293-303), a membranal ER ⁇ -related protein (De Wilde A. et al. J Cell Physiol, 2006 209: 786- 801) or a nuclear estrogen receptor of the ⁇ -type.
  • albumin includes human serum albumin, animal albumin, recombinant albumin, and fragments thereof. Additionally, the albumin may exist as a monomer, a dimer, a polymer, or may be enclosed in microspheres. Albumin as disclosed herein may also be treated with polyethylene glycol (PEG) by well-known techniques to increase its immunocompatibility. According to one embodiment, albumin comprises human serum albumin. According to another embodiment, albumin comprises bovine serum albumin (BSA). It is to be appreciated that species-specific serum albumin is necessary for compatibility reasons. Thus, when treating or diagnosing human patients, human serum albumin may preferably be used and when treating or diagnosing other animals, the serum albumin should likewise be species-specific, e.g., bovine serum albumin for treating or diagnosing cattle.
  • BSA bovine serum albumin
  • the albumin in the conjugate is used as a carrier.
  • the isoflavone carboxy derivatives and the bioactive moieties according to the invention may be conjugated to the albumin carrier by any means know in the art, non-limiting examples include: conjugation through the albumin's lysine residues (amino groups), conjugation through the N-terminus and conjugation through cy stein residues (thiol groups).
  • amino groups conjugation through the albumin's lysine residues
  • thiol groups conjugation through cy stein residues
  • amino, hydroxyl and hydrazine groups can each form a covalent bond with a reactive carbonyl group (e.g., a carboxylic acid chloride or activated ester such as an N-hydroxysuccinimide ester (NHS)).
  • a reactive carbonyl group e.g., a carboxylic acid chloride or activated ester such as an N-hydroxysuccinimide ester (NHS)
  • carboxy derivatives of isoflavones as well as bioactive moieties may be coupled to an amino group of albumin (e.g. lysine group) via N-hydroxysuccinimide ester.
  • N-sulfo succinimide and a water soluble carbodiimide may be used for coupling a carboxy group of the isoflavone or of the bioactive moiety to an amino group of albumin through the formation of a peptide linkage.
  • the conjugate of the invention comprises a bioactive moiety selected from a diagnostic agent and a therapeutic agent.
  • the bioactive moiety is a therapeutic agent selected from the group consisting of a cytotoxic compound, a cytostatic compound, an antisense compound, an anti-viral agent.
  • the cytotoxic compound is selected from, but not restricted to DNA synthesis and function inhibitory agents; microtubule (mitotic spindle) formation and function inhibitory agents; anti metabolites; alkylating agents; antibiotics; nitrosoureas; hormones and proteins.
  • the therapeutic agent is an anti-tumor agent.
  • anti-cancer reagent refers to any type of reagent that may be used in the treatment of cancer and/or cancer related conditions.
  • the anti-cancer reagent may include any naturally occurring or synthetically produced molecule that is capable of affecting directly or indirectly the growth and/or viability of cancer cells.
  • the anti-cancer reagent may include, for example, a naturally occurring protein or peptide, a modified protein or peptide, a recombinant protein, a chemically synthesized protein or peptide, a chemical molecule, a synthetic chemical molecule, a chemotherapeutic drug, a biologically therapeutic drug, and the like, or any combination thereof.
  • the anti-cancer reagent may be cytotoxic (toxic to cells) and/or cytostatic (suppress cell growth) and/or antiproliferative to the cancer cells and may exert its effect on cancer cells directly and/or indirectly.
  • Non limiting examples of anti-cancer agents and chemotherapeutic drugs may include such drugs as, but not limited to: Alkaloids, such as, but not limited to: Docetaxel, Etoposide, Irinotecan, Paclitaxel, Teniposide, Topotecan, Vinblastine, Vincristine, Vindesine; Alkylating agents, such as, but not limited to: Busulfan, Improsulfan, Piposulfan, Benzodepa, Carboquone, Meturedepa, Uredepa, Altretamine, triethylenemelamine, Triethylenephosphoramide,
  • Alkaloids such as, but not limited to: Docetaxel, Etoposide, Irinotecan, Paclitaxel, Teniposide, Topotecan, Vinblastine, Vincristine, Vindesine
  • Alkylating agents such as, but not limited to: Busulfan, Improsulfan, Piposulfan, Benzodepa, Carboquone, Meturede
  • taxanes including, but not limited to vincristine (Oncovin), vinblastine (Velban), paclitaxel (Taxol, Paxene), vinorelbine (Navelbine), epothilone B or D or a derivative of either, and discodermolide or its derivatives, nitrosoureas including, but not limited to procarbazine (Matulane), lomustine, CCNU (CeeBU), carmustine (BCNU, BiCNU, Gliadel Wafer), and estramustine (Emcyt); nucleoside analogs including, but not limited to mercaptopurine, 6-MP (Purinethol), fluorouracil, 5-FU (Adrucil), thioguanine, 6-TG (Thioguanine), hydroxyurea (Hydrea), cytarabine (Cytosar-U, DepoCyt), floxuridine (FUDR), fludarabine (Flu
  • the bioactive moiety is a diagnostic agent selected from the group consisting of paramagnetic particles; radioactive moieties; dyes, fluorophores, and infrared dyes.
  • Typical diagnostic radioactive moieties include " 01 Tc, 95 Tc, 111 In, 62 Cu, 64 Cu, 67 Ga, and 68 Ga; and therapeutic radioactive moieties include 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 212 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 67 Cu, 105 Rh, 111 Ag, and 192 Ir.
  • Typical paramagnetic particles include rare earth metals, typically, gadolinium, manganese, yttrium, ytterbium, europium, lutetium, gallinium and the like. Iron ions may also be used.
  • Preferred fluorophore moieties include fluorescent dyes having (a) high fluorescence intensity; (b) sufficiently long excitation and emission wavelength maxima so that interference from natural fluorescence of either diseased or normal tissue is minimized; (c) sufficiently long measured fluorescence decay time to allow accurate measurement of emitted light over background fluorescence and scattering (at least about 2, preferably at least about 10 nanoseconds); and (d) high degree of fluorescence polarization.
  • Fluorophores include macrocyclic fluorescent dye compounds, especially compounds having aromatic ⁇ -electron systems.
  • fluorophore moieties may comprise a substantially planar multidentate macrocyclic ligand and may be coordinated to a complexing central ion or atom such as aluminum, phosphorous, and the group FVB elements, e.g. silicon, germanium, and tin.
  • suitable fluorophores include coumarin dyes, nitrobenzoxazole dyes, cyanine dyes, dipyrrometheneboron dyes, xanthene dyes (including the benzo- and naphtho- xanthene dyes), phenoxazine dyes (as well as the benzo- and naphtho-phenoxazine dyes) and compounds from other classes of dyes well known to those of skill in the art.
  • fluorophores include the fluorophores in the following non- exclusive list: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7- dichlorofluorescein; 5-Carboxyfluorescein (5 -FAM); 5-Carboxynapthofluorescein; 5- Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5 -Hydroxy Tryptamine (HAT); 5 -ROX (carboxy-X- rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6- JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7- Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-meth
  • Infrared dyes include cyanine dyes.
  • cyanine dyes include l,r,3,3,3',3'-Hexamethylindotricarbocyanine iodide; 1,1',3,3,3',3'- Hexamethylindotricarbocyanine perchlorate; l,l'-Diethyl-2,2'-dicarbocyanine iodide; l,l'-Diethyl-2,2'-quinotricarbocyanine iodide; l,l'-Diethyl-4,4'-carbocyanine iodide; l,l '-Diethyl-4,4'-dicarbocyanine iodide; l,3-Bis[4-(dimethylamino)-2- hydroxyphenyl]-2,4-dihydroxycyclobutenediylium dihydroxide, bis(inner salt); 1 ,4,8, 11,15,
  • the infarared dye is CyTEE-777.
  • suitable cyanine dyes are described in U.S. Patent No. 7,514,069, the contents of the aforementioned reference are incorporated by reference herein in their entirety as if fully set forth herein.
  • the paramagnetic particles as well as the radioactive moieties can be provided within a chelator.
  • chelators include: tetraazacyclododecanetetraacetic acid (DOTA), mercaptoacetylglycylglycyl-glycine (MAG3), diethylenetriamine pentaacetic acid
  • DTTA l,4,7-triaza-cyclononane-N-N'-N"-triacetic acid
  • TETA tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid
  • the conjugate comprising at least one isoflavone derivative bound to albumin and at least one diagnostic compound is selected from the group of 7-(O)-carboxymethyl daidzein-albumin-CyTE-777, 7-(O)- carboxymethyl daidzein-albumin-GdDTPA, 7-(O)-carboxymethyl daidzein-albumin- FAM and 7-(O)-carboxymethyl daidzein-albumin-EuDTTA.
  • the present invention provides methods for the diagnosis and treatment of cancer in a subject comprising administering a conjugate comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety selected from a diagnostic agent and/or a therapeutic agent.
  • the conjugate of the invention is useful for targeting cells sensitive to isoflavone, preferably, cancer cells sensitive to isoflavone.
  • the cancer may include solid tumors, non-solid tumors, primary tumors, metastasis or any combination thereof.
  • the cancer may include carcinomas, sarcomas, myelomas, leukemias, lymphomas or any combination thereof.
  • the cancer may include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, a metastasis thereof or any combination thereof.
  • the cancer may include hepatocellular carcinoma, hematoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma
  • the cancer comprises cancer cells sensitive to isoflavone.
  • cancer may include ovarian carcinoma, colon cancer, breast cancer, melanoma, non-small lung cancer, leukemia, endometrial cancer and prostate cancer.
  • the cancer is ovarian cancer.
  • the cancer is breast cancer.
  • the cancer is colon cancer.
  • the cancer is prostate cancer.
  • detecting or imaging cancer cells in a subject comprising (a) administering to the subject an effective amount of a conjugate comprising at least one isoflavone derivative bound to albumin and at least one diagnostic agent; and (b) detecting or imaging cells that take up the conjugate to determine if cancer is present in a subject.
  • Detection of the conjugate can be performed using essentially any detection device to obtain an image of the cancerous cells.
  • diagnosis may be performed either directly or indirectly in an in vivo diagnostic imaging procedure e.g. by magnetic resonance imaging (MRI), near infrared (NIR), optical imaging, scintigraphy, SPECT, PET, X-ray, ultrasound imaging, electrical impedance or magnetometric procedures.
  • MRI magnetic resonance imaging
  • NIR near infrared
  • optical imaging scintigraphy
  • SPECT single photontigraphy
  • PET PET
  • X-ray X-ray
  • ultrasound imaging electrical impedance or magnetometric procedures.
  • the diagnostic imaging procedure is MRI. According to other embodiments, the diagnostic imaging procedure is NIR. According to some embodiments, in vivo diagnosis may be used to detect the presence of a disease state and/or monitor the progression of the disease. According to some embodiments, the conjugates of the invention are useful diagnostic contrast agents for in vivo imaging of a mammalian body.
  • the diagnostic conjugates of the present invention can be used in a method which comprises administering to a subject's body, or to selected regions thereof, effective amounts of the diagnostic conjugate.
  • the agent can be injected directly into the vicinity of the body to be imaged, or it can be intravenously, intradermaly, intralesionaly, intramusculary, intravesiculary, subcutaneously injections or gastrointestinally administered. Then, an image of the body or of the indicated region is obtained using conventional imaging equipment and techniques as described above.
  • the image thus produced by that equipment can optionally be fixed in hard copy form, in photographic negative or positive form, or stored within a computer memory for subsequent recall onto a screen or for subsequent conversion into hard copy.
  • a “diagnostically effective amount” as used herein refers to a dose of the diagnostic conjugate, or diagnostic agent that is sufficient for detection (e.g. tumor detection) using the various diagnostic methods described.
  • Typical doses of the diagnostic conjugates are in the range from about 0.001 to about 20 mmol/kg body weight, and preferably in the range from about 0.005 to about 5 mmol/kg body weight.
  • the diagnostically effective amount may be different depending on the diagnostic agent attached to the conjugate of the invention as well as the diagnostic apparatus used for detection.
  • the present invention provides a method for site targeted chemotherapy in a subject in need thereof comprising administering to the subject a therapeutical composition comprising a therapeutically effective amount of a conjugate comprising at least one isoflavone derivative bound to albumin and at least one cytotoxic compound for targeting of the cytotoxic compound to cancer cells.
  • a therapeutical composition comprising a therapeutically effective amount of a conjugate comprising at least one isoflavone derivative bound to albumin and at least one cytotoxic compound for targeting of the cytotoxic compound to cancer cells.
  • the cancer cells are sensitive to isoflavone.
  • therapeutically effective amount refers to that amount of the therapeutic conjugate of the invention which elicits a biological or medicinal response that will contribute to the cancer-treating ability of the therapeutic conjugate.
  • treating refers to partial or total inhibition of the growth, spreading, or metastasis of cancer, as well as partial or total destruction of the cancer cells.
  • treating includes the reduction or elimination of cancer, and also the reduction in the incidence of the disease.
  • the present invention provides pharmaceutical composition i.e., diagnostic and therapeutic compositions, comprising as an active ingredient a conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound selected from a diagnostic agent and a therapeutic agent according to embodiment of the invention and a pharmaceutically acceptable carrier, excipient or diluent.
  • a "diagnostic composition” refers to a preparation of one or more of the active ingredients described herein, e.g. a conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound which is a diagnostic agent, with other components such as physiologically suitable carriers and excipients.
  • the purpose of a diagnostic composition is to facilitate the administration of the conjugate of the invention to a subject for diagnostic purposes.
  • a "therapeutic composition” refers to a preparation of one or more of the active ingredients described herein, e.g. a conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound which is a therapeutic agent, with other components such as physiologically suitable carriers and excipients.
  • the purpose of a therapeutic composition is to facilitate the administration of the conjugate of the invention to a subject for treatment purposes.
  • therapeutically acceptable carrier and “pharmaceutically acceptable carrier”, which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • excipient refers to an inert substance added to a diagnostic or a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • a diagnostic or a pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • a carrier refers to any substance suitable as a vehicle for delivering a conjugate of the present invention to a suitable in vivo or in vitro site.
  • carriers can act as a pharmaceutically acceptable excipients of a diagnostic or a therapeutical composition containing a conjugate of the present invention.
  • Preferred carriers are capable of maintaining a conjugate of the present invention in a form that, upon arrival of the conjugate to a cell, the conjugate is capable of entering the cell.
  • Non limiting examples of suitable carriers include: water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters and glycols.
  • Aqueous carriers can contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity.
  • Suitable auxiliary substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer.
  • Auxiliary substances can also include preservatives, such as thimerosal, m- and o-cresol, formalin and benzol alcohol.
  • Preferred auxiliary substances for aerosol delivery include surfactant substances non-toxic to a subject, for example, esters or partial esters of fatty acids containing from about six to about twenty-two carbon atoms.
  • esters include: caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric, and oleic acids. Diagnostic and therapeutic compositions of the present invention can be sterilized by conventional methods.
  • composition for use in accordance with the present invention thus may be formulated in conventional manner using one or more acceptable diluents or carriers comprising excipients and auxiliaries as described, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Proper formulation is dependent on the route of administration chosen.
  • the diagnostic and therapeutic composition of the present invention may be formulated for administration by a route selected from the group consisting of intravenous injections, intravenous infusion, intradermal, intralesional, intramuscular, intravesicular and subcutaneous injections or depots.
  • the composition of the invention may be administered parenterally by means other than injection, for example, they could be introduced laparascopically, intravesicularly, or via any orifice not related to the gastrointestinal tract.
  • the compositions of the present invention may be formulated for oral administration.
  • the diagnostic and therapeutic compositions can take the form of solutions, suspensions, emulsions, syrups, gels, tablets, pills, capsules, powders, suppositories and the like.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • Such compositions will contain an effective amount of the conjugate of the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • composition of the present invention may be manufactured by processes well known in the art, e.g. by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • albumin conjugate isoflavone derivative bound to a bioactive moiety such as an imaging agent or a therapeutic agent for selective delivery to cells susceptible to isoflavone according to the present invention
  • BSA BSA
  • nystatin diethylenetriamine pentaacetic acid (DTPA) anhydride
  • Carboxyfluorescein succinimide ester (FAM-NHS)
  • Carboxy-X-rhodamin succinimide ester (ROX-NHS) - Molecular Probes, Invitrogen (California, USA).
  • the conjugates were prepared in a three-step procedure.
  • both the carboxy derivative of daidzein and the imaging agent were treated with N-hydroxysuccinimide and carbodiimide to form an active ester.
  • the activated ester of the imaging agent reacted at pH 8.5 with the amino group of lysine residues of BSA to form a BSA-imaging agent conjugate.
  • the activated ester of daidzein reacted at pH 8.5 with the amino group of lysine residues of BSA to form a daidzein-BSA-imaging agent conjugate
  • BSA-DTPA was synthesized as described (Dafni et al., 2003, Magn. Reson. Med.; 50:904-14). Briefly, DTPA anhydride 1.6 gr (suspended in 4 ml of dry DMF) was slowly added with stirring to BSA 1.3 gr (in 40 ml Hepes 0.1 M, pH 8.8) while the reaction was titrated with NaOH 5N and stirred for 2 hours.
  • the product was dialyzed against NaHCO 3 . Afterwards, the N- hydroxysuccinimide ester of 7-(O)-carboxymethyl (daidzein-NHS), synthesized according to Kohen et al, 2007, J Med. Chem.; 50:6405-10, (11 mg in 800 ⁇ l of anhydrous DMF) was added to BSA-DTPA (in 40 ml OfNaHCO 3 , 0.1M, pH 8.5) and stirred overnight. The product was dialyzed first against NaHCO 3 , followed by sodium citrate (0.1 M, pH 6.5).
  • GdCl 3 650 mg (in 5 ml sodium acetate 0.1M, pH 6.0) was added to Daidzein-B SA-DTPA in sodium citrate (0.1 M pH 6.5). The final product (Daidzein-BSA-GdDTPA) was dialyzed extensively against water and lyophilized.
  • CyTE-777-NHS (44 mg) prepared according to Hilderbrand et al., 2005, Bioconjug. Chem.; 16:1275-81, or FAM-NHS (35 mg) or ROX-NHS (35 mg) (in 200 ⁇ l of dry DMF) were added to BSA (450 mg in 15 ml NaHCO 3 , 0.1M, pH 8.5), slowly with stirring. The reaction was stirred overnight, and the product was dialyzed against NaHCO 3 , 0.1M, pH 8.5, followed by several changes of water and lyophilized.
  • N-hydroxysuccinimide ester of 7-(O)-carboxymethyl (daidzein-NHS), (8 mg in 600 ⁇ l of anhydrous DMF) was added to BSA-CyTE-777 (100 mg in 6 ml of NaHCO 3, 0.1M, pH 8.5) and stirred overnight.
  • the product was purified by dialyzed against NaHCO 3 0.1M, pH 8.5, followed by extensive dialysis against water and lyophilized.
  • Example 2 In vitro evaluation of the endocytic pathways in ovarian cancer cells: a. A fluorescence microscopy study:
  • MLS cells (2*10 5 ) were cultured on polylysine coated coverslips for 48h. Subsequently, the coverslips were incubated for 1 hour at 37 with daidzein-BSA- FAM (200 ⁇ g/ml) or BSA-FAM (200 ⁇ g/ml) or combination of both in the presence and absence of a blocking dose of nystatin (50 ⁇ g/ml). The excess of fluorescent material was washed 3 times with PBS, and the cells were fixed with 4% PFA, washed, stained with DAPI and mounted.
  • daidzein-BSA- FAM 200 ⁇ g/ml
  • BSA-FAM 200 ⁇ g/ml
  • nystatin 50 ⁇ g/ml
  • FIG. IA Upon addition of nystatin (50 ⁇ g/ml, 30 min), an elevated membrane staining and reduced uptake of BSA-FAM was observed (FIG. IA, B), whereas a significant enhanced internalization of daidzein-B SA-FAM by MLS cells was observed (FIG. IA).
  • MLS cells (10 6 ) were incubated for 30 minutes with daidzein-BSA-FAM (200 ⁇ g/ml) or BSA-ROX (200 ⁇ g/ml) or a combination of both in the presence and absence of nystatin (50 ⁇ g/ml).
  • the excess of fluorescent material was washed three times with PBS containing 0.01% of sodium azide.
  • the cellular uptake of fluorescent material was monitored by FACScan (Becoton Dickinson, USA).
  • the excitation produced by air-cooled argon laser 288 nm and the emission signal was collected by FLl filter (BP530/30) for FAM and FL2 filter (BP585/42) for ROX.
  • Example 3 In vivo targeted delivery of daidzein-BSA-CyTE-777 to ovarian carcinoma cells:
  • the NIR signal in the whole animal was monitored by IVIS 100 and IVIS Spectrum (Xenogen, Caliper) at 24, 48 and 72 hours.
  • mice were fed with alfalfa-free (chlorophyll-free) diet 72 hours before visualization.
  • the data was acquired by 710-760 excitation, 675-720 excitation background and 810- 860 emission filters, hi the IVIS Spectrum the data obtained for the daidzein-BSA- CyTE-777 was acquired by 745 nm excitation and 820 nm emission filters.
  • the pharmacokinetics of the fluorescent and MRI BSA based contrast media and their plasma concentration after intravenous administration were previously reported to be similar for all tags (Dafni et al. 2003, Magn. Reson. Med., 50:904-14).
  • mice were inoculated subcutaneously with 2.5* 10 6 MLS tumor cells.
  • mice administered with daidzein-BSA-CyTE777 or BSA-CyTE777 were inoculated subcutaneously with 2.5* 10 6 MLS tumor cells.
  • mice administered with daidzein-BSA-CyTE777 or BSA-CyTE777 were inoculated subcutaneously with 2.5* 10 6 MLS tumor cells.
  • mice administered with daidzein-BSA-CyTE777 or BSA-CyTE777 were administered with daidzein-BSA-CyTE777 or BSA-CyTE777.
  • mice injected with daidzein-BSA-CyTE777 showed more specific localization in the tumor and in higher concentration than mice administered with BSA-CyTE777.
  • Prolonged enhancement of the fluorescent signal in the tumor area was further observed in mice that were injected with both daidzein- BSA-CyTE-777 and the non-specific competitor - BSA-FAM.
  • the NIR signal in the tumors was detectable even 10 days after the administration.
  • mice were injected intravenously with BSA-Eu chelate (0.34 mg equivalent to 100000 counts of europium in 0.1 ml PBS/mouse) or daidzein-BSA-Eu chelate (0.24 mg of protein equivalent to 100000 counts of europium in 0.1 ml PBS/mouse).
  • the Eu chelates were used here due to their chemical similarity to the Gd chelates used for MRI. After 24 and 48 hours the mice were sacrificed and tumors as well as various tissues were removed.
  • the tissues were subsequently homogenized in RIPA buffer ((20 mM Tris, pH 7.4, 137 mM NaCl, 10% glycerol, 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) sodium dodecyl sulfate (SDS), 1% Triton X-100, 2 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail) and centrifuged in an airfuge.
  • the protein content in supernatant was first quantified by Bradford assay and then used for biodistribution studies and Western blot analysis.
  • the membrane was blocked over night at 4C with 2% BSA in 10 mM Tris- buffered saline containing 0.05% Tween (TBST), followed by incubation with anti- daidzein antibody IgG (1 mg/ml TBS) for 2 hours at 25C. The membrane was then washed three times in TBST, incubated with an HRP-conjugated goat anti mouse antibody (1:10000 in TBS) and re- washed. The protein bands were visualized by ECL.
  • TBST Tris- buffered saline containing 0.05% Tween
  • the tumor bearing mice were injected intravenously with BSA-GdDTPA (12 mg in 200 ⁇ L PBS/mouse) or daidzein-BSA-GdDTPA (12 mg in 200 ⁇ L PBS/mouse) or with combination of daidzein-BSA-GdDTPA and BSA-FAM (competition experiment).
  • Rl was measure 24, 48 and 72 hours after injection of the contrast material.
  • Tl weighted 3D gradient-echo (GE) images, with pulse flip angles of 5°, 15°, 30°, 50° and 70° were acquired for the determination of Rl values.
  • the acquisition parameters used TR 10 ms; TE 3.561 ms; 2 averages; field of view 4x4x4; 128x128x128 pixels.
  • Three-dimensional gradient-echo data sets were used for generation of Rl maps as well as for calculation of the average Rl values in selected regions of interest by nonlinear best fit to equation [H]:
  • I M 0 sin ⁇ (1 -e "TR*Rl ) / (1 - cos ⁇ e "TR*Rl ) (II)
  • I is the signal intensity as a function of the pulse flip angle. Student's t test (two tailed, equal variance) was used for statistical analysis of the significance of change in relaxation rate between control and labeled tumors.
  • Daidzein-BSA-GdDTPA showed significant relaxivity of 194 mM ' V 1 (per BSA; FIG. 7A). This relaxivity was similar to the relaxivity of BSA-GdDTPA 196 mM ' V 1 (per BSA).
  • Time course experiments showed specific localization and retention of daidzein-BSA- GdDTPA in the tumor site 24, 48 and 72 hours after injection as compared to controls or mice injected with BSA-GdDTPA.
  • Prolonged detection of daidzein-BSA- GdDTPA was feasible even for a lower administrate dose.
  • the specific localization of the targeted contrast agent in the tumor site was detected by MRI at 9.4T 24h after injection (FIG. 7D) and was still detectable and even enhanced 7 days after injection (FIG. 7E).

Abstract

The present invention relates to conjugates comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety including a diagnostic agent or a therapeutic agent, the conjugate being capable of delivering the bioactive moiety into a tumor cell. The invention further relates to the use of said conjugate for the diagnosis and treatment of cancer.

Description

MULTIFUNCTIONAL ALBUMIN CONJUGATES
FIELD OF THE INVENTION
The invention relates to conjugates comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety including a diagnostic agent or a therapeutic agent, the conjugate being capable of delivering the bioactive moiety into a tumor cell. The invention further relates to the use of said conjugate for the diagnosis and treatment of cancer.
BACKGROUND OF THE INVENTION
The detection of a target site benefits from a high signal-to-background ratio of detection agent. Therapy benefits from higher percentages of the administered therapeutic agent reaching the target site, as well as a reasonably long duration of uptake and binding. Furthermore, drug targeting spares normal cells and significantly diminishes drug-toxicity.
Chemotherapy constitutes one of the major therapeutic approaches for the treatment of cancer, along with surgery and radiotherapy. However, the usefulness of commonly used anti-cancer drugs such as daunomycin and adriamycin is severely limited by their toxicity towards normal tissues, particularly the myocardium and the rapidly proliferating cells of the gastrointestinal tract and bone marrow. In addition, these drugs are affected by the mechanisms of multi-drug resistance. Affinity targeting of these cytotoxic drugs to tumor cells offers an approach that might overcome some of these drawbacks.
The targeting ratio and amount of agent delivered to a target site can be improved using targeting agents conjugated to diagnostic or therapeutic agents for preferential localization. A variety of materials have been developed over the years for targeted delivery of diagnostic agents and drugs. Examples include specific targeting of tumor cells with monoclonal antibody-drug conjugates (U.S. Patents No.
5,846,545), avidin-biotin conjugates (U.S. Patent No. 5,525,338), the use of proteins or peptide hormones for which specific receptors are located on membranes of tumor cells as carriers or targetors of cytotoxic drugs, for example, liposomes coupled to glycoproteins (U.S. Patent No. 7,005,139), conjugates of a targeting protein such as transferrin and a drug (U.S. Patent No. 7,001,991), growth factors coupled to anthracycline drugs (WO 88/00837) or melanocyte-stimulating hormone (MSH) (Varga JM et al. 1977 Nature 276:56-58) and luteinizing hormone releasing hormone (LHRH) analogues (Nagy A. et al. 1996 Proc. Natal. Acad. ScL USA 93:7269-7273), conjugated to cytotoxic drugs for targeted chemotherapy of cancers that possess membranal receptors.
Site directed chemotherapy utilizing nuclear receptors (e.g. estrogen receptors) is not well documented. In fact, few studies have been described on the use of estrogen-cytotoxic drug conjugates (e.g. Estracyt, Leo 299; Heiman et al. 1980 J. Med. Chem. 23:994-1002) for affinity therapy, and success with such steroid-drug conjugates has been rather limited. Some inventors of the present invention have recently disclosed the use of carboxy derivatives of isoflavones such as biochanin A, daidzein, formononetin and genistein as selective targeting agents of therapeutic agents to cells having estrogen receptors (International publication WO 2003/079965). Specifically, it was shown that linking the cytotoxic drug daunomycin to the 7-(O)-carboxymethyl derivative of the isoflavone daidzein resulted in an active anti-cancer conjugate both in vitro and in vivo. This daidzein-daunomycin conjugate retained daunomycin' s cytotoxic effects in an ovarian xenograft while averting its toxicity in the myocardium (Somjen, D. et al. J Steroid Biochem. MoI. Biol. 2008 110:144-149).
The delivery of drugs by the intravascular route has also been limited to the administration of soluble drugs because of the danger of embolism formation when insoluble particles are injected. To circumvent some of these problems, methods of microencapsulating drugs in gelable hydrophilic colloids, incorporating active drug in the internal structure of a metabolizable "protein-like" carrier and formulations of polylactide and drug have been described. In yet another suggested method albumin was used as a carrier for drugs taking advantage of its central role in affecting the pharmacokinetics of chemotherapeutics and imaging agents. When using the albumin delivery system intravascularly no emboli formation was detected (U.S. Pat. No. 4147767). Furthermore, water insoluble drugs which formerly could not be administered in this manner because of the risk of embolus formation could be safely given by entrapping them in albumin spherules. Other advantages of albumin spherules as medicament carriers include their ease of preparation, complete removal from the body by metabolism, nonantigenicity, proven safety for intravascular administration and the capability of accommodating a wide variety of drug molecules in a relatively non-specific fashion. The use of albumin as a carrier of imaging probes has been described (U.S. Pat. No. 4,337,240; Kobayashi H and Brechbiel M.W. 2005 Adv. Drug Deliv. Rev. 57 2271- 2286). In a recent study a biotin-BS A-GdDTPA was used for tracking tumor stroma fibroblasts by MRI. Remarkably, histological staining by the biotinylated albumin based marker showed high specificity to myofibroblast stroma tracks. However subsequent in vivo studies pointed out the exclusion of the contrast medium from the tumor nodules, suggesting that the interaction of therapeutics or contrast medium with albumin could hinder their delivery to the tumor cells. It is also well known in the art that albumin is used as an immunogenic carrier for small molecules which are usually non immunogenic. Wang et al. J. Agric. Food. Chem., 1994, 42:1584-7, disclosed the preparation of a BSA-formononetin conjugate for the preparation of anti-formononetin antibodies.
There is a recognized need for more efficient approaches for the detection and treatment of cancer, and it would be highly advantageous to have improved probes, which can be used for affinity targeting of both therapeutic and imaging agents.
SUMMARY OF THE INVENTION
The present invention relates to conjugates comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety including but not limited to a diagnostic agent or a therapeutic agent, said conjugates being capable of delivering the bioactive moiety into a cell and their use for the diagnosis and treatment of cancer. The present invention also provides improved targeting methods where internalization and cellular retention of a bioactive moiety (e.g. a diagnostic or a therapeutic agent) are enhanced by binding of the bioactive moiety to an isoflavone- albumin conjugate. It is now disclosed that is the conjugates of the present invention are unexpectedly internalized in target cells to a greater extent and/or faster than without such internalization agent (e.g. isoflavone-albumin).
The present invention is based in part on the unexpected discovery that the conjugation of carboxy isoflavone derivatives to albumin significantly increase the uptake of the conjugate by cancerous cell lines sensitive to isoflavone. Specifically it was shown, that an imaging agent attached to a daidzein-BSA conjugate was specifically localized in ovarian carcinoma tumor cells, and displayed prolonged retention in the tumor of up to 7 days after administration as compared to the same imaging agent attached to BSA alone. Furthermore, unexpectedly, addition of nystatin, which usually inhibits BSA cellular uptake, resulted in significant augmentation of the cellular uptake of the daidzein-BSA conjugate. Without wishing to be bound to any theory or mechanism of action it is hypothesized that the conjugate is regulating endocytosis by cancerous cell lines sensitive to isoflavone, involving competition between receptor mediated internalization through the binding of the carboxy isoflavone derivative and caveolae mediated internalization via binding of albumin.
"Cells sensitive to isoflavone" as used herein refers to cells whose growth may be affected by isoflavones or by carboxy derivatives of isoflavones. At certain concentrations the isoflavones may enhance growth, while at other concentrations the isoflavones may result in inhibition of cancer cell growth. According to one embodiment, cells sensitive to isoflavone are cancer cells. Non limiting examples to cancer cells which are sensitive to isoflavones include ovarian carcinoma cells, colon cancer cells, breast cancer cells, melanoma, non-small lung cancer, leukemia, endometrial cancer and prostate cancer cells. According to one embodiment, cells sensitive to isoflavone may also be estrogen sensitive cells. According to another embodiment, cells sensitive to isoflavone are cells expressing an estrogen receptor. According to yet another embodiment the cells sensitive to isoflavone are cells expressing a nuclear or putative membranal estrogen receptor subtype β. According to a further embodiment, cell sensitive to isoflavone are cells susceptible to isoflavone.
According to one aspect, the present invention provides a conjugate comprising at. least one isoflavone derivative bound to albumin and at least one compound D, the conjugate represented by the structure of general formula (I): Albumin — Dn1
Figure imgf000006_0001
(I) wherein m is 1-20; ni is 0-10; n2 is 0 or 1 ;
R1 is selected from the group consisting of -OH, -OCH3, -OGIc, -OR7COOX and -OR7C(=O)-;
R2, R5 and R6 are each independently selected from the group consisting of -H, -R7COOX and -R7C(=0)-;
R3 is selected from the group consisting of -H, -OH, -R7COOX, -OR7COOX, -R7C(O)- and -0R7C(=0)-; R4 is selected from the group consisting of -H, -CH3, -R7COOX and -R7C(=0)-;
D is a active agent selected from the group consisting of a diagnostic agent (e.g. an imaging agent) and a therapeutic agent;
R7 is selected from the group consisting Of Cj-C6 alkylene, (C2-C2o) alkenylene; and (C2-C20) heteroalkylene;
X is selected from the group consisting of -H and -(CH2)n-Y wherein Y is -CH3 or -NH2 and n is 0-10; with the proviso that one of R1, R2, R3, R4, R5 or R6 comprises a carboxy group selected from -0R7C(=0)- and -R7C(=O)- which is bound to albumin, and at least one of ni and n2 is other than zero. According to one embodiment, the conjugate of the invention is capable of delivering the bioactive moiety into a cell sensitive to isoflavone. According to another embodiment, the conjugate displays improved properties having at least one of increased partition into a cancer cell (e.g. tumor cell), prolonged tumor retention or increased circulation time. The conjugates of the invention comprising an isoflavone bound to albumin and a bioactive moiety bound to the isoflavone, albumin or both, have improved qualities selected from partition to the tumor, tumor retention and circulation time compared to the bioactive moiety alone, the bioactive moiety bound to albumin, or the bioactive moiety bound to isoflavone. According to some embodiments of the present invention, the isoflavone derivative is selected from a derivative of diadzein, genistein, formononetin and biochanin A. According to one exemplary embodiment, the isoflavone derivative is daidzein derivative.
According to some embodiments the carboxy-isoflavone derivative is selected from the group consisting of 7-(O)-carboxymethyl daidzein, 6-carboxymethyl biochanin A, 2-carboxyalkyl biochanin A, 8-carboxymethyl biochanin A, 7-(O)- carboxymethyl formononetin, 2-carboxyalkyl genistein and 6-carboxymethyl genistein. According to one embodiment, the carboxy-isoflavone derivative is 2- carboxyalkyl biochanin A. According to other embodiment, the carboxy-isoflavone derivative is 6-carboxymethyl biochanin A. According to other embodiment, the carboxy-isoflavone derivative is 8-carboxymethyl biochanin A. According to an exemplary embodiment, the carboxy-isoflavone derivative is 7-(O)-carboxymethyl daidzein (i.e., R1 is -0-CH2-C(O)- and R2 - R6 are all -H).
According to some embodiments of the present invention, D is an imaging agent selected from, yet not restricted to paramagnetic particles (such as gadolinium, yttrium, lutetium, europium and gallinium); radioactive moieties (such as radioactive indium, rhenium and technetium); fluorescent dyes (such as fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), Cyan fluorescent protein
(CFP), rhodamine I, II, III and IV, rhodamine B, rosamine and infrared dyes (such as CyTEE-777)). According to certain embodiments the paramagnetic particles as well as the radioactive moieties can be provided within a chelator. Non-limiting examples for chelators include: tetraazacyclododecanetetraacetic acid (DOTA), mercaptoacetylglycylglycyl-glycine (MAG3), diethylenetriamine pentaacetic acid (DTPA) and N-l-φ-isothiocyanatopheny^diethylenetriamine-N^N^N3- tetraacetate(DTTA).
According to some embodiments of the present invention, D is a therapeutic agent selected from the group consisting of a cytotoxic compound, a cytostatic compound, an antisense compound and an anti-viral agent. According to some embodiments, D is a cytotoxic compound.
According to some embodiments, the cytotoxic compound D is selected from, but not restricted to DNA synthesis and function inhibitory agents (such as adriamycin, bleomycin, chlorambucil, cisplatin, carboplatin, oxaloplatin, daunomycin, ifosfamide and melphalan); microtubule (mitotic spindle) formation and function inhibitory agents (such as vinblastine, vincristine, vinorelbine, paclitaxel (taxol) and docetaxel); anti metabolites (such as cytarabine, fluorouracil, fluroximidine, mercaptopurine, methotrexate, gemcitabin and thioquanine); alkylating agents (such as mechlorethamine, chlorambucil, cyclophosphamide, melphalan and methotrexate); antibiotics (such as bleomycin and mitomycin); nitrosoureas (such as carmustine (BCNU) and lomustine); hormones (such as tamoxifen, leuprolide, flutamide and megestrol acetate) and proteins (such as interferon and asparaginase).
According to some embodiments, the cytotoxic substance D is an anti-tumor agent. According to some other embodiments, the anti-tumor agent D is selected from the group consisting of adriamycin, bleomycin, chlorambucil, cisplatin, daunomycin and melphalan. According to some embodiments, the anti-tumor agent is daunomycin.
According to some other embodiments, the conjugate of the present invention may further comprise an affinity molecule. According to one embodiment, the affinity molecule is attached to the albumin. According to some embodiments, the affinity molecule is selected from the group consisting of at least the antigen-binding portion of a specific antibody and biotin.
According to some embodiments of the present invention, nt is greater than 1 and D may be the same or different at each occurrence. According to other embodiments, n2 is other than zero where one of R1, R2, R3, R4, R5 or R6 comprises a carboxy group selected from -OR7C(=O)- and -R7C(=O)- which is bound to D. According to some embodiments, both n! and n2 are other than zero where one of R1, R2, R3, R4, R5 or R6 comprises a carboxy group selected from -OR7C(^O)- and - R7C(=O)- which is bound to D, wherein D may be the same or different at each occurrence. According to another aspect the present invention provides a diagnostic composition comprising an effective amount (e.g. an amount effective for enhancing image contrast in an imaging procedure) of a conjugate comprising at least one isoflavone derivative which is bound to albumin and at least one compound D which is an imaging agent. According to another aspect the present invention provides a pharmaceutical composition comprising an effective amount of a conjugate comprising at least one isoflavone derivative which is bound to albumin and at least one compound D which is a therapeutic agent.
According to a further aspect the present invention provides a method of targeting cells sensitive to isoflavone comprising administering to a subject in need thereof an effective amount of a conjugate comprising at least one isoflavone derivative which is bound to albumin and at least one bioactive moiety.
According to another aspect, the invention provides a method for generating enhanced images of a human or animal body and generation of an image of at least part of said body.
According to yet another aspect, the present invention provides a method for diagnosing cancer in a subject comprising administering to the subject a conjugate comprising at least one isoflavone derivative bound to albumin and at least one diagnostic compound for affinity targeting of the diagnostic compound to cells sensitive to isoflavone. According to one embodiment, the cells are cancer cells. According to another embodiment of the invention diagnosis of tumors may be performed in vitro or in vivo. According to various embodiments, diagnosis may be performed either directly or indirectly in an in vivo diagnostic imaging procedure e.g. by magnetic resonance imaging (MRI), near infrared (NIR), optical imaging, scintigraphy, SPECT, PET, X-ray, ultrasound imaging, electrical impedance or magnetometric procedures. According to some embodiments, the diagnostic imaging procedure is MRI. According to other embodiments, the diagnostic imaging procedure is NIR. According to some embodiments, in vivo diagnosis may be used to detect the presence of a disease state and/or monitor the progression of the disease. According to some embodiments, the conjugates (compositions of matter) of the invention are useful diagnostic contrast agents for in vivo imaging of a mammalian body. According to another aspect the present invention provides a method for treating cancer comprising administering to a subject in need thereof a therapeutical composition comprising a therapeutically effective amount of a conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound D being a therapeutic agent, thereby targeting and internalizing the conjugate to cells susceptible to isoflavone. According to some embodiments, cancer treated by methods of the invention is selected from the group consisting of ovarian cancer, colon cancer, breast cancer, melanoma, non-small lung cancer, leukemia, endometrial cancer and prostate cancer. According to some currently preferred embodiments, the present invention provides a method for treating ovarian cancer. According to some embodiments, the methods of the invention may be used to treat diseases other than cancer. According to some embodiments the disease other than cancer is an estrogen related condition such as osteoporosis, endometriosis, atherosclerosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy, obesity, menopausal syndromes, type-II diabetes, and Alzheimer's disease. The present invention will be better understood in conjunction with the description, figures and claims below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. IA shows the specific binding and endocytosis of daidzein-BSA-FAM by MLS ovarian carcinoma cells. Two-photon fluorescence microscopy of MLS human ovarian carcinoma cells incubated with Daidzein-BSA-FAM or BSA-FAM, in the presence or absence of a blocking dose of nystatin. Panel a. DAPI nuclear staining in gray; panel b. Daidzein-BSA-FAM or BSA-FAM in white; panel c. overlay of panels a and b. Fig. IB shows the specific binding and endocytosis of BSA-ROX and Daidzein-BSA-FAM by MLS ovarian carcinoma cells. Two-photon fluorescence microscopy of MLS human ovarian carcinoma cells incubated with Daidzein-BSA- FAM and BSA-ROX, with or without nystatin
FIG. 2 shows the binding and endocytosis of BSA-ROX and Daidzein-B SA-FAM by
MLS ovarian carcinoma cells as determined by FACS analysis. A. Histogram of Daidzein-B SA-FAM uptake (grey-control, black-Daidzein-B SA-FAM uptake, light gray- Daidzein-BSA-FAM uptake in presence of nystatin), collected by FLl filter (BP
530/30); B. BSA-ROX uptake (grey-control, black- BSA-ROX uptake, dark gray-
BSA-FAM uptake in presence of nystatin), collected by FL2 filter (BP 585/42); C.
Statistics median of uptake histogram analysis (c=control; n=nystatin; dbf=daidzein- BSA-FAM; br=BSA-ROX).
FIG. 3 Competition of Daidzein-BSA-FAM (light gray) by BSA-ROX (gray) in the absence (A) and presence of nystatin (B) (FLl filter for the collection of FAM fluorescence, FL2 filter for the collection of ROX fluorescence), C and D display control samples without and with nystatin respectively. E. Statistical analysis of the competition assay. Change in percent of positively stained fluorescent cells in the absence and presence of nystatin (n) (light gray-Daidzein-B SA-FAM (dbf), dark gray- BSA-ROX (br)).
FIG. 4 shows the in vivo targeted delivery of daidzein-BSA-CyTE-777 to ovarian carcinoma tumors followed by NIR Imaging. daidzein-BSA-CyTE-777 (A); Control BSA-CyTE-777 (B); daidzein-BSA-CyTE-777 + BSA-FAM after 48 hours (C) and 72 hours (D) respectively; E. In Vivo Imaging of daidzein-BSA-CyTE-777: localization in tumor-free CD-I nude mice. CD-I nude mice were injected intravenously with daidzein-BSA-CyTE-777 (left) or non injected (right). The NIR signal after 24 hours is shown. FIG. 5 shows the biodistribution of daidzein-BS A-EuDTTA versus BSA-EuDTTA in MLS tumor bearing mice. A. Europium signal 24h after injection (n=3-5). B. Europium signal 48h after injection (Tl-injected with BSA- EuDTTA, T2 and T3 - injected with daidzein-B SA-Eu). C. Western blot analysis with anti-daidzein antibody of the extracts of tumors (T), kidneys (K) and livers (L) of mice 48h after injection. FIG. 6 demonstrates that while Daidzein-BSA-FAM accumulates in tumor cells, BSA-FAM is localized in tumor blood vessels. A. Control. B. Section of a tumor from a mouse injected with BSA-FAM. C. Section of a tumor from a mouse injected with daidzein-BSA-FAM (FAM-white; DAPI-gray).
FIG. 7 demonstrates the advantages of using daidzein-B SA-GdDTPA as a contrast material to MRI. A. The specific R1 relaxivity of daidzein-B SA-GdDTPA was measured to be 194 mM'V1 per BSA; B. shows the specific localization and retention of the contrast material inside the tumor in MLS bearing mice administered intravenously with BSA-GdDTPA (left panel) or daidzein-BSA-GdDTPA (right panel). Tl weighted MRI images and R1 maps were obtained 24, 48 and 72h after injection, and used for derivation of mean R1 values of the tumor; C. shows the statistically significant elevation of Rl relaxation in tumors injected with daidzein- BSA-GdDTPA as compared with non injected animals or mice injected with BSA- GdDTPA; D, E. demonstrate prolonged detection of contrast material inside the tumor in MLS tumor bearing mice administered intravenously daidzein-BSA- GdDTPA after 24h and 7 days respectively.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise specified, "a" or "an" means "one or more".
The present invention relates to conjugates comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety including but not limited to a diagnostic agent (e.g. imaging agent) or a therapeutic agent and their use for the diagnosis and treatment of cancer.
According to some aspects the present invention relates to the use of the conjugate comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety selected from an imaging agent for targeting of the imaging agent to cancer cells for diagnostic purposes. According to some embodiments, the cancer cells are sensitive to isoflavone.
According to other aspects the present invention relates to the use of the conjugate comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety selected from a therapeutic agent for targeting of drugs to cancer cells for cancer treatment purposes. According to some embodiments, the cancer cells are sensitive to isoflavone. The term "targeting" as used herein means that the conjugate is able to induce the formation of a functional response (preferably the accumulation of a diagnostic agent or an anti-cancer drug in cells sensitive to isoflavone) in cancer cells preferentially over normal cells as compared with the diagnostic agents or drugs commonly used which are not given as part of the conjugate of the invention..
As used herein, a "diagnostic agent" is a molecule or atom which is conjugated to a targeting moiety (e.g. isoflavone or isoflavone-albumin), to produce a conjugate, which is useful for diagnosis. Non-limiting examples of diagnostic agents include paramagnetic particles; radioactive moieties; fluorescent dyes and infrared dyes. According to one embodiment, the diagnostic agent is a contrast agent. According to some embodiments, the diagnostic agent may be provided within a chelator. According to other embodiments, the conjugate of the invention comprising isoflavone derivative bound to albumin and a diagnostic agent may be used as a contrast agent. The term "therapeutic agent" as used herein refers to a molecule or atom which is conjugated to a targeting moiety (e.g. isoflavone or isoflavone-albumin conjugate) to produce a conjugate which is useful for therapy. Non limiting examples of therapeutic agents include cytotoxic compounds, cytostatic compounds, antisense compounds or anti-viral agents. Delivery of bioactive moieties to tumor cells:
Despite the high permeability of VEGF stimulated tumor neovasculature to plasma proteins, targeting of contrast media and delivery of therapeutics to tumors is frequently compromised leading to poor sensitivity for tumor detection and resistance to therapy. The low transfer rate is attributed to high interstitial pressure (Bouzin et al., 2007, Drug Resist. Updat.; 10: 109-20). The present invention is directed to a bifunctional targeted conjugate comprising isoflavone, albumin and an active agent (diagnostic and/or therapeutic agents) which effectively overcomes the tumor-blood barrier, thus increasing the delivery of the conjugate to the rumor cells. Without wishing to be bound by any theory or mechanism of action it is hypothesized that tumor cells escape from delivery of extravasated albumin based compounds, through caveolae mediated sequestration of the albumin based compound by perivascular myofibroblasts, thus generating an effective tumor-blood barrier. Evoking an alternate competing endocytic pathway using isoflavone (e.g. daidzein) resulted in significant partition of the isoflavone-albumin based compound (e.g. the conjugate of the invention) into the tumor. According to some embodiments, the partition of the conjugate of the invention into the tumor could be further enhanced in the presence of free albumin (non conjugated albumin). According to other embodiments, the partition of the conjugate of the present invention into the tumor could be further enhanced by the addition or co-administration of nystatin. Without wishing to be bound by any theory or mechanism of action, it is hypothesized that the free albumin or nystatin increase the partition of the conjugate through suppression of caveolae mediated endocytosis by competition with the conjugate, in the absence of direct modulation of the tumor interstitial pressure.
The term "partition into a cell" or "partition into a cancer cell" or "partition into a tumor cell" as used herein refers to the amount of bioactive moiety (diagnostic agent and/or therapeutic agent) found in the interior part of a cell versus the amount of the bioactive moiety found at the exterior part of the cell, specifically, within or on the cell's membrane. According to one embodiment, the partition into a tumor cell of the bioactive moiety is at least 10% higher than the partition into a tumor cell of the bioactive moiety when administered alone and/or bound to albumin alone and/or bound to isoflavone alone; alternatively, at least 20% higher; alternatively at least 30% higher; alternatively, at least 40% higher; alternatively, at least 50% higher; alternatively, at least 60% higher; alternatively, at least 70% higher; alternatively, at least 80% higher; alternatively, at least 90% higher; alternatively, at least 2 times higher; alternatively at least 3 times higher.
According to some embodiments, the conjugate of the present invention displays prolonged tumor retention and/or increased circulation time as compared to tumor retention and circulation time of the bioactive agent alone, the bioactive agent bound to albumin alone, or the bioactive agent bound to isoflavone alone.
The term "tumor retention" refers to the duration of the presence of a sufficient amount of a bioactive moiety inside a tumor cell since the time it was administered to a subject, or introduced to the cell, or internalized or up taken by the cell. The term "sufficient amount" implies a sufficient amount of a diagnostic agent that will enable detection (e.g. tumor detection) using various diagnostic methods, for example, the diagnostic imaging procedures described. Alternatively, "sufficient amount" implies an amount of a therapeutic agent which is sufficient to cause a desired therapeutic effect. According to some embodiments, the bioactive moiety of the conjugate is retained inside the tumor cell for at least 7 days; alternatively, for at least 5 days, alternatively, for at least 3 days, alternatively for at least 48 hours, alternatively, for at least 24 hours.
"Circulation time" as used herein refers to the duration of the presence of the conjugate of the invention in the blood or plasma; specifically, to the duration of the presence of the bioactive moiety, which is part of the conjugate, in the blood or plasma. The circulation time of the bioactive moiety is determined by its distribution, metabolism and its removal from the body via excretion. Alternatively, "circulation time" may be referred to as the half-life of the bioactive moiety in the plasma. "Half- life" refers to the period of time required for the concentration or amount of the bioactive moiety in the body to be reduced by one-half. A plasma half-life of a bioactive moiety depends on how quickly the bioactive moiety is eliminated from the plasma. A bioactive moiety that leaves plasma may have any of several fates. It can be eliminated from the body, or it can be translocated to another body fluid compartment such as the intracellular fluid or it can be destroyed in the blood. The removal of a the bioactive moiety from the plasma is known as clearance and the distribution of the bioactive moiety in the various body tissues is known as the volume of distribution. Both of these pharmacokinetic parameters are important in determining the circulation time or half life of a bioactive moiety.
Experimentally the half life can be determined by giving a single dose, usually intravenously, and then measure the concentration of the bioactive moiety in the plasma at regular intervals. The concentration of the drug will reach a peak value in the plasma and will then fall as the bioactive moiety is hydrolyzed or broken down and cleared from the blood.
According to some embodiments of the present invention, the circulation time or the half-life of a bioactive moiety is increased upon introduction or binding of the bioactive moiety to an isoflavone-albumin conjugate. According to one embodiment, the circulation time or half-life of the bioactive moiety is at least 10% higher than the circulation time or half-life of the bioactive moiety when administered alone, bound to albumin, or bound to isoflavone; alternatively, at least 20% higher; alternatively at least 30% higher; alternatively, at least 40% higher; alternatively, at least 50% higher; alternatively, at least 60% higher; alternatively, at least 70% higher; alternatively, at least 80% higher; alternatively, at least 90% higher; alternatively, at least 2 times higher; alternatively at least 3 times higher.
It should be appreciated that each of the following conjugates consisting of two constituents: isoflavone-albumin, albumin-bioactive moiety and isofiavone- bioactive moiety was known in the prior art, however a three constituents conjugate comprising isoflavone, albumin and a bioactive moiety had been disclosed for the first time in the present invention and shows superior properties such as high cellular partition and tumor retention over those achieved by the conjugates consisting of two constituents s described above, when these properties has been determined.
Chemical definitions:
The term "Cl to C6 alkylene" refers to a saturated, linear or branched hydrocarbon moiety, such as -CH2-, -CH2-CH2-; -CH2-CH2-CH2-; -CH2-(CH2)2-CH2; -CH2-(CH2)3-CH2; -CH2-(CH2)4-CH2;. The term "C2 to C20 alkenylene" as used herein, denotes a divalent group derived from a straight chain or branch hydrocarbon moiety containing from two to twenty carbon atoms having at least one carbon-carbon double bond. Alkenylene groups include, but are not limited to, for example, ethenylene, 2-propenylene, 2- butenylene, l-methyl-2-buten-l-ylene, and the like. The term "heteroalkylene" refers to an alkylene or alkenylene moiety having at least one heteroatom (e.g., N, O, or S). Preferred are heteroalkylenes having at least one O.
The term "GIc" denotes glucosyl or glucoside. Isoflavone compounds Epidemiological in vitro as well as in vivo animal studies indicate that isoflavones present in large quantities in soybeans and soy products (e.g., genistein, daidzein) and red clover (biochanin A) are promising agents for cancer therapy, chemoprevention and inhibition of tumor progression (Aggarwal, B. B. at al, Biochem. Pharmacol. 2006, 71, 1397). In estrogen-sensitive cancer cell lines (e.g., breast, colon, etc.) expressing estrogen receptor ERa, ERβ or both, these isoflavone compounds can act as weak estrogens and stimulate cell growth at concentrations ranging from 0.1 to 20 μM. The affinity of most isoflavones to the two subtypes of ER is low (Kuiper, G. G. at al, Endocrinology 1998, 139, 4252) with the exception of genistein, which shows a strong selectivity for Erβ over Era. Both genistein and daidzein display 100-fold greater sensitivity for activating transcription in transfected cells via Erβ compared to Era (Harris, D. M. at al., Exp. Biol. Med. 2005, 230, 558). Interestingly, isoflavones inhibit cell growth at concentrations greater than 20 μM. According to some embodiments, isoflavone compounds display estrogenic effects (e.g. stimulate cell growth). According to other embodiments, isoflavone compounds display anti-estrogenic effects (e.g. inhibit cell growth). Thus, according to some embodiments, isoflavone compounds may be referred to as modulators of cell growth.
In addition to the estrogenic and anti-estrogenic effects, isoflavones show a wide spectrum of biological activities not ascribed to activation of the ER such as regulation of cell-signaling pathways, and can inhibit proliferation and induce apoptosis in ER-negative breast cancer cell lines as well as in ER-positive cell lines. Examples include genistein which was shown to inhibit the protein-tyrosine kinase pathway and was further used in a treatment of choroidal neovascularization (U.S. Pat. No. 6,028,099). Genistein was also shown to display topoisomerase II activity, and to induce apoptosis and cell differentiation. Moreover, genistein has been shown to inhibit the proliferation of both cancer and normal cells, and was used for prophylactic treatment of cataract (WO 00/37066).
The 4-methoxy derivative of genistein, biochanin A, is equally potent to genistein as a growth inhibitor in breast cancer lines due to its conversion to genistein
(Peterson et al. Am. J. Clin. Nutr. 1998 68:1505S-1511S). However, when administered in equal doses, biochanin A, and not genistein, inhibited the growth of several tumors derived from the gastrointestinal tract and grown in nude mice.
Among the isoflavones daidzein exhibits unique properties. Daidzein is known to interact with the lipid interface on the cell surface (Lehtonen J.Y et al. Biochim Biophys Acta, 1996 1285: 91-100) thus facilitating endocytosis. Furthermore, acting as a weak estrogen, daidzein may recognize a putative plasma membrane estrogen receptor (Somjen D. et al. J Steroid Biochem MoI Biol, 2005 93: 293-303), a membranal ERβ-related protein (De Wilde A. et al. J Cell Physiol, 2006 209: 786- 801) or a nuclear estrogen receptor of the β-type. The use of the isoflavone ring as a template for designing isoflavone carboxy derivatives useful as selective estrogen receptor modulators (SERMs) has been described (US 2005/0096381 Al). The contents of the aforementioned reference are incorporated by reference herein in their entirety as if fully set forth herein. The synthesis and evaluation of the anti-proliferative activities of derivatives of carboxyalkyl isoflavones has been described by U.S. Patent Application No. 2005/0096381 and Kohen F. et al., J Med. Chem., 2007, 50, 6405-6410. Methods for the conjugation of a bioactive moiety to carboxy derivatives of isoflavones are described by U.S. Patent Application No. 2005/0096381. The contents of the aforementioned references are incorporated by reference herein in their entirety as if fully set forth herein. It is to be appreciated that the conjugation of a bioactive moiety to a carboxy derivative of isoflavone may be done by any other means known in the art.
Albumin As used herein, the term albumin includes human serum albumin, animal albumin, recombinant albumin, and fragments thereof. Additionally, the albumin may exist as a monomer, a dimer, a polymer, or may be enclosed in microspheres. Albumin as disclosed herein may also be treated with polyethylene glycol (PEG) by well-known techniques to increase its immunocompatibility. According to one embodiment, albumin comprises human serum albumin. According to another embodiment, albumin comprises bovine serum albumin (BSA). It is to be appreciated that species-specific serum albumin is necessary for compatibility reasons. Thus, when treating or diagnosing human patients, human serum albumin may preferably be used and when treating or diagnosing other animals, the serum albumin should likewise be species-specific, e.g., bovine serum albumin for treating or diagnosing cattle.
According to one embodiment the albumin in the conjugate is used as a carrier. The isoflavone carboxy derivatives and the bioactive moieties according to the invention may be conjugated to the albumin carrier by any means know in the art, non-limiting examples include: conjugation through the albumin's lysine residues (amino groups), conjugation through the N-terminus and conjugation through cy stein residues (thiol groups). For example, amino, hydroxyl and hydrazine groups can each form a covalent bond with a reactive carbonyl group (e.g., a carboxylic acid chloride or activated ester such as an N-hydroxysuccinimide ester (NHS)). Other suitable bond forming groups are well-known in the literature and can be used to prepare the conjugates of the present invention. Specifically, carboxy derivatives of isoflavones as well as bioactive moieties may be coupled to an amino group of albumin (e.g. lysine group) via N-hydroxysuccinimide ester. Alternatively N-sulfo succinimide and a water soluble carbodiimide may be used for coupling a carboxy group of the isoflavone or of the bioactive moiety to an amino group of albumin through the formation of a peptide linkage. Bioactive moieties
According to some embodiments, the conjugate of the invention comprises a bioactive moiety selected from a diagnostic agent and a therapeutic agent. According to some embodiments the bioactive moiety is a therapeutic agent selected from the group consisting of a cytotoxic compound, a cytostatic compound, an antisense compound, an anti-viral agent.
According to some embodiments, the cytotoxic compound is selected from, but not restricted to DNA synthesis and function inhibitory agents; microtubule (mitotic spindle) formation and function inhibitory agents; anti metabolites; alkylating agents; antibiotics; nitrosoureas; hormones and proteins. According to some embodiments, the therapeutic agent is an anti-tumor agent.
As referred to herein, the term "anti-cancer reagent" refers to any type of reagent that may be used in the treatment of cancer and/or cancer related conditions.
The anti-cancer reagent may include any naturally occurring or synthetically produced molecule that is capable of affecting directly or indirectly the growth and/or viability of cancer cells. The anti-cancer reagent may include, for example, a naturally occurring protein or peptide, a modified protein or peptide, a recombinant protein, a chemically synthesized protein or peptide, a chemical molecule, a synthetic chemical molecule, a chemotherapeutic drug, a biologically therapeutic drug, and the like, or any combination thereof. The anti-cancer reagent may be cytotoxic (toxic to cells) and/or cytostatic (suppress cell growth) and/or antiproliferative to the cancer cells and may exert its effect on cancer cells directly and/or indirectly. Non limiting examples of anti-cancer agents and chemotherapeutic drugs may include such drugs as, but not limited to: Alkaloids, such as, but not limited to: Docetaxel, Etoposide, Irinotecan, Paclitaxel, Teniposide, Topotecan, Vinblastine, Vincristine, Vindesine; Alkylating agents, such as, but not limited to: Busulfan, Improsulfan, Piposulfan, Benzodepa, Carboquone, Meturedepa, Uredepa, Altretamine, triethylenemelamine, Triethylenephosphoramide,
Triethylenethiophosphoramide, Chlorambucil, Chloranaphazine, Cyclophosphamide, Estramustine, Ifosfamide, Mechlorethamine, Mechlorethamine Oxide HcI, Melphalan, Novemebichin, Perfosfamide Phenesterine, Prednimustine, Trofosfamide, Uracil Mustard, Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, Semustine Ranimustine, Dacarbazine, Mannomustine, Mitobronitol, Mitolactol, Pipobroman, Temozolomide; Antibiotics and analogs, such as, but not limited to: Aclacinomycins, Actinomycins, Anthramycin, Azaserine, Bleomycins, Cactinomycin, Carubicin, Carzinophilin, Cromomycins, Dactinomycins, Daunorubicin, 6-Diazo-5-oxo-L- norleucine, Doxorubicin, Epirubicin, Idarubicin, Menogaril, Mitomycins, Mycophenolic Acid, Nogalamycine, Olivomycins, Peplomycin, Pirarubicin, Plicamycin, Porfiromycin, Puromycine, Streptonigrin, Streptozocin, Tubercidin, Zinostatin, Zorubicin; Antimetabolites, such as, but not limited to: Denopterin, Edatrexate, Methotrexate, Piritrexim, Pteropterin, Tomudex, Trimetrexate, Cladridine, Fludarabine, 6-Mercaptopurine, Pentostatine Thiamiprine, Thioguanine, Ancitabine, Azacitidine, 6-Azauridine, Carmofur, Cytarabine, Doxifluridine, Emitefur, Floxuridine, Fluorouracil, Gemcitabine, Tegafur; Platinum complexes, such as, but not limited to: Caroplatin, Cisplatin, Miboplatin, Oxaliplatin; alkylators including, but not limited to, busulfan (Myleran, Busulfex), chlorambucil (Leukeran), ifosfamide (with or without MESNA), cyclophosphamide (Cytoxan, Neosar), glufosfamide, melphalan, L-PAM (Alkeran), dacarbazine (DTIC-Dome), and temozolamide (Temodar); anthracyclines, including, but not limited to doxorubicin (Adriamycin, Doxil, Rubex), mitoxantrone (Novantrone), idarubicin (Idamycin), valrubicin (Valstar), and epirubicin (Ellence); antibiotics, including, but not limited to, dactinomycin, actinomycin D (Cosmegen), bleomycin (Blenoxane), daunorubicin, and daunomycin (Cerubidine, DanuoXome); aromatase inhibitors, including, but not limited to anastrozole (Arimidex) and letroazole (Femara); bisphosphonates, including, but not limited to zoledronate (Zometa); cyclo-oxygenase inhibitors, including, but not limited to, celecoxib (Celebrex); estrogen receptor modulators including, but not limited to tamoxifen (Nolvadex) and fiilvestrant (Faslodex); folate antagonists including, but not limited to methotrexate and tremetrexate; inorganic aresenatesincluding, but not limited to arsenic trioxide (Trisenox); microtubule inhibitors (e.g. taxanes) including, but not limited to vincristine (Oncovin), vinblastine (Velban), paclitaxel (Taxol, Paxene), vinorelbine (Navelbine), epothilone B or D or a derivative of either, and discodermolide or its derivatives, nitrosoureas including, but not limited to procarbazine (Matulane), lomustine, CCNU (CeeBU), carmustine (BCNU, BiCNU, Gliadel Wafer), and estramustine (Emcyt); nucleoside analogs including, but not limited to mercaptopurine, 6-MP (Purinethol), fluorouracil, 5-FU (Adrucil), thioguanine, 6-TG (Thioguanine), hydroxyurea (Hydrea), cytarabine (Cytosar-U, DepoCyt), floxuridine (FUDR), fludarabine (Fludara), pentostatin (Nipent), cladribine (Leustatin, 2-CdA), gemcitabine (Gemzar), and capecitabine (Xeloda); osteoclast inhibitors including, but not limited to pamidronate (Aredia); platinum containing compounds including, but not limited to cisplatin (Platinol) and carboplatin (Paraplatin); retinoids including, but not limited to tretinoin, ATRA (Vesanoid), alitretinoin (Panretin), and bexarotene (Targretin); topoisomerase 1 inhibitors including, but not limited to topotecan (Hycamtin) and irinotecan (Camptostar); topoisomerase 2 inhibitors including, but not limited to etoposide, VP- 16 (Vepesid), teniposide, VM-26 (Vumon), and etoposide phosphate (Etopophos); tyrosine kinase inhibitors including, but not limited to imatinib (Gleevec); various other proteins, peptides and enzymes, various other molecules, such as, for example, Super Oxide dismutase (SOD), leptin; flavanoids; or any combinations thereof.
According to some embodiments the bioactive moiety is a diagnostic agent selected from the group consisting of paramagnetic particles; radioactive moieties; dyes, fluorophores, and infrared dyes. Typical diagnostic radioactive moieties include "01Tc, 95Tc, 111In, 62Cu, 64Cu, 67Ga, and 68Ga; and therapeutic radioactive moieties include 186Re, 188Re, 153Sm, 166Ho, 177Lu, 149Pm, 90Y, 212Bi, 103Pd, 109Pd, 159Gd, 140La, 198Au, 199Au, 169Yb, 175Yb, 165Dy, 166Dy, 67Cu, 105Rh, 111Ag, and 192Ir. Typical paramagnetic particles include rare earth metals, typically, gadolinium, manganese, yttrium, ytterbium, europium, lutetium, gallinium and the like. Iron ions may also be used.
Preferred fluorophore moieties include fluorescent dyes having (a) high fluorescence intensity; (b) sufficiently long excitation and emission wavelength maxima so that interference from natural fluorescence of either diseased or normal tissue is minimized; (c) sufficiently long measured fluorescence decay time to allow accurate measurement of emitted light over background fluorescence and scattering (at least about 2, preferably at least about 10 nanoseconds); and (d) high degree of fluorescence polarization. Fluorophores include macrocyclic fluorescent dye compounds, especially compounds having aromatic π-electron systems. These fluorophore moieties may comprise a substantially planar multidentate macrocyclic ligand and may be coordinated to a complexing central ion or atom such as aluminum, phosphorous, and the group FVB elements, e.g. silicon, germanium, and tin. Other suitable fluorophores include coumarin dyes, nitrobenzoxazole dyes, cyanine dyes, dipyrrometheneboron dyes, xanthene dyes (including the benzo- and naphtho- xanthene dyes), phenoxazine dyes (as well as the benzo- and naphtho-phenoxazine dyes) and compounds from other classes of dyes well known to those of skill in the art. Other suitable fluorophores include the fluorophores in the following non- exclusive list: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7- dichlorofluorescein; 5-Carboxyfluorescein (5 -FAM); 5-Carboxynapthofluorescein; 5- Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5 -Hydroxy Tryptamine (HAT); 5 -ROX (carboxy-X- rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6- JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7- Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-rnethoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AutoFluorescent Protein-(Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor (various)™; Alizarin Complexon; Alizarin Red; AUophycocyanin (APC); AMC, AMCA-S; AMCA Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue 600; Anthrocyl stearate; APC (AUophycocyanin); APC-Cy7; APTRA- BTC=Ratio Dye, Zn2+; APTS; Astrazon Brilliant Red 4G, Orange R, Red 6B, and Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA and FQ; Auramine; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high or low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP; Blue Fluorescent Protein; blue shifted GFP (Y66H); BFP/GFP FRET; Bimane; Bisbenzamide; bis-BTC=Ratio
Dye, Zn2+; Blancophor FFG and SV; BOBO™-1 and -3; Bodipy (various); BO- PRO™- 1 and -3; Brilliant Sulphoflavin; BTC-Ratio Dye Ca2+; BTC-5N; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green (various) and Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP-Cyan Fluorescent Protein; Cyan Fluorescent Protein; CFP/YFP FRET; Chlorophyll; Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA; Coelenterazine Dye (various); Coumarin Phalloidin; C- phycocyanine; Methylcoumarin; Methylcoumarin CTC; CTC Formazan; Cy2™ and various others; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl (various); Dansyl (various); DAPI; Dapoxyl (various); DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4- ANEPPS; Di-8-ANEPPS; DiA (4-Di- 16- ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DiICl 8(5)); DIDS; Dihydorhodamine 123 (DHR); DiI (DiICl 8(3)); Dinitrophenol; DiO (DiOCl 8(3)); DiR (various); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; Enhanced Blue Fluorescent Protein; Enhanced Cyan Fluorescent Protein; Enhanced Green Fluorescent Protein; ELF; Eosin; Erythrosin; Erythrosin; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Enhanced Yellow Fluorescent Protein; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (various); Genacryl Brilliant Red B, Brilliant Yellow 10GF, Pink 3G, and Yellow 5GF; GeneBlazer (CCF2); GFP (various); Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258, 33342, and 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1 (various); Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-I; JO-JO-I; JO-PRO-I; LaserPro; Laurodan; LDS 751; Leucophor PAF, SF, and WS; Lissamine Rhodamine (various); Calcein/Ethidium homodimer; LOLO-I; LO-PRO-I; Lucifer Yellow; Lyso Tracker (various); LysoSensor (various); Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green and Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin; Maxilon Brilliant Flavin; Merocyanin; Methoxycoumarin; Mitotracker Green, Orange, and Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);
Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin; Oregon Green™ (various); Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5 and Cy7; PerCP; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite (various); Phosphine; PhotoResist; Phycoerythrin B and R; PKH26 and PKH67; PMIA; Pontochrome Blue Black; POPO-I and -3; PO-PRO-I and -3; Primuline; Procion Yellow; Propidium lodid (PI); PYMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin; QSY; Quinacrine Mustard; Red 613 [PE- TexasRed]; Resorufin; Rhodamine (various); Rose Bengal; R-phycocyanine; R- phycoerythrin (PE); red shifted GFP (various); Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red, Orange, and Yellow L; SuperGlo™GFP (various); SITS (various); SNAFL (various); SNARF; Sodium Green; SpectrumAqua, Green, Orange, and Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B and G; SYTO (various); SYTOX Blue, Green, and Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5, S, and TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-I, -3 and -5; TOTO-I and -3; TriColor (PE-Cy5); TRITC; TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; X-Rhodamine; XRITC; Xylene Orange; Y66F, H, and W; Yellow shifted Green Fluorescent Protein; Yellow Fluorescent Protein; YO-PRO-I and -3; and; YOYO-I and -3.
Infrared dyes include cyanine dyes. Non limiting examples of cyanine dyes include l,r,3,3,3',3'-Hexamethylindotricarbocyanine iodide; 1,1',3,3,3',3'- Hexamethylindotricarbocyanine perchlorate; l,l'-Diethyl-2,2'-dicarbocyanine iodide; l,l'-Diethyl-2,2'-quinotricarbocyanine iodide; l,l'-Diethyl-4,4'-carbocyanine iodide; l,l '-Diethyl-4,4'-dicarbocyanine iodide; l,3-Bis[4-(dimethylamino)-2- hydroxyphenyl]-2,4-dihydroxycyclobutenediylium dihydroxide, bis(inner salt); 1 ,4,8, 11,15,18,22,25-Octabutoxy-29H,3 lH-phthalocyanine; 1,8,15,22-
Tetrakis(phenylthio)-29H,3 lH-phthalocyanine; 2,11 ,20,29-Tetra-tert-butyl-2,3- naphthalocyanine; 2,3 ,9, 10, 16, 17,23,24-Octakis(octyloxy)-29H,31 H-phthalocyanine; 2,3-Naphthalocyanine; 2,9, 16,23-Tetrakis(phenylthio)-29H,31 H-phthalocyanine; 2,9, 16,23 -Tetra-tert-butyl-29H,31 H-phthalocyanine; 2,9, 16,23-Tetraphenoxy-
29H,31 H-phthalocyanine; 29H,31H-Phthalocyanine β-form; 3,3'- Diethylthiadicarbocyanine iodide; 3,3'-Diethylthiatricarbocyanine iodide; 5,9,14,18,23,27,32,36-Octabutoxy-2,3-naphthalocyanine; Aluminum
1 ,4,8, 11,15,18,22,25-octabutoxy-29H,31 H-phthalocyanine triethylsiloxide;
Aluminum phthalocyanine chloride; Boron sub-2,3-naphthalocyanine chloride; Cobalt(II) 1,2,3,4,8,9,10,1 l,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H- phthalocyanine; Cobalt(II) 2,3-naphthalocyanine; Cobalt(II) phthalocyanine β-form; Copper phthalocyanine-3,4',4",4'"-tetrasulfonic acid tetrasodium salt; Copper(II) 3, 10, 17,24-tetra-tert-butyl- 1 ,8, 15,22-tetrakis(dimethylamino)-29H,31 H- phthalocyanine; Copper(II) 5,9,14,18,23,27,32,36-octabutoxy-2,3-naphthalocyanine; Copper(II) phthalocyanine; Dilithium phthalocyanine; Gallium(III) 2,3- naphthalocyanine chloride; Gallium(III)-phthalocyanine chloride; IR-1048; IR-1061; IR- 140; IR-27; IR-676 iodide; IR-676; IR-775 chloride; IR-780 iodide; IR-783; IR- 786; IR-786 perchlorate; IR-792 perchlorate; IR-797 chloride; IR-800; IR-806; IR- 813 perchlorate; IR-820; IR-895; Indocyanine green Dye; Iron(II) phthalocyanine; Lead(II) phthalocyanine; Magnesium phthalocyanine; Manganese(II) phthalocyanine; Methylsilicon(IV) phthalocyanine chloride; Nickel(II) phthalocyanine Dye; Propyl Astra Blue Iodide; Silicon 2,3-naphthalocyanine dichloride; Tetrakis(4- cumylphenoxy)phthalocyanine; Tin(II) phthalocyanine; Titanium(IV) phthalocyanine dichloride; Vanadyl 2,3-naphthalocyanine; Zinc phthalocyanine and Zinc(II) tetranitrophthalocyanine. According to currently preffered embodiments, the infarared dye is CyTEE-777. Other examples of suitable cyanine dyes are described in U.S. Patent No. 7,514,069, the contents of the aforementioned reference are incorporated by reference herein in their entirety as if fully set forth herein.
According to certain embodiments the paramagnetic particles as well as the radioactive moieties can be provided within a chelator. Non-limiting examples for chelators include: tetraazacyclododecanetetraacetic acid (DOTA), mercaptoacetylglycylglycyl-glycine (MAG3), diethylenetriamine pentaacetic acid
(DTPA), N- 1 -(p-isothiocyanatophenyl)diethylenetriamine-N1 ,N2,N3 -tetraacetate
(DTTA), l,4,7-triaza-cyclononane-N-N'-N"-triacetic acid (NOTA) and tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA). The preparation of metal chelates may be carried out by any means know in the art
According to some embodiments, the conjugate comprising at least one isoflavone derivative bound to albumin and at least one diagnostic compound is selected from the group of 7-(O)-carboxymethyl daidzein-albumin-CyTE-777, 7-(O)- carboxymethyl daidzein-albumin-GdDTPA, 7-(O)-carboxymethyl daidzein-albumin- FAM and 7-(O)-carboxymethyl daidzein-albumin-EuDTTA.
Diagnosis and treatment of cancer In yet another aspect the present invention provides methods for the diagnosis and treatment of cancer in a subject comprising administering a conjugate comprising at least one isoflavone derivative bound to albumin and at least one bioactive moiety selected from a diagnostic agent and/or a therapeutic agent. According to some embodiments, the conjugate of the invention is useful for targeting cells sensitive to isoflavone, preferably, cancer cells sensitive to isoflavone.
The present methods find broad applicability in the detection and treatment of a number of cancers. According to some embodiments, the cancer may include solid tumors, non-solid tumors, primary tumors, metastasis or any combination thereof. The cancer may include carcinomas, sarcomas, myelomas, leukemias, lymphomas or any combination thereof. The cancer may include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, a metastasis thereof or any combination thereof. The cancer may include hepatocellular carcinoma, hematoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma including small cell, non- small and large cell lung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon carcinoma, rectal carcinoma, hematopoietic malignancies including all types of leukemia and lymphoma including: acute myelogenous leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or any combination thereof. According to some embodiments, the cancer comprises cancer cells sensitive to isoflavone. According to some embodiments, cancer may include ovarian carcinoma, colon cancer, breast cancer, melanoma, non-small lung cancer, leukemia, endometrial cancer and prostate cancer. According to some embodiment, the cancer is ovarian cancer. According to some embodiments, the cancer is breast cancer. According to some embodiments, the cancer is colon cancer. According to some embodiments, the cancer is prostate cancer. According to the present invention provides methods for detecting or imaging cancer cells in a subject, comprising (a) administering to the subject an effective amount of a conjugate comprising at least one isoflavone derivative bound to albumin and at least one diagnostic agent; and (b) detecting or imaging cells that take up the conjugate to determine if cancer is present in a subject. Detection of the conjugate can be performed using essentially any detection device to obtain an image of the cancerous cells. According to various embodiments, diagnosis may be performed either directly or indirectly in an in vivo diagnostic imaging procedure e.g. by magnetic resonance imaging (MRI), near infrared (NIR), optical imaging, scintigraphy, SPECT, PET, X-ray, ultrasound imaging, electrical impedance or magnetometric procedures. According to some embodiments, the diagnostic imaging procedure is MRI. According to other embodiments, the diagnostic imaging procedure is NIR. According to some embodiments, in vivo diagnosis may be used to detect the presence of a disease state and/or monitor the progression of the disease. According to some embodiments, the conjugates of the invention are useful diagnostic contrast agents for in vivo imaging of a mammalian body.
The diagnostic conjugates of the present invention can be used in a method which comprises administering to a subject's body, or to selected regions thereof, effective amounts of the diagnostic conjugate. The agent can be injected directly into the vicinity of the body to be imaged, or it can be intravenously, intradermaly, intralesionaly, intramusculary, intravesiculary, subcutaneously injections or gastrointestinally administered. Then, an image of the body or of the indicated region is obtained using conventional imaging equipment and techniques as described above. The image thus produced by that equipment can optionally be fixed in hard copy form, in photographic negative or positive form, or stored within a computer memory for subsequent recall onto a screen or for subsequent conversion into hard copy.
A "diagnostically effective amount" as used herein refers to a dose of the diagnostic conjugate, or diagnostic agent that is sufficient for detection (e.g. tumor detection) using the various diagnostic methods described. Typical doses of the diagnostic conjugates are in the range from about 0.001 to about 20 mmol/kg body weight, and preferably in the range from about 0.005 to about 5 mmol/kg body weight. The diagnostically effective amount may be different depending on the diagnostic agent attached to the conjugate of the invention as well as the diagnostic apparatus used for detection.
According to a further aspect the present invention provides a method for site targeted chemotherapy in a subject in need thereof comprising administering to the subject a therapeutical composition comprising a therapeutically effective amount of a conjugate comprising at least one isoflavone derivative bound to albumin and at least one cytotoxic compound for targeting of the cytotoxic compound to cancer cells. According to some embodiments, the cancer cells are sensitive to isoflavone.
The term "therapeutically effective amount" as used herein refers to that amount of the therapeutic conjugate of the invention which elicits a biological or medicinal response that will contribute to the cancer-treating ability of the therapeutic conjugate.
The term "treating" as used herein refers to partial or total inhibition of the growth, spreading, or metastasis of cancer, as well as partial or total destruction of the cancer cells. The term "treating" includes the reduction or elimination of cancer, and also the reduction in the incidence of the disease.
Diagnostic and therapeutic compositions
According to another embodiment, the present invention provides pharmaceutical composition i.e., diagnostic and therapeutic compositions, comprising as an active ingredient a conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound selected from a diagnostic agent and a therapeutic agent according to embodiment of the invention and a pharmaceutically acceptable carrier, excipient or diluent. As used herein, a "diagnostic composition" refers to a preparation of one or more of the active ingredients described herein, e.g. a conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound which is a diagnostic agent, with other components such as physiologically suitable carriers and excipients. The purpose of a diagnostic composition is to facilitate the administration of the conjugate of the invention to a subject for diagnostic purposes.
As used herein, a "therapeutic composition" refers to a preparation of one or more of the active ingredients described herein, e.g. a conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound which is a therapeutic agent, with other components such as physiologically suitable carriers and excipients. The purpose of a therapeutic composition is to facilitate the administration of the conjugate of the invention to a subject for treatment purposes.
Hereinafter, the phrases "therapeutically acceptable carrier" and "pharmaceutically acceptable carrier", which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
Herein, the term "excipient" refers to an inert substance added to a diagnostic or a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
In another embodiment of the present invention, a diagnostic or a pharmaceutical composition further comprises a pharmaceutically acceptable carrier. As used herein, a "carrier" refers to any substance suitable as a vehicle for delivering a conjugate of the present invention to a suitable in vivo or in vitro site. As such, carriers can act as a pharmaceutically acceptable excipients of a diagnostic or a therapeutical composition containing a conjugate of the present invention. Preferred carriers are capable of maintaining a conjugate of the present invention in a form that, upon arrival of the conjugate to a cell, the conjugate is capable of entering the cell. Non limiting examples of suitable carriers include: water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters and glycols. Aqueous carriers can contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity.
Suitable auxiliary substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances can also include preservatives, such as thimerosal, m- and o-cresol, formalin and benzol alcohol. Preferred auxiliary substances for aerosol delivery include surfactant substances non-toxic to a subject, for example, esters or partial esters of fatty acids containing from about six to about twenty-two carbon atoms. Examples of esters include: caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric, and oleic acids. Diagnostic and therapeutic compositions of the present invention can be sterilized by conventional methods.
Pharmaceutical composition for use in accordance with the present invention thus may be formulated in conventional manner using one or more acceptable diluents or carriers comprising excipients and auxiliaries as described, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Proper formulation is dependent on the route of administration chosen.
The diagnostic and therapeutic composition of the present invention may be formulated for administration by a route selected from the group consisting of intravenous injections, intravenous infusion, intradermal, intralesional, intramuscular, intravesicular and subcutaneous injections or depots. Alternatively, the composition of the invention may be administered parenterally by means other than injection, for example, they could be introduced laparascopically, intravesicularly, or via any orifice not related to the gastrointestinal tract. Alternatively, the compositions of the present invention may be formulated for oral administration.
The diagnostic and therapeutic compositions can take the form of solutions, suspensions, emulsions, syrups, gels, tablets, pills, capsules, powders, suppositories and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions will contain an effective amount of the conjugate of the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
Pharmaceutical composition of the present invention may be manufactured by processes well known in the art, e.g. by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
The principles of the invention, using an albumin conjugate isoflavone derivative bound to a bioactive moiety such as an imaging agent or a therapeutic agent for selective delivery to cells susceptible to isoflavone according to the present invention, may be better understood with reference to the following non-limiting examples.
EXAMPLES
Reagents: BSA, nystatin, diethylenetriamine pentaacetic acid (DTPA) anhydride, GdCl3 and 4',6-diamidino-2-phenylindole (DAPI) - sigma-Aldrich (St. Louis, MO).
Carboxyfluorescein succinimide ester (FAM-NHS)), Carboxy-X-rhodamin succinimide ester (ROX-NHS) - Molecular Probes, Invitrogen (California, USA).
N- 1 -(p-isothiocyanatophenyl)diethylenetriamine-N ' ,N2,N3-tetraacetate(DTTA) chelated with Eu 3+- Perkin-Elmer (Turku, Finland).
Example 1: Synthesis of daidzein-BSA-imaging agent conjugate:
The conjugates were prepared in a three-step procedure. In the first step of the reaction, both the carboxy derivative of daidzein and the imaging agent were treated with N-hydroxysuccinimide and carbodiimide to form an active ester. In the second step the activated ester of the imaging agent reacted at pH 8.5 with the amino group of lysine residues of BSA to form a BSA-imaging agent conjugate. In the third step the activated ester of daidzein reacted at pH 8.5 with the amino group of lysine residues of BSA to form a daidzein-BSA-imaging agent conjugate The synthesis of Daidzein-BSA-GdDTPA: BSA-DTPA was synthesized as described (Dafni et al., 2003, Magn. Reson. Med.; 50:904-14). Briefly, DTPA anhydride 1.6 gr (suspended in 4 ml of dry DMF) was slowly added with stirring to BSA 1.3 gr (in 40 ml Hepes 0.1 M, pH 8.8) while the reaction was titrated with NaOH 5N and stirred for 2 hours. The product was dialyzed against NaHCO3. Afterwards, the N- hydroxysuccinimide ester of 7-(O)-carboxymethyl (daidzein-NHS), synthesized according to Kohen et al, 2007, J Med. Chem.; 50:6405-10, (11 mg in 800 μl of anhydrous DMF) was added to BSA-DTPA (in 40 ml OfNaHCO3, 0.1M, pH 8.5) and stirred overnight. The product was dialyzed first against NaHCO3, followed by sodium citrate (0.1 M, pH 6.5). GdCl3 650 mg (in 5 ml sodium acetate 0.1M, pH 6.0) was added to Daidzein-B SA-DTPA in sodium citrate (0.1 M pH 6.5). The final product (Daidzein-BSA-GdDTPA) was dialyzed extensively against water and lyophilized.
The synthesis of daidzein-BSA-CvTE-777/FAM/ROX: CyTE-777-NHS (44 mg) prepared according to Hilderbrand et al., 2005, Bioconjug. Chem.; 16:1275-81, or FAM-NHS (35 mg) or ROX-NHS (35 mg) (in 200 μl of dry DMF) were added to BSA (450 mg in 15 ml NaHCO3, 0.1M, pH 8.5), slowly with stirring. The reaction was stirred overnight, and the product was dialyzed against NaHCO3, 0.1M, pH 8.5, followed by several changes of water and lyophilized. The preparation of N- hydroxysuccinimide ester of 7-(O)-carboxymethyl (daidzein NHS) has been reported previously (Kohen, et al. 2007, J Med. Chem. 50:6405-6410; Dafni et al. 2002. NMR Biomed 15:120-131).
N-hydroxysuccinimide ester of 7-(O)-carboxymethyl (daidzein-NHS), (8 mg in 600 μl of anhydrous DMF) was added to BSA-CyTE-777 (100 mg in 6 ml of NaHCO3, 0.1M, pH 8.5) and stirred overnight. The product was purified by dialyzed against NaHCO3 0.1M, pH 8.5, followed by extensive dialysis against water and lyophilized.
Example 2: In vitro evaluation of the endocytic pathways in ovarian cancer cells: a. A fluorescence microscopy study:
MLS cells (2*105) were cultured on polylysine coated coverslips for 48h. Subsequently, the coverslips were incubated for 1 hour at 37 with daidzein-BSA- FAM (200 μg/ml) or BSA-FAM (200 μg/ml) or combination of both in the presence and absence of a blocking dose of nystatin (50 μg/ml). The excess of fluorescent material was washed 3 times with PBS, and the cells were fixed with 4% PFA, washed, stained with DAPI and mounted. The images were monitored by two-photon microscopy (2PM; Zeiss LSM 510 META NLO; equipped with a broadband Mai Tai- HP-fentosecond single box tunable Ti-sapphire oscillator, with automated broadband wavelength tuning 700-1020 nm from Spectraphysics, for two-photon excitation). In this experiment the uptake of BSA-FAM, targeting cellular caveolae, and daidzein- BSA-FAM, targeting both caveolae and cell surface receptors with affinity to daidzein, by MLS human epithelial ovarian carcinoma cells was detected by fluorescence microscopy. It was shown that the fluorescent probes internalized the interacellular vesicles (FIG. IA). Upon addition of nystatin (50 μg/ml, 30 min), an elevated membrane staining and reduced uptake of BSA-FAM was observed (FIG. IA, B), whereas a significant enhanced internalization of daidzein-B SA-FAM by MLS cells was observed (FIG. IA).
Without being bound by theory or mechanism of action it is hypothesized that albumin binding mediated by caveolae interferes with the internalization of daidzein- BSA-FAM into the cells, despite the role of caveolae uptake of BSA-FAM.
To further test this hypothesis, the ability of BSA to compete with daidzein-BSA- FAM on the albumin binding site in caveolae, thus facilitating the internalization of the material into the cells, was tested (FIG. IB). Here again, internalization of daidzein-BSA-FAM was augmented by addition of nystatin. b. A flow cytometry study:
MLS cells (106) were incubated for 30 minutes with daidzein-BSA-FAM (200 μg/ml) or BSA-ROX (200 μg/ml) or a combination of both in the presence and absence of nystatin (50 μg/ml). The excess of fluorescent material was washed three times with PBS containing 0.01% of sodium azide. The cellular uptake of fluorescent material was monitored by FACScan (Becoton Dickinson, USA). The excitation produced by air-cooled argon laser 288 nm and the emission signal was collected by FLl filter (BP530/30) for FAM and FL2 filter (BP585/42) for ROX.
In this experiment the treatment of cells with nystatin resulted in a significant 4 fold enhancement in the fluorescence intensity of cells incubated with daidzein-BSA-FAM (FIG. 2A, B). On the other hand, binding and uptake of BSA-ROX were only slightly reduced by nystatin. Incubation of the cells with both BSA-ROX and daidzein-BSA- FAM resulted in differential response to nystatin, with increased labeling of cells with daidzein-BSA-FAM (3 fold induction in the intensity and increase of 20% in the population of labeled cells) and decreased fluorescence of BSA-ROX (1.3 fold in intensity and 51% decrease in the labeled population) (FIG. 3 A-E).
Example 3: In vivo targeted delivery of daidzein-BSA-CyTE-777 to ovarian carcinoma cells:
All in vivo experiments were approved by the Animal Care and Use Committee of the Weizmann Institute. CD-I nude mice were inoculated s.c. with 2.5*106 MLS rumor cells. Tumors were allowed to grow until 5-7 mm in diameter (for approximately 14- 21 days). a. A NIR imaging study:
Tumor bearing mice were injected intravenously with daidzein-BSA-CyTE-777 (i.v.; lmg (for IVIS 100) or 0.5 mg (for IVIS Spectrum) in 0.1 ml PBS/mouse; n=5 and n=2 respectively); or with BSA-CyTE-777 (1 mg or 0.5 mg in 0.1 ml PBS/mouse; n=2 and n=2) as control or with and BSA-FAM (lmg in 0.1 ml PBS/mouse) as a non specific competitor. The NIR signal in the whole animal was monitored by IVIS 100 and IVIS Spectrum (Xenogen, Caliper) at 24, 48 and 72 hours. The mice were fed with alfalfa-free (chlorophyll-free) diet 72 hours before visualization. In the IVIS 100 the data was acquired by 710-760 excitation, 675-720 excitation background and 810- 860 emission filters, hi the IVIS Spectrum the data obtained for the daidzein-BSA- CyTE-777 was acquired by 745 nm excitation and 820 nm emission filters. The pharmacokinetics of the fluorescent and MRI BSA based contrast media and their plasma concentration after intravenous administration were previously reported to be similar for all tags (Dafni et al. 2003, Magn. Reson. Med., 50:904-14). Systemic delivery of daidzein-BSA-CyTE-777 resulted in tumor selective enhancement of NIR fluorescent for subcutaneous ovarian carcinoma tumor xenografts. Mice were inoculated subcutaneously with 2.5* 106 MLS tumor cells. Ten days after tumor inoculation, mice administered with daidzein-BSA-CyTE777 or BSA-CyTE777. Already in the first 30 minutes after administration, mice injected with daidzein-BSA-CyTE777 showed more specific localization in the tumor and in higher concentration than mice administered with BSA-CyTE777. Competition of daidzein-BSA-CyTE777 with BSA-ROX overload increased the NIR signal of daidzein-BSA-CyTE777 in the tumor. This experiment further demonstrated different pharmacokinetic behavior in the elimination of the contrast materials from the tumor. Control mice injected with BSA-CyTE777 showed first order clearance of the contrast media. Daidzein-BSA-CyTE777 was eliminated with rapid initial kinetics, however it was followed by residual retention of daidzein-BSA-CyTE777 with very slow clearance. Thus, after the initial elimination mice administered with daidzein- BSA-CyTE777 showed specific NIR signal in the tumor area with maximal tumor to background ratio 48 hours after administration (n=8; FIG. 4A). Control mice that were injected with BSA-Cy TE-777 did not show any residual NIR signal 48 hours after administration (n=2; FIG. 4B). Prolonged enhancement of the fluorescent signal in the tumor area was further observed in mice that were injected with both daidzein- BSA-CyTE-777 and the non-specific competitor - BSA-FAM. In these mice the NIR signal could be detected even 72 hours after administration (n=2-4; FIG. 4C and D). The NIR signal in the tumors was detectable even 10 days after the administration. b. A biodistribution study:
The tumor bearing mice were injected intravenously with BSA-Eu chelate (0.34 mg equivalent to 100000 counts of europium in 0.1 ml PBS/mouse) or daidzein-BSA-Eu chelate (0.24 mg of protein equivalent to 100000 counts of europium in 0.1 ml PBS/mouse). The Eu chelates were used here due to their chemical similarity to the Gd chelates used for MRI. After 24 and 48 hours the mice were sacrificed and tumors as well as various tissues were removed. The tissues were subsequently homogenized in RIPA buffer ((20 mM Tris, pH 7.4, 137 mM NaCl, 10% glycerol, 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) sodium dodecyl sulfate (SDS), 1% Triton X-100, 2 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail) and centrifuged in an airfuge. The protein content in supernatant was first quantified by Bradford assay and then used for biodistribution studies and Western blot analysis.
For biodistribution studies an aliquot (20μg) from each of the supernatants was added to a microtiter plate. Enhancement solution (200μl) was added to each aliquot and the plate was shaken for 15 minutes on a plate shaker. The Europium signal in the wells was measured by time resolved fluorescence using Victor (Perkin-Elmer, USA). For Western blot analysis, aliquots (100 mg/lane) from the supernatants were subjected to 8% SDS-PAGE. Proteins were electrophoretically transferred from the SDS-polyacrilamide gel to a nitrocellulose membrane (Protan BA 85, Schleicher & Schuell). The membrane was blocked over night at 4C with 2% BSA in 10 mM Tris- buffered saline containing 0.05% Tween (TBST), followed by incubation with anti- daidzein antibody IgG (1 mg/ml TBS) for 2 hours at 25C. The membrane was then washed three times in TBST, incubated with an HRP-conjugated goat anti mouse antibody (1:10000 in TBS) and re- washed. The protein bands were visualized by ECL. These experiments demonstrated that 24 and 48 hours after injection, a 2 to 3 fold increase in Eu in tumors of mice injected with daidzein-BSA-Eu chelate was observed as compared to those injected with BSA-Eu chelate (FIG. 5A, B). Western blot analysis of tissues isolated 48 hours after injection from these mice showed a specific protein band of daidzein-BSA in the tumor, liver and kidney of the daidzein-BSA-Eu chelate treated animal, but not in the BSA-Eu chelate treated animal (FIG. 5C).
The distribution of contrast material inside the tumors was visualized by fluorescence microscopy, in tumors isolated 24 hours after injection with daidzein-B SA-FAM or BSA-FAM. Daidzein-BSA-FAM was localized in the tumor cells areas, while BSA- FAM was localized to the tumor blood vessels and their surrounding stroma cells (Fig. 6A - C). c. An MRI study:
MRI studies were performed on a horizontal 4.7T Bruker Biospec spectrometer using an actively radio-frequency decoupled 1.5 cm surface coil embedded in a Perspex board and a birdcage transmission coil. For in-vitro experiments: R1 measurements spin echo images were acquired at 8 different repetition times ranging between 2000 and 100 ms; 2 averages, field of view 4x4 cm, slice thickness 1 mm, matrix 128x128). R] relaxation rates for the in vitro experiments were derived by non linear single exponential fitting of images acquired at different repetition times according to equation I: I = M0 (l-e"TR*Rl) (I) Where I is the measured signal intensity for each TR and Ri is derived from optimization of the curve fitting; Mo is the steady state signal intensity in fully relaxed images.
For in vivo experiments: The tumor bearing mice were injected intravenously with BSA-GdDTPA (12 mg in 200 μL PBS/mouse) or daidzein-BSA-GdDTPA (12 mg in 200 μL PBS/mouse) or with combination of daidzein-BSA-GdDTPA and BSA-FAM (competition experiment). Rl was measure 24, 48 and 72 hours after injection of the contrast material. Tl weighted 3D gradient-echo (GE) images, with pulse flip angles of 5°, 15°, 30°, 50° and 70° were acquired for the determination of Rl values. The acquisition parameters used: TR 10 ms; TE 3.561 ms; 2 averages; field of view 4x4x4; 128x128x128 pixels. Three-dimensional gradient-echo data sets were used for generation of Rl maps as well as for calculation of the average Rl values in selected regions of interest by nonlinear best fit to equation [H]:
I = M0 sin α (1 -e"TR*Rl) / (1 - cos α e"TR*Rl) (II) Where I is the signal intensity as a function of the pulse flip angle. Student's t test (two tailed, equal variance) was used for statistical analysis of the significance of change in relaxation rate between control and labeled tumors.
Daidzein-BSA-GdDTPA showed significant relaxivity of 194 mM'V1 (per BSA; FIG. 7A). This relaxivity was similar to the relaxivity of BSA-GdDTPA 196 mM'V1 (per BSA). MRI data acquired from MLS tumor bearing mice 24 hours after administration of daidzein-BSA-GdDTPA or BSA-GdDTPA, showed significantly higher contrast enhancement, consistent with accumulation of daidzein-BSA- GdDTPA (targeted contrast agent) in the tumor area as compared to vehicle injected mice or mice injected with BSA-GdDTPA (non-targeted contrast agent) (FIG. 7B). Time course experiments showed specific localization and retention of daidzein-BSA- GdDTPA in the tumor site 24, 48 and 72 hours after injection as compared to controls or mice injected with BSA-GdDTPA. Statistically significant elevation of Ri relaxation was visualized in tumors injected with daidzein-BSA-GdDTPA as compared to non injected mice or mice injected with BSA-GdDTPA (for 24h p=0.01 ; 48h p-0.016, 72h p=0.0488) (FIG. 7C). Prolonged detection of daidzein-BSA- GdDTPA was feasible even for a lower administrate dose. MLS tumor bearing mice were injected with daidzein-BSA-GdDTPA (4 mg/200 μl; n=3). The specific localization of the targeted contrast agent in the tumor site was detected by MRI at 9.4T 24h after injection (FIG. 7D) and was still detectable and even enhanced 7 days after injection (FIG. 7E).

Claims

1. A conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound D, the conjugate represented by the structure of general formula (I):
Albumin — Dn1
Figure imgf000039_0001
I wherein m is 1-20; ni is 0-10; n2 is 0 or 1 ;
R1 is selected from the group consisting of -OH, -OCH3, -OGIc, -OR7COOX and -OR7C(=O)-;
R2, R5 and R are each independently selected from the group consisting of -H, -
-R7COOX and -R7C(O)-;
R3 is selected from the group consisting of -H, -OH, -R7COOX, -OR7COOX, -
R7C(K))- and -OR7C(=O)-;
R4 is selected from the group consisting of -H, -CH3, -R7COOX and -R7C(=0)-;
D is a bioactive moiety selected from a diagnostic agent and a therapeutic agent;
R7 is selected from the group consisting of Cj-C6 alkylene, (C2-C20) alkenylene; and (C2-C20) heteroalkylene;
X is selected from the group consisting of -H and -(CH2V Y wherein Y is -CH3 or -NH2 and n is 0-10; with the proviso that one of R1, R2, R3, R4, R5 or R6 is comprises a carboxy group selected from -OR7C(O)- and -R7C(O)- which is bound to albumin, where at least one of ni and n2 is other than zero.
2. The conjugate of claim 1, wherein the conjugate is capable of delivering the bioactive moiety into a cell sensitive to isoflavone.
3. The conjugate of claim 2, wherein the cell is a cancer cell.
4. The conjugate of claim 3, wherein the cancer cell is selected from the group consisting of an ovarian carcinoma cell, a colon cancer cell, a breast cancer cell, a melanoma cell, a non-small lung cancer cell, a leukemia cell, an endometrial cancer cell and a prostate cancer cell.
5. The conjugate of claim 1 , wherein n\ is other than zero and when n\ is greater than 1 , D is the same or different at each occurrence.
6. The conjugate of claim 1, wherein n2 is other than zero, wherein one of R1, R2, R3, R4, R5 or R6 comprises a carboxy group selected from -OR7C(=O)- and -R7C(=O)- which is bound to D.
7. The conjugate of claim 6, wherein n2 is other than zero, wherein one of R1, R2, R3, R4, R5 or R6 comprises a carboxy group selected from -OR7C(=O)- and -R7C(O)- which is bound to D, wherein D is the same or different at each occurrence.
8. The conjugate of claim 1 wherein the isoflavone derivative is selected from a derivative of daidzein, genistein, formononetin and biochanin A.
9. The conjugate of claim 1 wherein the isoflavone derivative is a derivative of daidzein.
10. The conjugate of claim 1 wherein the isoflavone derivative is selected from the group consisting of 7-(O)-carboxymethyl daidzein, 6-carboxymethyl biochanin A, 2-carboxyalkyl biochanin A, 8-carboxymethyl biochanin A, 7-(O)-carboxymethyl formononetin, 2-carboxyalkyl genistein and 6-carboxymethyl genistein.
11. The conjugate of claim 1 wherein the isoflavone derivative is 7-(O)- carboxymethyl daidzein.
12. The conjugate of claim I5 wherein D is an diagnostic agent selected from the group consisting of paramagnetic particles such as gadolinium, yttrium, lutetium, europium and gallinium; radioactive moieties such as radioactive indium, rhenium and technetium; fluorescent dyes such as fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), Cyan fluorescent protein (CFP), rhodamine I, II, III and IV, rhodamine B and infrared dyes such as CyTE-777 and rosamine.
13. The conjugate of claim 12, wherein the paramagnetic particle is gadolinium.
14. The conjugate of claim 12, wherein the paramagnetic particles are provided within a chelator.
15. The conjugate of claim 14, wherein the chelator is selected from the group consisting of: diethylenetriamine pentaacetic acid (DTPA), tetraazacyclododecanetetraacetic acid (DOTA), mercaptoacetylglycylglycyl- glycine (MAG3) and N-l-φ-isothiocyanatophenyOdiethylenetriamine-N^N^N3- tetraacetate(DTTA).
16. The conjugate of claim 15, wherein the chelator is diethylenetriamine pentaacetic acid (DTPA).
17. The conjugate of claim I5 wherein D is a therapeutic agent selected from the group consisting of cytotoxic compounds, cytostatic compounds, antisense compounds and anti-viral agents.
18. The conjugate of claimlό, wherein D is a cytotoxic compound selected from the group consisting of: DNA synthesis and function inhibitory agents such as adriamycin, bleomycin, chlorambucil, cisplatin, carboplatin, oxaliplatin, daunomycin and melphalan; microtubule (mitotic spindle) formation and function inhibitory agents such as vinblastine, vincristine, vinorelbine, paclitaxel (taxol) and docetaxel; anti metabolites such as cytarabine, fluorouracil, fluroximidine, mercaptopurine, methotorexate, gemcitabin and thioquanine; alkylating agents such as mechlorethamine, chlorambucil, cyclophosphamide, melphalan ifosfamide and methotrexate; antibiotics such as bleomycin and mitomycin; nitrosoureas such as carmustine (BCNU) and lomustine; hormones such as tamoxifen, leuprolide, flutamide and megestrol acetate and proteins such as interferon and asparaginase.
19. The conjugate of claim 1, further comprising an affinity molecule selected from biotin and at least the antigen binding portion of a specific antibody.
20. A diagnostic composition comprising as an active ingredient a conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound D, having the general formula (I): Albumin — Dn1
Figure imgf000042_0001
wherein m is 1-20; ni is 0-10; n2 is 0 or 1 ;
R1 is selected from the group consisting of -OH, -OCH3, -OGIc, -OR7COOX and -OR7C(=O)-;
R2, R5 and R6 are each independently selected from the group consisting of -H, -
R7COOX and -R7C(=O)-; R3 is selected from the group consisting of -H, -OH, -R7COOX, -OR7COOX, -
R7C(O)- and -OR7C(=O)-;
R4 is selected from the group consisting of -H, -CH3, -R7COOX and -R7C(=0)-;
D is a bioactive moiety selected from a diagnostic agent and a therapeutic agent;
R7 is selected from the group consisting of Ci-C6 alkylene, (C2-C20) alkenylene; and (C2-C20) heteroalkylene;
X is selected from the group consisting of -H and -(CH2)n-Y wherein Y is -CH3 or -NH2 and n is 0-10; with the proviso that one of R1, R2, R3, R4, R5 or R6 comprises a carboxy group selected from -OR7C(=O)- and -R7C(=0)- which is bound to albumin and at least one of n\ and n2 is other than zero; and with the proviso that at least one D is a diagnostic agent.
21. The diagnostic composition of claim 20, wherein ni is other than zero and when ni is greater than 1, D is the same or different at each occurrence.
22. The diagnostic composition of claim 21, wherein n2 is other than zero, wherein one of R1, R2, R3, R4, R5 or R6 comprises a carboxy group selected from -
OR7Q=O)- and -R7C(=O)- which is bound to D.
23. The diagnostic composition of claim 21, wherein n2 is other than zero, wherein one of R1, R2, R3, R4, R5 or R comprises a carboxy group selected from - OR7C(O)- and -R7C(=O)- which is bound to D, wherein D is the same or different at each occurrence.
24. The diagnostic composition of claim 20, wherein the isoflavone derivative is a derivative of daidzein, genistein, formononetin and biochanin A.
25. The diagnostic composition of claim 24, wherein the isoflavone derivative is daidzein derivative.
26. The diagnostic composition of claim 20, wherein the isoflavone derivative is selected from the group consisting of 7-(O)-carboxymethyl daidzein, 6- carboxymethyl biochanin A, 2-carboxyalkyl biochanin A, 8-carboxymethyl biochanin A, 7-(O)-carboxymethyl formononetin, 2-carboxyalkyl genistein and 6- carboxymethyl genistein.
27. The diagnostic composition of claim 26, wherein the isoflavone derivative is 7- (O)-carboxymethyl daidzein.
28. The diagnostic composition of claim 20, wherein D is an diagnostic agent selected from the group consisting of paramagnetic particles such as gadolinium, yttrium, lutetium, europium and gallinium; radioactive moieties such as radioactive indium, rhenium and technetium; fluorescent dyes such as fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), Cyan fluorescent protein
(CFP), rhodamine I, II, III and IV, rhodamine B and infrared dyes such as CyTE- 777 and rosamine.
29. The diagnostic composition of claim 28, wherein the paramagnetic particle is gadolinium.
30. The diagnostic composition of claim 28, wherein the paramagnetic particles is provided within a chelator.
31. The diagnostic composition of claim 30, wherein the chelator is selected from the group consisting of: diethylenetriamine pentaacetic acid (DTPA), tetraazacyclododecanetetraacetic acid (DOTA), mercaptoacetylglycylglycyl- glycine (MAG3) and N-l-φ-isothiocyanatophenytydiethylenetriamine-N'jN^N3- tetraacetate(DTTA).
32. The diagnostic composition of claim 31, wherein the chelator is diethylenetriamine pentaacetic acid (DTPA).
33. The diagnostic composition of claim 20, further comprising nystatin.
34. A method for diagnosing a disease or disorder in a subject comprising administering to the subject a diagnostic composition according to claim 20.
35. The methods according to claim 34, wherein the disease or disorder is associated with cells which are sensitive to isoflavone.
36. The method according to claim 34, wherein the disease or disorder is cancer.
37. The method according to claim 35, wherein cancer is selected from ovarian cancer, breast cancer, colon cancer, melanoma, non-small lung cancer, leukemia, endometrial cancer and prostate cancer.
38. The method according to claim 34, wherein the disease or disorder is an estrogen related condition selected from the group consisting of osteoporosis, endometriosis, atherosclerosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy and Alzheimer's Disease.
39. A method for enhancing image contrast in in vivo imaging procedure comprising administering a diagnostic composition according to claim 20.
40. The method of claim 39, wherein the in vivo imaging procedure is selected from the group consisting of MRI, NIR, optical imaging, scintigraphy, SPECT, PET, X- ray, ultrasound imaging, electrical impedance and magnetometric procedures.
41. The method of claim 40, wherein the in vivo imaging procedure is MRI.
42. The method of claim 40, wherein the in vivo imaging procedure is NIR.
43. A pharmaceutical composition comprising as an active ingredient a conjugate comprising at least one isoflavone derivative bound to albumin and at least one compound D, the conjugate represented by the structure of general formula (I): Albumin — Dn1
Figure imgf000045_0001
I wherein m is 1-20; n! is 0-10; n2 is 0 or 1 ;
R1 is selected from the group consisting of -OH, -OCH3, -OGIc, -OR7COOX and -OR7C(O)-;
R2, R5 and R6 are each independently selected from the group consisting of -H, -
R7COOX and -R7C(O)-; R3 is selected from the group consisting of -H, -OH, -R7COOX, -OR7COOX, -
R7C(K))- and -0R7C(=0)-;
R4 is selected from the group consisting of -H, -CH3, -R7COOX and -R7C(=0)-;
D is a bioactive moiety selected from a diagnostic agent and a therapeutic agent;
R7 is selected from the group consisting Of C1-C6 alkylene, (C2-C20) alkenylene; and (C2-C20) heteroalkylene;
X is selected from the group consisting of -H and -(CH2)n-Y wherein Y is -CH3 or -NH2 and n is 0-10; with the proviso that one of R1, R2, R3, R4, R5 or R6 comprises a carboxy group selected from -OR7C(O)- and -R7C(=0)- which is bound to albumin, where at least one of n\ and n2 is other than zero; and, with the proviso that at least one D is a therapeutic agent.
44. The pharmaceutical composition of claim 43, wherein ni is other than zero and when n\ is greater than 1 , D may be the same or different at each occurrence.
45. The pharmaceutical composition of claim 43, wherein n2 is other than zero, wherein one of R1, R2, R3, R4, R5 or R6 comprises a carboxy group selected from -
0R7C(=0)- and -R7C(O)- which is bound to D.
46. The pharmaceutical composition of claim 44, wherein n2 is other than zero, wherein one of R1, R2, R3, R4, R5 or R6 comprises a carboxy group selected from - OR7C(=O)- and -R7C(=O)- which is bound to D, wherein D is the same or different at each occurrence.
47. The pharmaceutical composition of claim 43, wherein the isoflavone derivative is selected from a derivative of daidzein, genistein, formononetin and biochanin A.
48. The pharmaceutical composition of claim 47, wherein the isoflavone derivative is daidzein derivative.
49. The pharmaceutical composition of claim 43, wherein the isoflavone derivative is selected from the group consisting of 7-(O)-carboxymethyl daidzein, 6- carboxymethyl biochanin A, 2-carboxyalkyl biochanin A, 8-carboxymethyl biochanin A, 7-(O)-carboxymethyl formononetin, 2-carboxyalkyl genistein and 6- carboxymethyl genistein.
50. The pharmaceutical composition of claim 49, wherein the isoflavone derivative is 7-(O)-carboxymethyl daidzein.
51. The pharmaceutical composition of claim 43, wherein D is a therapeutic agent selected from the group consisting of cytotoxic compounds, cytostatic compounds, antisense compounds and anti-viral agents.
52. The pharmaceutical composition of claim 51, wherein D is a cytotoxic compound selected from the group consisting of: DNA synthesis and function inhibitory agents such as adriamycin, bleomycin, chlorambucil, cisplatin, carboplatin, oxaliplatin, daunomycin and melphalan; microtubule (mitotic spindle) formation and function inhibitory agents such as vinblastine, vincristine, vinorelbine, paclitaxel (taxol) and docetaxel; anti metabolites such as cytarabine, fluorouracil, fluroximidine, mercaptopurine, methotorexate, gemcitabin and thioquanine; alkylating agents such as mechlorethamine, chlorambucil, cyclophosphamide, melphalan ifosfamide and methotrexate; antibiotics such as bleomycin and mitomycin; nitrosoureas such as carmustine (BCNU) and lomustine; hormones such as tamoxifen, leuprolide, flutamide and megestrol acetate And proteins such as interferon and asparaginase.
53. The pharmaceutical composition of claim 43, further comprising nystatin.
54. A method for treating a disease or disorder in a subject in need thereof comprising administering to the subject a pharmaceutical composition according to claim 41.
55. The method according to claim 54, wherein the disease or disorder is associated with cells which are sensitive to isoflavone.
56. The method according to claim 55, wherein the disease or disorder is cancer.
57. The method according to claim 56, wherein cancer is selected from ovarian cancer, breast cancer, colon cancer, melanoma, non-small lung cancer, leukemia, endometrial cancer and prostate cancer.
58. The method according to claim 54, wherein the disease or disorder is an estrogen related condition selected from the group consisting of osteoporosis, endometriosis, atherosclerosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy and Alzheimer's Disease.
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