WO2014107419A1 - Utilisation de gradients d'oscillation de champ magnétique intense pour une destruction spécifique de cellules marquées au moyen de nanoparticules magnétiques - Google Patents

Utilisation de gradients d'oscillation de champ magnétique intense pour une destruction spécifique de cellules marquées au moyen de nanoparticules magnétiques Download PDF

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WO2014107419A1
WO2014107419A1 PCT/US2013/078196 US2013078196W WO2014107419A1 WO 2014107419 A1 WO2014107419 A1 WO 2014107419A1 US 2013078196 W US2013078196 W US 2013078196W WO 2014107419 A1 WO2014107419 A1 WO 2014107419A1
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cells
nanoparticles
magnetic field
population
magnetic
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Dmitri Artemov
Yoshinori Kato
Hapuarachchige SUDATH
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The Johns Hopkins University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5094—Microcapsules containing magnetic carrier material, e.g. ferrite for drug targeting
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • 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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • 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
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1875—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle coated or functionalised with an antibody

Definitions

  • Immunotherapeutics fall into at least three classes: (1) deployment of antibodies that, themselves, target growth receptors, disrupt cytokine pathways, or induce complement or antibody-dependent cytotoxicity; (2) direct arming of antibodies with a toxin, a radionuclide, or a cytokine; (3) indirect arming of antibodies by attaching them to immunoliposomes used to deliver a toxin or by attaching them to an immunological cell effector (bispecific antibodies).
  • thermotherapy temperatures in a range from about 40° C to about 46° C. (hyperthermia) can cause irreversible damage to disease cells.
  • healthy cells are capable of surviving exposure to temperatures up to around 46.5° C. Elevating the temperature of individual cells in diseased tissue to a lethal level (cellular thermotherapy) may provide a superior treatment option.
  • Pathogens implicated in disease and other undesirable matter in the body can also be destroyed via exposure to locally high
  • Temperatures greater than 46° C. may also be effective for the treatment of cancer and other diseases by causing an instantaneous thermo-ablative response.
  • accurate and precise targeting is necessary to ensure that a minimal amount of healthy tissue is exposed to such temperatures. Failure to achieve such a level of targeting may produce increased detrimental side effects, and thereby reducing the benefits of the treatment.
  • Hyperthermia may hold promise as a treatment for cancer and other diseases because it induces instantaneous necrosis (typically referred to as "thermo-ablation") and/or a heat-shock response in cells (classical hyperthermia), leading to cell death via a series of biochemical changes within the cell.
  • thermal-ablation instantaneous necrosis
  • RF radio frequency
  • APAS annular phased array systems
  • a strategy for treating a disease by generating heat within a tumor using superparamagnetic particles (having characteristic relaxation time-lCT 9 sec) that are suspended in a suitable medium, referred to as magnetic fluids, and exposing the patient to an alternating magnetic field (AMF) has been proposed (see e.g., U.S. Pat. No. 6,541,039 to Lesniak et al. and U.S. Pat. No. 6,470,220 to Kraus, et al).
  • the methods disclosed in the prior art involve the introduction of the magnetic fluid directly into the region to be treated and heating the particles by exposing a significant portion of the patient to low amplitude (less than 16 kA/m) alternating magnetic fields with frequency of between 50 kHz and 200 kHz, including the region of interest. It is well established that exposing a significant portion of a patient to an AMF will increase tissue temperature over the whole region exposed, and even the core body temperature, significantly because of the eddy currents generated by the interaction of the AMF with tissues.
  • Hyperthermia for treatment of disease using magnetic fluids exposed to RF fields has been recognized for several decades. However, a major problem with magnetic fluid hyperthermia has been the inability to selectively deliver a lethal dose of particles to the cells or pathogens of interest, particularly when the composition is limited to particles possessing characteristic relaxation times much shorter than the period of the applied RF.
  • the present invention provides a
  • composition comprising one or more magnetic nanoparticles characterized in that the nanoparticles are capable of binding to a target cell or population of cells or the magnetic nanoparticles are capable of being introduced into the cytoplasm of the target cell or population of cells in a subject, and the nanoparticles are administered to the subject in conjunction with exposing the subject to an oscillating gradient magnetic field while in the presence of a static magnetic field for a sufficient time to induce cytotoxicity in the target cell or population of cells.
  • the present invention provides a method for inducing cytotoxicity in a target cell or population of cells comprising: a) contacting the target cell or population of cells with one or more magnetic nanoparticles such that the magnetic nanoparticles are either bound to the surface of the target cell or population of cells, or the magnetic nanoparticles are introduced into the cytoplasm of the target cell or population of cells; b) positioning the target cells in a static magnetic field for certain time to enable the formation of magnetic nanoparticle clusters; and c) exposing the target cells of b) to an oscillating gradient magnetic field while in the presence of a static magnetic field for a sufficient time to induce cytotoxicity in the target cell or population of cells.
  • the present invention provides a method for treating a proliferative disease in a subject comprising: a) administering to the subject a pharmaceutical composition comprising one or more magnetic nanoparticles which are either capable of binding to a target cell or population of cells or the magnetic nanoparticles are introduced into the cytoplasm of the target cell or population of cells in the subject; b) allowing the one or more magnetic nanoparticles to bind the target cell or population of cells such that the magnetic nanoparticles are bound to the surface of the target cell or population of cells or are internalized into the target cell or population of cells in the subject; c) exposing the subject to an oscillating gradient magnetic field while in the presence of a static magnetic field for a sufficient time to induce cytotoxicity in the target cell or population of cells.
  • Figure 1 illustrates the gradient pulse sequence used for treatment of breast cancer cells in vivo and in vitro on a Bruker 9.4T Biospec small animal MR scanner.
  • Figure 2 is a graph showing changes in the temperature of agarose samples exposed to variable gradient field at 9.4T magnetic field.
  • the samples were positioned in a water-cooled chamber used for irradiation of animals and cells with no direct contact between the samples and the chamber walls. Slight variations in temperature ⁇ 0.2
  • Figure 3 shows a series of photomicrographs of Prussian blue staining of BT-474 breast cancer cells labeled with iron oxide: (A) unlabeled control cells; (B) BNF
  • nanoparticles/PLL labeled overnight (C) specific labeling of cell membranes with biotin- trastuzumab and streptavidin-SPIO at 4 C; and (C) internalization of trastuzumab-SPIO complexes at 37 C.
  • Figure 4 is another series of photomicrographs showing BT-474 cells subjected to high-frequency gradient magnetic field: (A) control unlabeled cells (24h); (B) cells loaded with BNF nanoparticles (24h); (C) cells loaded with Feridex ® nanoparticles; (D) cells labeled with Herceptin-SPIO (24h); and (E) BNF labeled cells immediately after application of gradients. Rounding and detaching of the cells is immediately visible in the image.
  • Figure 5 depicts T2 weighted MR imaging of tumor bearing mice injected with SPIO particles intratumorally (yellow arrows) (A and B).
  • C and D are planar X-ray imaging of the injected tumor. Arrow indicates the radioopaque injection area.
  • Figure 6 shows (A) bioluminescence imaging of the control and treated animals with iron oxide and saline-injected MDA-MB-231//wc xenografts before (left) and 24 hours after GIFT (right) (animals are noted as ml to m6 from left to right in the figure), ml : iron oxide (FeO) w/o GIFT; m2: FeO w/o GIFT; m3:FeO with GIFT; m4: FeO with GIFT; m5: saline with gradients; m6: FeO with GIFT (right on the mouse), saline with gradient (left on the mouse) (from left to right).
  • FeO iron oxide
  • saline-injected MDA-MB-231//wc xenografts before (left) and 24 hours after GIFT (right)
  • ml iron oxide (FeO) w/o GIFT
  • m2 FeO
  • FIG. 7 is a graph of BLI intensity ratios for FeO/Saline injected tumors at 24 hours post GIFT in comparison to control injected but untreated tumors.
  • Figure 7 depicts spatially colocalized H&E staining of cell necrosis (A) and iron distribution by Prussian blue staining (B) was observed in tumor sections 48 hours after GIFT therapy (yellow arrows).
  • Figure 8 shows (A) EM micrographs of MDA-MB-231 cells fixed and embedded into the matrix at a 4.7T magnetic field. Formation of linear structures of BNF nanoparticles parallel to the magnetic field is apparent on the 65,000x field of view (bar line is 100 nm). Inset on the right shows distribution of BNF nanoparticles in cells fixed and processed under non-magnetic conditions. (B) Optical microscopy demonstrates aligning of SPIO in cells fixed in the presence of 4.7T magnetic field (arrow). Random iron distribution was detected in non-magnetized cells.
  • the present invention provides methods for specific cytotoxic therapy, including, but not limited to, specific non-thermal killing of targeted cells, for example, cancer cells or stem cells, by labeling, binding or introducing into the cytoplasm of the cells, one or more magnetic nanoparticles, such as superparamagnetic iron oxide (SPIO) nanoparticles for example, followed by exposing the cells to oscillating gradients of a magnetic field in the presence of strong static magnetic field such as B 0 field generated by modern MRI systems.
  • SPIO superparamagnetic iron oxide
  • Specific labeling of cells can be achieved either by various means known in the art, including modifying the cell surface with targeted magnetic nanoparticles (wherein the magnetic nanoparticles are conjugated to one or more antibodies, peptides, or other high affinity ligands), or by loading the cells with nanoparticles internalized by endocytosis, pinocytosis, or through the use of standard cell transfection reagents and techniques.
  • the static magnetic field as well as oscillating gradients can be produced by various known apparatus used to generate such fields, including, standard MRI systems. The methods provided herein make the technology applicable for clinical as well as in vitro use.
  • the methods of the present invention include the use of dedicated MR pulse sequences that are used for efficient kill of the SPIO-labeled cells.
  • GIFT gradient-induced Fe therapy, as in one embodiment Fe nanoparticles are used
  • the term "indication”, as used herein, refers to a medical condition, such as a disease. Breast cancer is an exemplary indication.
  • ligand or label or lableing refers to a molecule or compound that attaches to the magnetic nanoparticles and targets and attaches to a biological marker.
  • a monoclonal antibody specific for HER-2 an epidermal growth factor receptor protein
  • the magnetic nanoparticles are labeled with trastuzumab which is an antibody specific for the HER-2/neu receptor found on breast cancer cells.
  • linker or "linker molecule,” as used herein, refer to an agent that targets particular functional groups on a ligand and on a magnetic particle or a coating, and thus forms a covalent link between any two of these.
  • magnetic nanoparticle refers to aggregates of magnetically coupled superparamagnetic particles or SPIOs.
  • marker refers to an antigen or other substance to which the magnetic nanoparticles' ligand is specific.
  • HER-2 protein is an exemplary marker.
  • target cell or population of cells refers to the matter for which deactivation, rupture, disruption or destruction is desired, such as a diseased cell, a pathogen, or other undesirable matter.
  • a marker may be attached to the target cell or population of cells.
  • the methods provided herein are the combined use of magnetic nanoparticles that are targeted to tumor cells with a specific ligand, which are on located on the target cell or population of cells.
  • the patient is positioned and subjected to a static Bo field, using, for example, a MRI machine for a period of time.
  • a static Bo field using, for example, a MRI machine for a period of time.
  • the oscillating gradient magnetic field is administered to the subject and/or to the targeted cell or population of cells, and will cause the nanoparticles to move or vibrate on the cell surface or within the cytoplasm of the cell while avoiding heating to targeted areas.
  • the magnetic nanoparticles of the present invention comprise a specific ligand or ligands or a targeting agent or agents.
  • ligand or “targeting agent” is meant any object that enables specific interaction with a target.
  • the targeting agent can migrate and integrate into a defined target tissue or population of cells, for example, in response to signaling from the target cell population. For example, growing tumor expresses cytokines (growth factors EGF, VEGF, TGF, etc.). Cell-surface molecules that are cancer specific antigens (or disease-specific antigens) can also serve as targets.
  • the methods of the present invention can be used to clear blood clots.
  • oscillating gradient magnetic field means a pulse sequence having, in one or more embodiments a series of gradient pulses repeated as for example square or sine waveform gradients, applied in three channels x, y, and z with a predetermined magnetic field strength, pulse duration, and frequency.
  • An example of such an oscillating gradient magnetic field is depicted in Fig. 1.
  • the magnetic nanoparticles can be labeled with a ligand and a dye.
  • the medicament for treating a proliferative disease in a subject can encompass many different formulations known in the pharmaceutical arts, including, for example, intravenous and sustained release formulations.
  • the proliferative disease or target cell or population of cells can include cancer.
  • Cancer can be any cancer, including any solid tumor type, such as alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, nasopharynx cancer, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer,
  • the magnetic nanoparticles can be injected into a tumor.
  • the target cells may also refer to tissue or cells of the immune system, such as tissue or cells effected by AIDS, pathogen-borne diseases, which can be bacterial, viral, parasitic, or fungal.
  • pathogen-borne diseases include HIV, tuberculosis, and malaria; hormone-related diseases, such as obesity, vascular system diseases, central nervous system diseases, such as multiple sclerosis, and undesirable matter, such as adverse angiogenesis, restenosis, amyloidosis, toxic reaction by-products associated with organ transplants, and other abnormal cell or tissue growth.
  • the nanoparticles of the present invention comprise magnetic nanoparticles which can be detected and in some embodiments, may also be visualized using magnetic resonance imaging (MRI), and these particles are also capable of cytotoxicity via being placed in an oscillating gradient magnetic field.
  • MRI magnetic resonance imaging
  • the nanoparticles should be magnetic or superparamagnetic in character. Examples include magnetic iron oxides Fe 3 0 4 (magnetite) and ⁇ -Fe 2 03 (maghemite) which have been proved to be well tolerated by the human body.
  • the nanoparticles of the invention may be metal or metal oxide nanoparticles and may for example contain cobalt, iron, cobalt and platinum or gold.
  • the nanoparticles should be biocompatible or, at least, be of an acceptable level of toxicity at therapeutic dosage levels.
  • the nanoparticles used in the methods of the invention are iron oxide
  • the nanoparticles are magnetic nanoparticles.
  • Magnetic nanoparticles that can be used in the invention include
  • the magnetic nanoparticles are superparamagnetic iron oxide (SPIO) nanoparticles.
  • SPIO superparamagnetic iron oxide
  • the present invention provides a
  • composition comprising one or more magnetic nanoparticles characterized in that the nanoparticles are capable of binding to a target cell or population of cells or the magnetic nanoparticles are capable of being introduced into the cytoplasm of the target cell or population of cells in a subject, and the nanoparticles are administered to the subject in conjunction with exposing the subject to an oscillating gradient magnetic field while in the presence of a static magnetic field for a sufficient time to induce cytotoxicity in the target cell or population of cells.
  • the magnetic nanoparticles can me visualized using MRI, optical, PET or SPECT imaging.
  • the nanoparticles of the present invention comprise bionized nanoferrite (BNF) particles prepared via the core-shell method with a core of 75-80% (w/w) magnetite and a shell of hydroxy ethyl starch, and which are available with particle diameters of about 80 nm and 100 nm.
  • BNF bionized nanoferrite
  • Other functions in addition to cytotoxicity and imaging are also contemplated by the nanoparticles of the present invention.
  • the nanoparticles can be labeled with other imaging agents, such as radionuclides, such as positron emitters, like 18 F or n C. Fluorescent and near-infra red labeling and functionalizing the nanoparticles with monoclonal antibodies and other functional groups (such as those that could promote clustering) are also
  • the term "localization” as used herein, means that the magnetic nanoparticles of the present invention have sufficient time post-administration, to migrate through the tissues or the body of the subject and arrive at the site of the target cell or population of cells, such as a tumor or tumors for example.
  • the term "detection” as used herein, means that the magnetic nanoparticles of the present invention are scanned with a magnetic resonance imaging (MRI) device or machines which are known and available in the art.
  • MRI magnetic resonance imaging
  • the whole body of the subject or the local area where the tumor is suspected of being located is placed in the MRI machine and the nanoparticles loaded on the stem cells are detected in the machine and their location is identified.
  • the types of MRI that may be used include T; weighted scans, T 2 weighted scans and T 2 * weighted scans. Both positive and negative MR contrast methods can be used to detect SPIO nanoparticles.
  • the magnetic nanoparticles can be administered to the subject via any number of routes which allow administration of viable cells, including, for example, intravenous, intrathecal, local and intra-tumor injection, implants, systemic, parenteral, subcutaneous, intravascular, intramuscular, intraperitoneal, topical, transdermal, buccal, intravaginal, ocular, inhalation, depot injection, and through various medical devices.
  • routes which allow administration of viable cells including, for example, intravenous, intrathecal, local and intra-tumor injection, implants, systemic, parenteral, subcutaneous, intravascular, intramuscular, intraperitoneal, topical, transdermal, buccal, intravaginal, ocular, inhalation, depot injection, and through various medical devices.
  • a pharmaceutically acceptable carrier can be any of those conventionally used, and is limited only by physico-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration.
  • the carriers described herein for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the carrier be one which is chemically inert to the active agent(s), and one which has little or no detrimental side effects or toxicity under the conditions of use.
  • the carriers include soluble carriers such as known buffers which can be physiologically acceptable (e.g., phosphate buffer) as well as solid
  • compositions such as solid-state carriers or latex beads.
  • the carriers or diluents used herein may be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.
  • Solid carriers or diluents include, but are not limited to, extracellular matrix, any scaffolds, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • any scaffolds e.g., gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • starches e.g., corn starch, pregelatinized starch
  • sugars e
  • pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, or suspensions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include, for example, water, alcoholic/aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles for subcutaneous, intravenous, intraarterial, or intramuscular injection
  • parenteral vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • fluid and nutrient replenishers such as those based on Ringer's dextrose, and the like.
  • electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • the choice of carrier will be determined, in part, by the particular magnetic nanoparticle compositions, as well as by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions of the invention.
  • the following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and interperitoneal administration are exemplary, and are in no way limiting. More than one route can be used to administer the magnetic nanoparticle compositions of the present invention, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
  • injectable formulations are in accordance with the invention.
  • the requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ⁇ SHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)).
  • pharmaceutically active compound or “therapeutically active compound” means a compound useful for the treatment or modulation of a disease or condition in a subject suffering therefrom.
  • pharmaceutically active compounds can include any drugs known in the art for treatment of disease indications.
  • a particular example of a pharmaceutically active compound is a chemotherapeutic agent.
  • chemotherapeutic agent generally includes pharmaceutically or therapeutically active compounds that work by interfering with DNA synthesis or function in cancer cells. Based on their chemical action at a cellular level, chemotherapeutic agents can be classified as cell-cycle specific agents (effective during certain phases of cell cycle) and cell-cycle nonspecific agents (effective during all phases of cell cycle). Without being limited to any particular example, examples of chemotherapeutic agents can include alkylating agents, angiogenesis inhibitors, aromatase inhibitors, antimetabolites, anthracyclines, antitumor antibiotics, monoclonal antibodies, platinums, topoisomerase inhibitors, and plant alkaloids.
  • the methods of the present invention can include wherein a chemotherapeutic agent is administered to the subject before, during or after administration of the magnetic nanoparticles using the methods of the present invention.
  • chemotherapeutic agents used in accordance with one or more embodiments of the present invention, typically, an attending physician will decide the dosage of the composition with which to treat each individual subject, taking into
  • inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.
  • the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • the term "subject” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
  • mammals of the order Rodentia such as mice and hamsters
  • mammals of the order Logomorpha such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is
  • the methods of the present invention can be used in cell transplantation that might require a rapid destruction of the implanted cells.
  • a method would include inoculation of SPIO nanoparticle labeled pluripotent stem cells (originated from embryonic or adult tissues) to treat brain, heart, or spinal cord damage or degeneration, if an acute rejection reaction or uncontrollable growth of the inoculated cells is detected.
  • the magnetic force generated by magnetic field gradients on such structures is significantly amplified compared to that of a single nanoparticle due to the larger linear dimensions and significantly reduced magnetic demagnetizing factor, which, for an ellipsoid of rotation with the ratio of short to long axis of -1/4, is L/4 « 0.079 along the long axis (compared to 0.33 for a sphere).
  • Optical microscopy of the labeled cells presented in Fig. 8B demonstrates that the SPIO structures can be easily resolved by visible light of fluorescent confocal microscopy.
  • Heating effects Heating of conductive magnetic particles in the variable magnetic field is due to three different mechanisms including hysteresis heating, eddy current or induction heating, and ferromagnetic resonance. Hyperthermia with magnetic
  • nanoparticles is typically achieved because of the hysteresis heating and the cyclic increase in the internal energy can be defined as:
  • Eddy current heating can be estimated using equation: where P (W/kg) is power per unit mass, B p is the amplitude of the magnetic field, d is characteristic size of the object, is the frequency ( ⁇ /2 ⁇ ), p and D are the resistivity and the density of the material, and k is an empirical factor between 1 and 2 for different geometries of the object.
  • Specific cell surface labeling was achieved by treating Her-2/neu receptor positive BT-474 human breast cancer cells with biotinylated trastuzumab humanized monoclonal antibodies (Herceptin®, Genentech) followed by streptavidin-conjugated SPIO nanoparticles (MiltenyiBiotec). The labeling and following incubation was performed both at 4 °C to prevent internalization. Labeling at 37 °C resulted in rapid internalization of SPIO into the target cell.
  • An alternative protocol included complexing of BNF nanoparticles with poly-L-lysine transfection agent for 1 hour at 37 °C and overnight incubation of cultured breast cancer cells with the nanoparticles in the incubator.
  • GIFT Gradient magnetic field treatment procedure.
  • GIFT was initiated 24 hours after microinjection of SPIO suspension (10 ⁇ ⁇ of 20 mg/mL in saline) into tumors.
  • Animal body temperature was stabilized at 37 C using a cylindrical water-cooled animal holder. All mice were sacrificed and tumors were excised 48 hours after the treatment. Tumors were fixed in 10% PFA and paraffin embedded section prepared by the pathology lab.
  • GIFT Gradient magnetic field treatment procedure.
  • GIFT was initiated 24 hours after microinjection of SPIO suspension (10 ⁇ ⁇ of 20 mg/mL in saline) into tumors.
  • Animal body temperature was stabilized at 37 C using a cylindrical water-cooled animal holder. All mice were sacrificed and tumors were excised 48 hours after the treatment. Tumors were fixed in 10% PFA and paraffin embedded section prepared by the pathology lab.
  • Bioluminescence imaging of MDA-MB-231//wc xenografts was performed with a Xenogen IVIS 200 system approximately 10 minutes after i.p. administration of D-luciferin solution (200 ⁇ ⁇ of 15 mg/mL) before and after the treatment.
  • BLI only registers signals from viable tissues expressing the Firefly luciferase enzyme and therefore is a sensitive marker of tumor viability.
  • variable gradient fields using the methods of the present invention induce irreversible damage to magnetic nanoparticle labeled cells, and that the efficient killing of SPIO labeled cancer cells and significant reduction in the viable tumor volume following GIFT therapy observed in our studies is likely due to the mechanical destruction of cell membranes and/or organelles, and not related to radiofrequency induced hyperthermia.

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Abstract

Selon un ou plusieurs modes de réalisation, la présente invention concerne des compositions pharmaceutiques et des méthodes de thérapie cytotoxique spécifique, comprenant, mais sans y être limitées, l'extermination non thermique spécifique de cellules ciblées, par exemple, de cellules cancéreuses ou de cellules souches, par marquage, liaison ou introduction dans le cytoplasme des cellules d'une ou de plusieurs nanoparticules magnétiques, telles que, par exemple, des nanoparticules d'oxyde de fer superparamagnétique (SPIO), puis par exposition des cellules à des gradients d'oscillation d'un champ magnétique en la présence d'un puissant champ magnétique statique tel qu'un champ B0 généré par des systèmes d'IRM modernes. Selon l'invention, on peut obtenir un marquage spécifique de cellules soit par divers moyens connus dans la technique, comprenant la modification de la surface cellulaire par des nanoparticules magnétiques ciblées (les nanoparticules magnétiques étant conjuguées à un ou plusieurs anticorps, peptides, ou autres ligands à affinité élevée), soit par charge des cellules avec des nanoparticules internalisées par endocytose, pinocytose, ou par l'utilisation de réactifs et de techniques de transfection cellulaire standard. Le champ magnétique statique de même que les gradients d'oscillation peuvent être produits par divers appareils connus utilisés pour générer ces champs, comprenant des systèmes d'IRM standard. Les méthodes de la présente invention permettent d'appliquer la technologie à une utilisation clinique de même qu'à une utilisation in vitro.
PCT/US2013/078196 2013-01-02 2013-12-30 Utilisation de gradients d'oscillation de champ magnétique intense pour une destruction spécifique de cellules marquées au moyen de nanoparticules magnétiques WO2014107419A1 (fr)

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US20120259205A1 (en) * 2002-02-14 2012-10-11 Peyman Gholam A Method and composition for hyperthermally treating cells
US20080268061A1 (en) * 2005-04-12 2008-10-30 Andreas Jordan Nanoparticle/Active Ingredient Conjugates
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