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WO2011151631A1 - Nanoparticules et leurs utilisations en imagerie moléculaire - Google Patents

Nanoparticules et leurs utilisations en imagerie moléculaire Download PDF

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Publication number
WO2011151631A1
WO2011151631A1 PCT/GB2011/000847 GB2011000847W WO2011151631A1 WO 2011151631 A1 WO2011151631 A1 WO 2011151631A1 GB 2011000847 W GB2011000847 W GB 2011000847W WO 2011151631 A1 WO2011151631 A1 WO 2011151631A1
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Prior art keywords
nanoparticles
binding
imaging
nanoparticle composition
radionuclide
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Inventor
Philip John Blower
Mark Green
Maite Jaureghi-Osoro
Rafael Torres Martin De Rosales
Peter Williamson
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Kings College London
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Kings College London
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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
    • A61K49/001—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
    • A61K49/0052—Small organic molecules
    • 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
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • 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
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric 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
    • A61K49/06—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0489—Phosphates or phosphonates, e.g. bone-seeking phosphonates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
    • A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates nanoparticles and their uses in molecular imaging, and in particular to nanoparticle compositions formed from an inorganic ionic material capable of binding to an imaging probe and optionally linked to a primary targeting group for binding to a target in a biological system, for example an in vivo location in a subject undergoing molecular imaging,
  • Molecular imaging may be defined as the three-dimensional mapping of molecular processes, such as gene expression, blood flow, physiological changes (pH, [0 2 ] etc.), immune responses, and cell trafficking, in vivo. It can be used to detect and diagnose disease, select optimal treatments, and to monitor the effects of treatments to obtain an early readout of efficacy.
  • a number of distinct technologies can in principle be used for molecular imaging, including positron emission tomography (PET), single photon emission tomography (SPET) , optical (01 ) and magnetic resonance imaging (MRI ⁇ .
  • PET positron emission tomography
  • SPET single photon emission tomography
  • MRI ⁇ magnetic resonance imaging
  • PET/CT and SPET/CT are now routine clinical
  • Radionuclide imaging with PET and SPET has the advantage of extreme! y high sensitivity and small amounts of administered contrast agents (e.g. picomolar in vivo) , which do not perturb the in vivo molecular processes .
  • the targeting principles for radionuclide imaging can be applied also in targeted delivery of radionuclide therapy .
  • the resolution of radionuclide imaging is low ( ⁇ 1 cm) .
  • MRI can deliver better anatomical resolution, but is less suitable for molecular imaging because much larger amounts of contrast agents (mi Hi to micromolar in vivo) are required, which perturb molecular processes and may be toxic .
  • Imaging with optical targeted contrast agents offers both high sensi tivity and high resolution, but is limited to imaging processes close to the body surface.
  • Radionuclides useful for SPET are largely metals (such as Tc-99m) , do not include elements that are native to most biological processes, and cannot be incorporated into small metabolic intermediates. They are more suitable for labelling larger molecules, such as peptides and proteins.
  • Typical PET isotopes e.g. F-18, Oil
  • FDG F-18-2-fluoro-2-deoxyglucose
  • PET tracers such as FDG requi e complex, multi-step syntheses and purification procedures that are not well-suited to the very short half-lives of these isotopes.
  • FDG requi e complex multi-step syntheses and purification procedures that are not well-suited to the very short half-lives of these isotopes.
  • Each new tracer for clinical use requires its own hot cell and automated synthesis unit, increasing costs and placing severe limitations on broadening the
  • F-18 farnesoid ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • MRi contrast agents are primarily either derivatised
  • biotinylated antibodies can be administered and allowed to clear from non-target tissues, followed by avidin, which binds to the pretargeted antibodies, and is also allowed co clear, and finally radiolabelled biotin is administered and may bind selectively to tissues containing the antibody-bio in-avidin con ugates by virtue of the multiple biotin-binding sites on the avidin molecule .
  • the high specificity and strong affinity o avidin for biotin enhance both the tumour- o-non-tumour ratio and tumour uptake of radioactivity (Ref 1, 3-6).
  • Avidin-biotin pre- targeting radioimmunotherapy has been used in various tumours and, although not yet clinically routine, has achieved promising clinical results. Since avidin has four biotin-binding sites there is a degree of target amplification associated with this approach of up to four- fold. However, there is also the problem with this approach that avidin is known to be highly immunogenic.
  • Target amplification is one of the aims of a large body of work aimed at use of nanoparticles for targeted delivery of contrast agents, especially for MRI .
  • a single nanoparticle e.g. surface- modified superparamagnetic iron oxide, or a liposome
  • a targeting molecule can in principle carry multiple drug or contrast molecules to a single target.
  • Several nanoparticle targeted delivery systems are currently used or under development for MRU, See, for example, Ref 9-11.
  • particulate materials have played a large part in radionuclide imaging (e.g. large particles such as macroaggrega ed albumin for lung perfusion imaging, and small particles such as "nanocoll” for imaging the reticuloendothelial system)
  • nuclear medicine has not yet benefited from improvements in design and synthesis of nanopar icles (e.g. control of size uniformity, morphology, surface derivatisation, porosity etc . ) that
  • Nanoparticles incorporating positron-emit ing metals such as 6i Cu have been prepared. See, for example, Ref 12, 13. Recently, Pulkkinen et al. have reported a nanoparticle based pre-targeting therapy in which cancer cells were incubated with biotinylated transferrin, then neutravidin and finally paclitaxel-loaded biotinylated nanoparticles formed from PLA-PEG (Ref 14) .
  • WO 2008 / 008917 describes the use of hydroxyapa i e particles that are pre-loaded with bisphosphonate drugs or phosphonate labelled radionuclides, and the use of these particles for targeting the delivery of radiation or drugs to the liver.
  • the particles may addi ionally include targeting molecules, such as antibodies capable of binding tumour associated antigens or ligands for receptors on tumour cells, either attached directly to the particles or via acetate or citrate linkers,
  • WO 2005 / 07986? describes the pre-labelling of hydroxyapatite particles with alpha-emitting radionuclides that may additionally include immobilised ligands for targeting the particles to biological structures such as tumours.
  • Ashokan et al (Biomaterials, 31: 2606-2616 , 2010 ) describes multimodal contrast agents based on hydroxyapa i e nanocrystals that are pre-labelled with Gd 3* and/or Eu 3* ions and include a folic acid group linked to the nanocrystals via a polyethylenimine linker for targeting cells expressing folate receptor.
  • the present invention relates to nanoparticle compositions and their uses for molecular imaging in which the nanopa ticles are formed from an inorganic ionic material
  • the nanoparticles a e optionally linked to a primary targe ing group which is capable of specifically binding to a target of interest in a biological system, for example a subject or patient being imaged, so that in use the nanoparticle binds to the target of interest via the primary targeting group and the imaging probe binds to the nanoparticle.
  • a primary targe ing group which is capable of specifically binding to a target of interest in a biological system, for example a subject or patient being imaged, so that in use the nanoparticle binds to the target of interest via the primary targeting group and the imaging probe binds to the nanoparticle.
  • nar.opart.icle-based imaging reagent that can then be introduced into the biological system for molecular imaging.
  • the present invention is generally applicable for molecular imaging of disease and/or for the delivery of targeted therapy, for example radionuclide based therapy in the treatment of cancer, and the target of interest in a biological system is generally a target in a subject undergoing diagnosis or treatment for a disease. While preferred embodiments relate to the use of imaging probes that are radionuclide imaging probes, the present invention is also applicable to paramagnetic or optical imaging probes.
  • the present invention differs from the prior art examples of pre-targeting approaches in that it employs inorganic ionic materials that are intrinsically capable of binding to the imaging probe, as compared to the prior art use of biological materials that have specific binding interactions, such as biotin and avidin or antibodies and antigens, for associating the retargeted reagent with imaging probe.
  • the use of nanoparticles formed from inorganic ionic materials also differs from the prior art use of nanoparticles formed from clusters of metal atoms, polymeric materials or more covalent materials such as silica and quantum dots, and may have the advantage that (a) the
  • nanoparticles have a comparatively high capacity for binding the imaging probes and/or (b) radiolabelling the nanoparticles prior to use can be done under GMP conditions in a simple, one-step process, unlike most PET radiotracer syntheses.
  • nanoparticles formed from the materials disclosed herein, such as hydroxyapacite or aluminium hydroxide are more biocornpatible than silica particles or quantum dots.
  • the present invention provides a nanoparticle composition for molecular imaging, the composition comprising nanoparticles formed from an inorganic ionic material intrinsically capable of binding to an imaging probe, the imaging probe comprising a radionuclide, an optical label or a
  • the nanoparticle is linked to a primary targeting group which is capable of specifically binding to a target of interest in a biological system, wherein in use the nanoparticle binds to the target of interest via the primary targeting group or the inorganic ionic material and the imaging probe binds to nanoparticle, so that the target of interest can be imaged by detecting the radionuclide, the optical label or the paramagnetic label.
  • the present invention addresses a problem that exist in the art in producing reagents for use in molecular imaging that comprise a fluorine radionuclide. Accordingly, the present invention provides a nanoparticle composition for use in a method of molecular imaging, the composition comprising
  • nanoparticles formed from an inorganic ionic material that is capable of directly binding to a fluorine radionuclide, wherein the nanoparticle is linked to a primary targeting group which is capable of specifically binding to a target of interest in a biological system, wherein in use the nanoparticle binds to the target of interest via the primary targeting group and the fluorine radionuclide acts as an imaging probe for imaging the target of interest .
  • These nanoparticle compositions have the advantage of a comparatively high capacity for binding the fluorine radionuclide imaging probes.
  • the inorganic ionic material is preferably hydroxyapatite , aluminium oxide, an aluminium oxide-hydroxide phase, or one of these materials adsorbed on the surface of nanoparticles formed from another material ,
  • the present invention provides a method of producing a nanoparticle composition for use in a method of molecular imaging, the method comprising:
  • radiolabelling reaction is close to quantitative, the present invention allows the labelled nanoparticles to be easily separated by centrifugation .
  • the methods of the present invention may comprise the step of:
  • the present invention provides a nanoparticle composition for use in a method of molecular imaging, the
  • the present invention provides the use of nanoparticles in the preparation of a nanoparticle composition for use in molecular imaging, wherein the composition comprises nanoparticles formed from an inorganic ionic material
  • the imaging probe comprising a radionuclide, an optical label or a
  • the nanoparticle is linked to a primary targeting group which is capable of specifically binding to a target of interest in a biological system, wherein in use the nanoparticle binds to the target of interest via the primary targeting group and the imaging probe binds to the nanoparticle, so that the target of interest can be imaged by detecting the radionuclide, the optical label or the paramagnetic
  • the present invention relates to the use of conjugates comprising bisphosphona e groups for linking functional groups to nanoparticles formed from inorganic ionic materials that are capable of binding to bisphosphonate groups, wherein the conjugate may further comprise one or more of a primary targeting group, an imaging probe comprising a radionuclide, an optical label or a paramagnetic label,
  • the present invention provides a bi functional linker comprising a bisphosphonate group covalently linked to a protein-reactive functional group or an amino acid, wherein the bisphosphonate group is capable of binding to a nanoparticle formed from an inorganic ionic material and (a) the protein- reactive functional group is for coupling to biomolecules , such as proteins, peptides or antibodies or (b) the amino acid is a precursor for the synthesis of a peptide.
  • the protein-reactive functional group may be a maleimide group (e.g. for linkage to thiol groups) , an ester, or an amino acid.
  • linkers may be used to produce the nanoparticle compositions of the present invention.
  • bisphosphonate linkers of the present invention include the linkers disclosed in the examples below, such as the raalei ide-bisphosphonate or
  • the present invention provides a conjugate comprising a bisphosphonate group capable of binding to a
  • conjugates further comprises an imaging probe, a primary targeting group or a further functional group, as described herein, example of these conjugates include the fluorescein-bisphosphonate conjugate disclosed in Example 6, the rhenium-quinolina e
  • the present invention provides a nanoparticle formed from an inorganic ionic material such as hydroxyapatite or a related material described herein, wherein the surface of the nanoparticle is deri at i sec with an aminobisphosphonate linker, e.g. for linkage to an imaging probe, a targeting group or a biomolecule, as disclosed in Example 6 below.
  • an aminobisphosphonate linker e.g. for linkage to an imaging probe, a targeting group or a biomolecule, as disclosed in Example 6 below.
  • the present invention provides a kit for use in a method of molecular imaging comprising
  • nanoparticle composition comprising nanoparticles formed from an inorganic ionic material intrinsically capable of binding to an imaging probe, the imaging probe comprising a radionuclide, an optical label or a paramagnetic label, wherein optionally the nanoparticle is linked to a primary targeting group which is capable of specifically binding to a target of interest in a biological sys em;
  • an imaging probe comprising a radionuclide, an optical label or a paramagnetic label, wherein the imaging probe is intrinsically capable of binding to the nanoparticles,-
  • the present invention provides a method of molecular imaging of a target of interest in a biological system, the method comprising:
  • nanoparticle composition comprising nanoparticles formed from an inorganic ionic material intrinsically capable of binding to an imaging probe, the imaging probe comprising a radionuclide, an optical label or a
  • paramagnetic label wherein optionally the nanoparticle is linked to a primary targeting group which is capable of specifically binding to a target of interest in a biological system;
  • an imaging probe comprising a radionuclide, an optical label or a paramagnetic label, wherein the imaging probe is intrinsically capable of binding to the nanoparticles specifically bound to the target of interest;
  • the present invention provides a pre- targeting system for use in a method of molecular imaging a target of interest in a biological system, the system comprising:
  • a nanoparticle composition for molecular imaging comprising nanoparticles formed from an inorganic ionic material intrinsically capable of binding to an imaging probe, the imaging probe comprising a radionuclide, an optical label or a paramagnetic label, wherein optionally the nanoparticle is linked to a primary targeting group which is capable of speci fically binding to a target of interest in a biological system;
  • an imaging probe comprising a radionuclide, an optical label or a paramagnetic label, wherein the imaging probe is intrinsically capable of binding to the nanoparticles , ⁇
  • the present invention provides a method of producing a nanoparticle composition for molecular imaging, the method comprising:
  • ⁇ b) optionally contacting the nanoparticle with a primary targeting group which is capable of specifically binding to a target of interest in a biological system so that the primary targeting group becomes linked to the nanoparticles;
  • the present invention provides a nanoparticle composition as obtainable by the method set out above, i.e. a nanoparticle composition in which the imaging probes or other labels are pre-assembled so that they are linked to the
  • nanoparticles before being introduced into the biological system.
  • the nanoparticle generally has an intrinsic and high binding capacity for the radionuclide probe, i.e. the binding of the probes to the nanoparticles is a consequence of the material (s) from which the nanoparticles are formed, rather than a result of a secondary interaction of a specific binding pair on the pre- argeted reagent and the probe.
  • the present invention is capable of achieving greater levels of amplification than are possible in the prior art through the use of pre-targeting antibodies
  • radionuclide probe is capable of acting as a radiotracer for ca rying ou PET, SPET or radionuclide therapy, but in addition can also act as a magnetic resonance contrast agent which requires much higher probe concentration (typically 1 million fold) at the target than radionuclide imaging.
  • the present invention also means that the incorporation of the imaging probe into the nanoparticles in embodiments where this is done before
  • administration can achieved in a simple one-step reaction easily performed under GMP conditions .
  • the present invention may employ radionuclide probes that are readily available or are
  • radionuclide probe and exploit shorter half-life radionuclides. This in turn means that patients administered with the reagents receive comparatively less radiation absorbed dose, while the imaging technique is able to exploit relatively slow targeting processes. Also the medical facility performing the imaging requires less complex radiochemis ry facilities because of the simpl icity of the probe synthesis compared to having to label a protein .
  • a further advantage over other (non-pretargeted) particulate targeting agents is lower background signal.
  • the main mechanism of clearance from blood and no mal tissues is by phagocytosis in the reticuloendothelial system. If these particles carry signal (paramagnetism or radionuclides) the reticuloendothelial system will dominate the image, obscuring targets in the liver, spleen and bone marrow. In the pre- argeted case, however, particles cleared by this mechanism will be invisible as they will be intracellular and inaccessible by the secondary probe.
  • the present invention allows the nanoparticles and/or radionuclide probes to be designed to include other useful diagnostic and/or therapeutic groups , for example o include a further label to enable multi-modal imaging to be carried out or a drug for treating a subject diagnosed with a particular disease or condition.
  • the present invention exploits the ideal availability and properties of the F-18 fluoride ion and of simple, readily available Tc-99m bisphosphonate complexes, especially their high affinity for a number of polar
  • nanoparticulate materials such as hydroxyapa 1 e , alumina, aluminium hydroxides, such as aluminium hydroxide itself, as well as aluminium oxide-hydroxide phases, and aluminium hydroxide adsorbed on other nanoparticles ⁇ e.g. hydroxyapatite, silica), zeolites, calcium phosphates, calcium oxalates, calcium carbonates and other calcium minerals, insoluble lanthani.de salts, insoluble salts and oxides and oxide- ydroxides of hard transition metals such as zirconium, titanium, iron, manganese, chromium and zinc.
  • hard transition metals such as zirconium, titanium, iron, manganese, chromium and zinc.
  • These materials can be synthesised in nanoparticulate form and conjugated with biornolecules for pre- argeting, such as RGD peptides, while leaving part of the particle surface or interior pore surface available for radiotracer binding in vivo.
  • biornolecules for pre- argeting such as RGD peptides
  • FIG. 1 A schematic representation of pre- targeting strategy .
  • pre-targeting agent is adminis ered.
  • pre- argeting nanoparticle agent accumulates slowly at the target cell .
  • adionuclide imaging probe seeks out and targets its high affinity binding sites.
  • FIG. 2b In vitro binding study of ("Cu(dtcbp) 2] in 50mm TRIS pH 7 at room tempera u e to various inorganic materials ( 1 mg ml/ 1 ) after 1 h incubation.
  • Figure 3 In vitro calcium salt binding study in 50 mM TRIS pH 6.
  • Figure 4 Gel electrophoresis of samples obtained from incubation of C2Ac-bisphosphonate conjugate Y and controls with nanoparticles
  • nanoparticles control, top
  • nanoparticles E(centre) and with nanoparticles P (bottom) .
  • Endosomes containing high-density particles can be seen in the nanoparticle-treated macrophages.
  • the present invention employs nanoparticle compositions for binding imaging probes, both as pre-targeting reagents in which the nanoparticle composition is introduced into the biological system for binding to the target of interest where the imaging probe binds to the pre-targeted nanoparticles in the biological system, or to pre-assemble an imaging reagent using the
  • the present invention uses nanoparticles that are formed from an inorganic ionic material intrinsically capable of binding to the imaging probe, typically via an interaction that comprises ionic bonding.
  • suitable inorganic materials include calcium salts, e.g. hydroxyapatites , calcium phosphates, calcium oxalates, calcium carbonates and other calcium minerals, insoluble insoluble salts and oxides of hard metals, such as alumina, zeolites, lanthanide salts, zirconium salts, titanium salts, iron salts , manganese salts, chromium and zi c salts.
  • Suitable inorganic materials also include aluminium hydroxides, including aluminium hydroxide itself, as well as aluminium oxide- hydroxide phases, and aluminium hydroxide adsorbed on other nanoparticles (e.g. hydroxyapatite, silica) .
  • nanoparticles according to the present invention such as aluminium hydroxide nanoparticles, have a high affinity for radionuclides such as F-18.
  • the experiments disclosed herein also demonstrate that nanoparticles formed from inorganic ionic materials are capable of directly binding to fluorine radionuclides, in contrast to previous chemistry reliant on bisphosphonate-based radionuclide probes or nanoparticles formed from radiolabelled cross-linked polymers.
  • a fluorine radionuclide imaging reagent in which (a) the nanoparticles have a comparatively high capacity for binding the fluorine imaging probes and/or (b) the process of radiolabelling the nanoparticles prior to use can be done under CMP conditions in a simple, one-step process, unlike most PET radiotracer syntheses .
  • hydroxyapatite is used as it has the advantage of being highly biocompatible as it is the mineral from which bone is constructed and is ultimately soluble to produce biocompatible soluble calcium and phosphate salts.
  • the nanoparticles may have any suitable geometry and may be solid, or, more preferably, porous or hollow as set out in detail below.
  • the material used to form the nanoparticle for interacting with the imaging probe may be coated or adsorbed on other nanoparticles (e.g. hydroxyapatite or silica).
  • the binding properties of the inorganic ionic materials may be used to link other functionalities to the nanoparticles, before or after introduction into the biological system, including the primary targeting group and/or further labels or probes, as explained further below.
  • the material forming the surface or internal pores of the nanoparticle is capable of binding to the imaging probe, either as part of a pre- targeting approach in which the nanoparticles initially bind to the target of interest in the biological system or in a pro- assembly step in which the imaging probe is linked to the
  • a component of the imaging probe for example a radionuclide, has an intrinsic ability to bind to the inorganic ionic material forming the surface of the nanoparticles.
  • probes comprising 19 F as a radionuclide are capable of binding to hydroxyapa ite, aluminium hydroxide and alumina .
  • the imaging probe may be a conjugate that comprises a
  • radionuclide an optical label or a paramagnetic label and a functional group or moiety that is capable of binding to the surface of the nanoparticles.
  • this approach include the use of bisphosphonate and bisphosphonate conjugates , or polycarboxylic acid conjugates that are capable of chelating metallic radionuclides and binding to hydroxyapati te ; catechol and hydroxypyridinone con ugates and polycarboxylic acid conjugates that are capable of binding to the surface of iron oxides, insoluble aluminium salts and insoluble lanthani.de salts ; or bisphosphonate conjugates incorporating a covalently bound halogen radionuclide such as 1-131, 1-123, At-211 or F-18.
  • the present invention is capable of providing amplification of the signal from the imaging probe, e.g. from a radionuclide, an optical label or a paramagnetic label, as a plurality of imaging probes can bind to each nanoparticle.
  • the amplification involves an average of at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 50, and more preferably at least 100 imaging probes binding sites on any given nanoparticle.
  • the level of amplification provided by the interaction of the nanoparticles and the imaging probes can be determined by those skilled in the art using the techniques described in the examples, such as the saturation experiment shown in Example 5. This means that the present invention is capable of achieving greater levels of amplification, and preferably significantly greater amplification (e.g.
  • radionuclide imaging probes at higher levels of amplification, e.g. where more than 10 4 , more than 10 5 , or 10 6 radionuclide probes or more are concentrated at the target of interest, possibly through the target binding to a plurality of nanoparticles, it becomes possible to carry out multi-modal studies using the radionuclide as a single primary radionuclide probe. This arises because the radionuclide can be used for carrying out PET, SPET or radionuclide therapy, and in addition can also act as a magnetic resonance contrast agent which requires much higher probe
  • the high binding capacity in this approach could amplify the MR contrast signal by a factor of 200 even with small ( 40 nm particles ⁇ , providing the possibility of using the
  • the nanoparticles of the present invention may be formed from a single material, e.g. from hydroxyapatite, or alternatively they may be formed from more than one material, for example by coating a nanoparticle formed form a first material with one or more further materials.
  • An example of the latter approach is the use of iron oxide nanoparticles coated with hydroxyapati e.
  • the materials from which the nanoparticle is formed may introduce further useful properties to the nanoparticle.
  • iron oxide nanoparticles, optionally coated with hydroxyapa ite can be used for dual modality imaging with both PET (or SPET) and magnetic resonance imaging, conferring the complementary advantages of PET or SPET (sensitivity,
  • nanoparticles can be made using a template such as a hydroxyapa ite particle and depositing a second material on the particle's surface, followed by dissolving away the hydroxyapa ite core using acid, thereby producing hollow nanoparticles formed from the second material capable of use for other applications, e.g. in drug delivery.
  • the nanoparticles of the present invention may have a number of forms including micro- and nanoporous structures and with various sizes , morphologies and surface chemistries .
  • Solid or hollow nanoparticles may be employed.
  • Porous ⁇ e.g. zeolites) and hollow nanoparticles have the potential advantage that whilst large molecular targeting molecules such as proteins may bind to the exterior surface to confer molecular targeting properties to the nanoparticles , the small molecules or ions serving as radionuclide probes may penetrate the interior and hence have access to greatly enlarged surface area, improving the capacity and kinetics of binding in
  • the nanoparticles have mean largest dimensions
  • nanoparticles can be determined by those skilled in the art using electron microscopy, dynamic light scattering, size exclusion chromatography, or electrophoresis. In general, small
  • nanoparticles have the advantage that they will have higher surface area-to-mass ratio, better penetration into tissues from vascular compartment and be of a suitable size to be recognised by macrophages, enabling nanoparticles that do not bind to the target of interest to be invisible when imaging is carried out. Larger nanoparticles may offer the advantage of higher signal
  • nanoparticle compositions are reasonably monodisperse, for example having a coefficient of variation of the mean dimension of less than 20% and more preferably less than 10% .
  • a wide range of primary targeting groups may be linked to
  • nanoparticles to direct the nanoparticle to different targets of interest in the biological system in question.
  • the present invention also allows each nanoparticle to be linked to a
  • the primary targeting group or a conjugate that comprises it binds to the material from which the nanoparticle is formed in a similar manner to the imaging probe.
  • conjugates that include bisphosphonate groups for achieving this is described in more detail below.
  • the nanoparticles do not need to include a primary targeting group, in which case the inorganic ionic material or the size of the nanoparticle is used to determine the distribution of the nanoparticles in the biological system, e.g. by accumulation of the nanoparticle composition in sites of increased vascular permeability, as in tumours or inflammation.
  • a primary targeting group is present.
  • the target of interest will comprise a member of a specific binding pair that is capable of specifically binding to the primary targeting group in the system, such that they will be members of a pair of molecules which have particular specificity for each other and which in normal conditions bind to each other in preference to binding to other molecules .
  • speci £ic binding pairs are well known in the art and include receptors and ligands, agonists or antagonists, enzymes and substrates,
  • primary targeting groups may be peptides, proteins or other biological molecules, such as
  • aptamers or small molecule ligands, that bind to specific in vivo molecular targets .
  • Classes of targets of interest include ligands or receptors or transporters expressed on diseased cells or tissue, cell surface antigens associated with disease states, or tumour markers, e.g. cancer specific markers or tissue specific markers .
  • One group of primary targeting groups are polypeptides capable of binding to phosphatidyl serine (PS) so that the nanoparticles can be employed in apoptosis or cell death imaging studies.
  • polypeptides include Annexin V and the C2 domain of a synaptotagmin.
  • Polypeptides that comprise one or more C2 domains are well known in the art. While some polypeptides have only one C2 domain, others have two or more C2 domains, and the domains are generally described by attaching a letter (in alphabetical order) to the end of the name (e.g. , C2A, C2B, and so on).
  • C2 domain For a protein that contains only one C2 domain, the domain is simply referred to as C2 domain, while the examples below use the C2A domain of rat synaptotagmin I, other C2 domains that are capable of binding to PS could be employed instead, for example a C2A domain of a synaptotagmin of another species. Further examples of proteins that contain a C2 domain include but are not limited to
  • Human synaptotagrains include synaptotagmin 1-7 , 12 and 13.
  • the present invention can employ an anti-CD33 antibody, or fragment thereof, for imaging cancer cells expressing CD33 such as cells of myelomonocytic lineage and leukae ic cells, see Emberson et al . , J. Immunol. Methods. 305 ( 2 ) : 135-51 , 2005.
  • a further example is the use of a tissue inhibitor of metal loproteinases (TIMPs), such as TIMP-2, for imaging matrix metalloproteinase expression, as expression of metal loproteinases has been
  • a further example of a polypeptide that can be used to make conjugates according to the present invention is complement receptor 2 (CR2 ) .
  • Antibodies capable of binding to the glycoprotein carcinoembryonic antigen (CEA) may also be used as primary targeting groups as members of this family of glycoproteins are expressed on colorectal cancer cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells, medullary thyroid cancer cells and breast cancer cells.
  • a further example may exploit the affinity or the peptide sequence arginine-glycine-aspartic acid (RGD) for the integrin
  • angiogenesis as is commonly seen in tumours, atherosclerotic plague and repairing diseased issue such as infracted myocardium, by linking an RGD peptide derivative to the nanoparticles surface by means of a bi sphosphonate or other suitable linker.
  • a further example may exploit the affinity of the peptide
  • octreotide or other related analogue of somatostatin which may bind to the somatostatin receptor expressed highly at the surface of cancer cells e.g. in carcinoid, medullary thyroid carcinoma and other neuroendocrine tumours , by linki ng a somatostatin analogue peptide to the nanoparticles surface by means of a bisphosphona e or other suitable linker .
  • the skilled person will be able to use other targeting groups such as bombesin , gastrin or VCAM targeting peptide.
  • the primary targeting group may be linked to the nanoparticle as a conjugate having a function l group for binding o the nanoparticle and a reactive group for linking to the primary targeting group.
  • a bi functional conjugate may be employed that
  • a b sphosphonate group for binding to the nanoparticle e.g. nanoparticles formed from hydroxyapati te and other insoluble calcium salts such as calcium phosphate , tricalcium phosphate, iron oxides and other inorganic materials consisting of insoluble salts of hard metals, and a reac ive group for site-specific linkage to the primary targeting group, for example a maleimide group or site-specific linkage to thiol groups in a biomolecule such as a peptide, polypeptide or antibody.
  • the maleimide group can then be site- specifically linked to a thiol group, for example of a cysteine residue incorporated site- specifically into the peptide or protei for the purpose .
  • Another example would be a bisphosphona e conjugated to an aldehyde or ketone group able to react site-specifically with a protein or peptide to which a
  • hydrazine or similarly reactive group has been site-specifically incorporated (e.g. using a hydrazinonicotinic acid derivative) to form a hydrazone or similar link.
  • the prima y targeting group may comprise a suitable polypeptide or protein, or a fragment or domain thereof .
  • polypeptides generally described by reference to “polypeptides”, this should be taken to include shorter sequences of amino acids (e.g., from 5 or 10 amino acids in length to 30, 40 or 50 amino acids in length), sometimes referred to in the art as peptides.
  • the term should also be taken to include polypeptides having secondary, tertiary or quaternary structure, generally referred to as proteins, as well as multidomain proteins .
  • Protein domains are fragments of a full length protein that have the ability to retain structure independent of the full length protein, typically forming a stable and folded three-dimensional structure. Many proteins consist of several structural protein domains and it is common for a particular domain to be found in a range of related proteins . Protein domains vary in length from between about 25 amino acids up to 500 amino acids in length, or from 50 amino acids to 250 amino acids, or from 75 amino acids to 150 amino acids .
  • polypeptide is an antibody
  • this term describes an immunoglobulin whe her natural or partly or wholly synthetically produced.
  • the term also covers any combination thereof
  • polypeptide or protein comprising an an body binding domain .
  • An ibody fragments which comprise an antigen binding domain are such as Fab, scFv, Pv, dAb, Fd; and diabodies . It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody . Such techniques may involve introducing DNA encoding the
  • immunoglobulin variable region or the complementarity determining regions (CDRs ) , of an antibody to the constant regions, or
  • immunoglobulin See, for instance, EP 0 184 187 A, GB 2,188,638 A or EP 0 239 400 A.
  • Antibodies can be modified in a number of ways and the term
  • antibody molecule should be construed as covering any specific binding member or substance having an antibody antigen-binding domain with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP 0 120 694 A and EP 0 125 023 A. It has been shown that fragments of a whole antibody can perform the f nction of binding antigens .
  • binding fragments are ( i ) the Fab fragment consisting of VL , VH , CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VI, and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al . , Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv
  • diabodies multivalent or multispecif ic fragments constructed by gene fusion (WO 94/13804; Holliger et al, P.N.A.S. USA, 90: 6444- 6448, 1993) .
  • Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al, Nature Biotech, 14: 1239-1245, 1996).
  • Minibodies comprising a scPv joined to a CH3 domain may also be made CHu et al , Cancer Res., 56: 3055-3061 , 1996).
  • the imaging probes that are capable of binding to the inorganic ionic nanoparticles comprise a radionuclide, an optical label or a paramagne ic label.
  • the present invention may also involve the use of further labelled probes that may be linked to or associated with the nanoparticles, for example to enable multi-modal imaging to be carried out, and the following discussion relating to imaging probes is applicable so the further probes, mutatis mutandis .
  • the imaging probes used in accordance with the present invention have the property of binding to the nanoparticles either in vitro or in vivo, in either the pre- targeted or pre-assembled
  • radionuclides that have an intrinsic binding affinity for the nanopar icles, and especially for one or more of the materials forming the surface of the nanoparticles.
  • F- 18- fluoride is capable of binding to materials such as
  • the radionuclide probe can comprise a radionuclide and a functional g roup that is capable of binding to the materials from which the nanoparticle is made. Examples of this include bisphosphonate-based radionuclide probes as
  • bisphosphonate groups are capable of binding nanoparticles formed from hydroxyapatite and other insoluble calcium salts such as calcium phosphate , tri calcium phosphate, iron oxides and other inorganic materials consisting of insoluble salts of hard metals .
  • PCT/GB2010/000692 which are bifunctional and have functional groups designed for efficiently chelating radionuclides.
  • a further class of bisphosphonate reagent includes the
  • a further class of bisphosphonate conjugates are aromatic
  • bisphosphonate derivatives containing a suitably derivatised benzene ring covalently linked to a bisphosphonate group in which the aromatic ring can be labelled with radionuclides such as radioiodine, At-211 or F-18 by covalent attachment of the halogen to the aromatic group by nucleophilic or electrophilic
  • radionuclides may form part of the probes of the present invention include technetium, rhenium, copper, cobalt, gallium, yttrium, lutetium, indium, zirconium, carbon, iodine, fluorine and astatine isotopes such as Tc-99m, Ga-67 , In-Ill , 1-123 (SPET) , Cu- 64, Cu-60, Cu-61, Cu-62, Tc-94m, Ga-68, Co-55, F-18, C-ll, 1-124, Zr-89 ⁇ PET) , Cu-67, Re-186, Re-188, Y-90, Lu-177, 1-131
  • radionuclide therapy radionuclide therapy
  • the present invention may emplo the radionuclides alone or in combinations .
  • technetium isotopes are employed or imaging purposes
  • rhenium isotopes for therapeutic purposes
  • copper and halogen isotopes for both imaging and therapy.
  • optical probes examples include fluorophores such as fluorophores
  • fluorescein e.g. the specific molecule fluorescein-bisphosphona e conjugate in Example 6 and luminescent molecules and complexes such as lanthanide complexes and the rheniu -qui nolinate
  • radionuclides and MR contrast agents provides the opportunity to combine modalities to enhance diagnosis and detection, for example the location of disease at the whole body level can be identified by whole body scan ing with PET or SPECT while the same nanoparticulate tracer may be detected visually during surgery to assist identification of diseased tissue.
  • combined PET and MR imaging can provide the advantage of high sensitivity (PET, SPET) , quantification of signal ⁇ PET) and anatomical resolution (MR) and measurement of the microenvironment ⁇ MR contrast
  • the nanoparticles and imaging probes of the present invention may use a range of different chemistries and techniques to enable imaging probes to bind to the surface of the nanoparticle, for linking the primary targeting groups to a nanoparticle and/or for introducing further groups and properties into the nanoparticle and probes of the present invention.
  • One particularly useful approach that can be used to enable imaging probes, and especially radionuclide probes, to bind to the nanoparticles and/or for attaching the primary targeting group to the surface of the nanoparticles and/or for attaching further conjugates to the nanoparticles is based on bisphosphonate
  • R is hydrogen or hydroxyl
  • n is an integer between 1 and 6;
  • R 2 is ⁇ (CH a ) 0 -R4, where o is 1, 2 or 3;
  • R 3 is -(C3 ⁇ 4) p -R s , where p is 1, 2 or 3 ;
  • R « and R 5 are independently selected from:
  • a sp2 hybridised heteroaryl group comprising a nitrogen, oxygen or sulphur heteroatom, typically present in the ortho position relative to the covalent bond to the E 2 or R 3 group; or b) a - R R 7 group, wherein R 6 and R 7 are independently hydrogen or an optionally substituted C 3- « alkyl group;
  • Preferred examples of these bisphosphonate compounds may be represented by the Formula la ;
  • Ri is hydrogen or hydroxy1 ;
  • R 2 is hydrogen, methyl or ethyl
  • the bisphosphonate group is a paroldronate, alendronate or neridronate .
  • the compounds disclosed in PCT/GB2010/000692 may be employed as complexes formed between the R 2 and R 3 substituents of a compound of Formula I and a chelatable radionuclide, such as isotopes of technetium, rhenium or copper ( 9, Tc , 186 Re, l88 Re» 6 Cu, 60 Cu, 6i Cu, S2 Cu or * 7 Cu) . These complexes then have free
  • Some compounds of Formula I may also be used as linkers for attaching the primary targeting group and/or a further functional group to the surface of the nanoparticles.
  • the bisphosphonate groups bind to the surface of the nanoparticle and the remaining functional groups of the compounds are available for coupling to the primary targeting group or a further group. This coupling reaction can take place before of after linking the
  • a further class of linkers that can be used for conjugating the primary targeting ligand to the nanoparticle and/or for
  • nanoparticle pre- argeting reagent are based on the bioconjugates disclosed in co-pending and co-owned GB-A-0914446.0 filed on 18 August 2009. See also Ref 17. when used in accordance with the present i nvent i on , these bioconjugates comprise a pr imary
  • linker sequence comprises (a) a free cysteine residue which is capable of site-specific linkage to a label and (b) a
  • polyhistidine sequence which is capable of site-specific labelling with a radionuclide.
  • the bioconjugates provide a way of coupling the primary targeting group to the nanopar icle, for example using a coupling reagent comprising a bisphosphonate (to bind to the nanoparticles ) and a maleimide group ( to bind to the thiol side chain of cysteine in the protein/pept ide sequence) and additionally provide a polyhistidine sequence for linkage to other labels use ul in multi-modal imaging (such as Tc-99m> or in radionuclide therapy ⁇ such as Re-188 ) .
  • a coupling reagent comprising a bisphosphonate (to bind to the nanoparticles ) and a maleimide group ( to bind to the thiol side chain of cysteine in the protein/pept ide sequence) and additionally provide a polyhistidine sequence for linkage to other labels use ul in multi-modal imaging (such as Tc-99m> or in radionuclide therapy ⁇ such as Re-188 ) .
  • linker sequences used in the biocon ugates are designed so that they comprise a free cysteine for site-specific covalent modification , e.g. with a bisphosphonate, and a polyhistidine tag for site-specific labelling with Tc-99m or Re-188 for example .
  • the skilled person can conveniently be able to determine whether site-specific labelling has occurred by
  • the function of the bioconjugate will typically be the binding interaction of the polypeptide with the target component presen in a biological system that the biocon ugate is used to image or label.
  • "not substantially interfering with the function of the bioconjugate” preferably means that the radiolabelled bioconjugate will retain at least 85%, more
  • the binding of the polypeptide or bioconjugate to the target component can be determined using techniques well known in the art for determining a binding affinity between a ligand and a receptor or a ligand and a target and include competitive ELISA, Biacore assay, cell binding assay, isothermal calorimetry or differential scanning calorimetry.
  • linkers may have two particular advantages.
  • the linkers enable the conjugate to be radiolabelled though the interaction of the polyhistidine tag with a
  • Multi-modal imaging means that a single target component in a biological system, whether present i a sample or in vivo in a living organism, can be exposed to the bioconjugate in one experiment and two different types of imaging experiments carried out based on the detection of the radionuclide and the second label. This has the advantage that the polypeptide portion of the bioconjugate localises two labels at a site of interest in the biological system, enabling different information to be determined using the two labels.
  • the linker sequences will be between 6 and 25 amino acids in length, more preferably between 9 and 16 amino acids in length, and comprise a free cysteine residue, a polyhistidine sequence and, optionally * a sequence of amino acid residues (e.g. between 5 and 10 amino acid residues) between the free cysteine and polyhistidine sequence or at either end of the linker.
  • free cysteine means that the cysteine residue does not participate in the formation of a disulphide bond with another cysteine present in the polypeptide sequence and is therefore capable of undergoing reactions to become covalently linked to the label and/or to interact with the complex comprising the
  • the linker sequence can be provided at either or both of the N- or C-termini of the polypeptide, although it is often preferable to conjugate the linker to the C-terminus of the polypeptide as this reduces the tendency for the linker to affect the tertiary structure of the polypeptide part of the
  • optical probes include optical probes, MR probes iGd ⁇ complexes with pendant bisphosphonate) , optical probes (fluorescent molecules with pendant bisphosphonate) , or other radionuclides (e.g. Ga-68 , In- 111 , 1-123, Lu -177 , Y-90 etc . chelated and derivatised with bisphosphonate.
  • radionuclides e.g. Ga-68 , In- 111 , 1-123, Lu -177 , Y-90 etc .
  • the nanoparticles of the present invention may be used for the molecular imaging of diseases, such as cancer, cardiovascular disease, immunological disease and associated conditions, such as transplant , inflammation , leucocyte tracking, stem cell tracking, allergy and infection.
  • diseases such as cancer, cardiovascular disease, immunological disease and associated conditions, such as transplant , inflammation , leucocyte tracking, stem cell tracking, allergy and infection.
  • the nanoparticles may also be employed for radionuclide therapy for treating cancer and arthritis .
  • the applications of the nanoparticles and imaging probes of the present invention include a wide range of imaging and
  • the nanoparticles are particularly useful for in vivo imaging applications such as cell death imaging, for example using nanoparticles for the detection of apoptosis.
  • This might be useful in a number of different medical or research applications, for example in the fields of oncology (e.g. in monitoring response to chemotherapy), cardiovascular medicine (e.g. in imaging damaged myocardium post myocardial infarction) or graft rejection (e.g. in imaging cardiac allograft rejection) .
  • the present invention is particularly relevant to nuclear medicine imaging techniques, such as Single Photon Emission Tomography
  • SPET radionuclide
  • Positron Emission Tomography an imaging technique that provides three-dimensional images by detecting pairs of gamma rays emitted indirectly by a positron-emitting radionuclide introduced into a sample or subject.
  • PET Positron Emission Tomography
  • SPET studies can be carried out using 9,m Tc and PET studies using 94ra Tc, The skilled person, however, will be aware of other suitable SPET and PET radionuclides that can be employed in the presen invention .
  • the present invention may be employed for positron emission tomography (PET), single photon emission tomography
  • SPET optical (01) and/or magnetic resonance imaging (MRI ) by appropriate selection of imaging probes .
  • MRI magnetic resonance imaging
  • the nanoparticles of the present invention may be used in methods of multi-modal imaging , that is where information or images are derived from two different techniques, either by the detection of the imaging probe capable of detection using two different
  • Multi-modal studies will be co-registered and may entail simultaneous imaging with two modalities or may need to take place in two steps, but generally employ the same sample so that spatial information obtained using the two techniques can be compared .
  • multi-modal imaging include PET/CT, SPET/CT, PET/MR and SPET/MR.
  • the nanoparticles are directly bound to a fluorine radionuclide, enabling them to be used as reagents for PET. Examples
  • Example 1 Binding of F-18-fluoride and Tc-bisphosphonate complexes to varioas inorganic materials
  • Nanoparticulate materials C-K and W-Hl were purchased from
  • Example 2 University of Kent. F was synthesised as described in Example 2. Samples 0 - R and II were synthesised as described in Example 4. Samples S - V were synthesised as described in Example 6. Jl was commercially available from Brenntag Biosector and is commonly used as an adjuvant in vaccines.
  • nanoparticles were prepared via the well defined method of Stober et al involving the hydrolysis and condensation of tetraethyl orthosilicate (TEDS) in an ethanol/water mixture, in the presence of ammonia giving spherical silica nanoparticulates with an average size of 2?0 nm (measured via TEM) .
  • aqueous ammonium hydroxide (25% ) (1.4 mh) and H 2 0 (1.6 mL) .
  • TEOS 0.8 mL
  • the reaction mixture was stirred for 30 mins before the white precipitate was washed and centrifuged.
  • the precipitate was washed with ethanol (10 mL x 4 @ 8000rpm) .
  • the silica particles were stored in an aqueous media (pH4-5 ) adjusted by 0.1M HCl .
  • F-18-fluoride binding to particulate materials .* Particles were suspended at the nominated concentration in water (1 mL) .
  • the suspension was placed in microcentrifuge tubes.
  • F-18-fluoride iaq .. 200 nL , 4-9 MBq) was added and samples were shaken for 20 mins at r.t. and then centrifuged for 5 min at 10,000 rpm.
  • a blank sample containing only water and F-18 was prepared similarly (STD1 and STD2 ) .
  • the supernatant was removed and the pellet and/or supernatant was counted for activity.
  • the percentage binding was calculated with respect to the input activity. All experiments were performed in trip icate.
  • hydroxyapatite, aluminium hydroxide (Jl) and Er 2 0 3 had a considerably higher binding affinity for 18 F- fluoride in these simple conditions. For example, 1 mg/mL hydroxyapatite in 3 ⁇ 40 gave a binding efficiency of >90%. In terms of radioactivity per gram of pellet/supernatant, this represents a concentration ratio of >2.9 x 10 4 .
  • hydroxyapatite and aluminium hydroxide were selected as a preferred material for further evaluation for binding affinity studies with [ 19 F] -fluoride . 9fa Tc-bisphosphonate and [ 64 Cu (dtcbp) 2 1 binding to particulate materials other than Al (OH) ]
  • Tc-MDP prepared by addition of [ 993 ⁇ 4 Fc ] -pertechnetate from a ""Tc-generator, according to the manufacturer's instructions) from a MDP kit or S Cu (dtcbp) 2 , prepared according to (Torres Martin de Rosales et al . , Synthesis of 6 Cu- Bis (dithiocarbamatebisphosphonate) and its
  • this example shows that certain ionic inorganic nanoparticulate materials, and especially hydroxyapatite,
  • aluminium hydroxide (Jl ) and alumina are intrinsically capable of binding to the radionuclide probes F-18-fluoride and Tc-99m ⁇
  • Tc-99m- chelate-bisphosphonate conjugates are suitable radionuclide probes for further evaluation as described below.
  • Bxainple 2 Synthesis of hydroxyapatite CHAP) nanoparticles without hydrothernial treatment (material F)
  • HAP is the predominan crystalline phase, confirmed in comparison wi h standard JCPDS files.
  • the material is highly crystalline, particularly indicated by the presence of a peak corresponding to (300) Millers plane.
  • the intense peaks and narrow peak width are also an indication of increased crystallinity (larger crystallite size) .
  • the TEM images reveal particulates with an irregular morphology, in transition between spherical and needle like with an average grain size of 39 nm. The morphology is in agreement with literature results.
  • the average particle sizes measured via TEM for A and F were 66nm and 39nm respectively.
  • Hydrothermal treatment has an effect on surface properties and morphology, producing particles that are less elongated and less prone to aggregation compared to the non-hydrothermal ly treated particles . Accordingly, this example demonstrates the preparation of nano- scale hydroxyapatite crystals used for experiments in the other examples, and shows that the hydroxyapatite particle morphology can be varied by control of the preparation conditions.
  • Example 4 Synthesis of hydroxyapatite nanoparticles without calcining or hydrothermal treatment, and stabilisation of HAP nanopartic1es against aggregation
  • surfactant is to control the morphology of the nanoparticles and to reduce the tendency of the particles to aggregate into larger assemblies.
  • the particles were characterized by transmission electron microscopy (TEM) .
  • TEM transmission electron microscopy
  • the general procedure entailed precipitation of calcium and phosphate ions in a basic aqueous medium wi hout any thermal treatment, neither calcining nor hydrothermal treatment.
  • NPs nanoparticles
  • V F 15 mL.
  • the ratio Ca/P was fixed to 1.68, suitable for the synthesis of the hydroxyapati e phase. See Table 2 for the conditions.
  • hydroxyapatite solution (0.1 mg/mL - 1.0 mg/mb) in a chosen medium (water, PBS) was placed in an Eppendorf tube. Each sample was tested in triplicate. In each experiment, standards were used, also in triplicate. These consisted of 1 mL of the medium of choice, without nanoparticles. The samples were placed in a shaker, and 100 ⁇ of the radiotracer was added to all the eppendorf tubes. The samples were shaken for 5-10 minutes, and then centrifuged. 100 ⁇ , of the supernatant was taken from all the samples, and measured in the gamma counter, and used to estimate the total supernatant activity by multiplying by 11. The hydroxyapatite-bound activity was calculated as the difference between the initial added activity and the supernatant activity.
  • F-18 labelling was carried out in serum at 3? °C using a series of HAP samples A, F, 0, P, Q, R at HA concentration of 0.5 mg/mL. Af er 5 minutes, the samples were centrifuged and the binding was checked . The samples were then re-suspended, and kept at 3? °C. F-18 binding was checked again 3 hours and 6 hours post-incubation . The results showed that binding was initially slow, but continued to increase up to 6 h (c . f . in the absence of serum equilibrium had been reached by 5 minutes) , eventually reaching levels comparable with labelling in water.
  • HAP particles were incubated with human se um albumin (which is present at high concentration in serum) and with fetuin ia protein that binds strongly to HAP and is present in serum; obtained from
  • the albumin concentration was 4 mg/ml (c.f.
  • K d is the concentration of HA that corresponds to half of the maximum % binding of the tracer.
  • K d is a useful measure of binding affinity for each type of particles under a given set of conditions .
  • Nanoparticles P in water gave the following parameters:
  • inhibition/delay may either be caused by a native serum component or by a high concentration of citrate (10 mM) that had been added to the commercial sample.
  • HA nanoparticles were labelled with F- 18 in water as described above. After the F-18 binding was measured, NaF 0.01M ( ⁇ was added to each sample. The samples were then shaken for 5 minutes , and the F-18 binding was measured again . The experiment was carried out using two HA samples (E and F) and two different concentrations (0.5 mg/mL and 0.1 mg/mL). The results show that fluoride initially bound to HAP is
  • exchangabili y is specific to fluoride-fluoride exchange since other ions with an affinity for HAP do not displace previously- bound fluoride, Stability of F-18-.labelled HAP nanoparticles in various media.
  • HAP nanoparticles previously labelled with [ 18 F] -fluoride could be repeatedly washed with water, centrifuged and the pellets
  • HAP nanoparticles were labelled with 16 F as described above, the binding was measured by sampling the supernatant and then alendronate was added (100 ph of 0.01 M added to 1 ml) for 10 minutes followed by measurement of bound radioactivity. No significant loss of radioactivity from the particles was observed.
  • this example demonstrates the condi ions under which fluoride ions and bisphosphon e derivatives may bind to different forms of hydroxyapa ite, and shows that the binding may occur in the presence of a range of compe ing molecules relevant to the in vivo application.
  • binding of the radionuclide probes F-18-fluoride or Tc-99m ⁇ bisphosphonate conjugates
  • HAP has previously been derivatised by treatment with aminoalkyl triethoxysilane, providing free amine groups at the surface may be coupled with molecules such as peptides .
  • the efficacy of binding of the bisphosphonate radiopharmaceuticals to HAP demonstrated in Examples 1 and 5, especially when the bisphosphonate group is free and not engaged in metal chelation (i.e. as in [ 99m Tc] -DPA-Ale and not as in [ 99m Tc] -MDP) suggests that bisphosphonate conjugates would be ideal for derivatisati on of the HAP surface with
  • This example describes the synthesis of novel bisphosphonate conjugates and their utility to derivatise the surface of HAP nanoparticles.
  • HAP (F) (20 mg> in H 2 0 ⁇ 5 mL) was added pamidronate (10 mg) in H 2 0 (5 mL) .
  • the reaction mixture was stirred for 2 h a r.t. and washed with water via centrifugation until no further
  • fluorescent particles V were subsequently used for macrophage phagocytic uptake experiments .
  • Lysine and glutamic acid derivatised nanoparticles were prepared similarly from S by reaction of the amine groups with suitably protected and activated lysine and glutamic acid respectively.
  • the complex can also be radiolabelled with Re ⁇ 188 or Tc-99ra for radionuclide imaging .
  • Nanoparticles S synthesised as above (10.1 mg) was dissolved in H 2 0
  • Methanol solutions of the rhenium complex were prepared with optical densities no higher than 0.2 at 366 nm.
  • the dimethyl POPOP standard was used to measure the quantum yield and diluted to an optical density of 0.01 measured at 366 nm. Samples were flushed with nitrogen before use.
  • a concentrated stock solution of the complex was prepared in ethanol . Emission was monitored from 480 to 700 ran with an excitation waveleng h of 366 nm in 1 nm increments with an integration time of 0.25 s and bandwidths of 6 nm. A quantum yield of 0.3% was measured.
  • Example 7 Synthesis of a bisphosphoaate-maleimide derivative and its HCl adduct X for use in coajugatiag biomolecules to the surface of HAP.
  • C2A is a phosphatidyl serine-binding domain of synaptotagmin , which has utility for in vivo imaging cell death when labelled with a radionuclide (Ref 17) .
  • a modified form of the protein containing C2Ac an engineered cysteine residue and a hexahistidine tag has been produced (Re£ 17) .
  • the cysteine residue can be used for site-specific modification to attach an imaging probe.
  • the bisphosphonate-maleimide conjugate w and its HCl adduct x can be site-specifically coupled to the cysteine thiol group of this engineered form of C2A to give bioconjugate Y .
  • the application of the nanoparcicles for molecular imaging requires derivatisation of the nanoparticles with a molecular targeting group such as a peptide or protein.
  • a molecular targeting group such as a peptide or protein.
  • Binding of Y was compared to nonspecific binding of C2A protein.
  • Conjugate Y (Example 8, 10 pL at 2 mg/mL) was diluted to 20 uL with PBS (resulting in a 1 mg/mL solution) . The solution was added to nanoparticles E (100 pg ⁇ and incubated for 30 roin at r.t.
  • Lane 1 (labelled C2A) is the standard C2Ac sample, in the absence of HAP. It has two bands corresponding to monomeric protein and dimeric protein (due to disulfide bond formation) .
  • Lane 2 (labelled C2ABP-P) shows the dissolved pellet after incubation of Y with E; the monomeric protein is present indicating that it has associated with the HAP, but dimeric protein is absent since it could not be derivatised and would not have bound to HAP.
  • Lane 3 (labelled C2A-BP-S) shows the supernatant from the latter incubation, with dimeric protein present and monomeric protein also present (this may be unmodified monomeric protein or Y that has not bound to HAP.
  • Lane 4 shows the supernatant from the latter incubation, with dimeric protein present and monomeric protein also present (this may be unmodified monomeric protein or Y that has not bound to HAP.
  • Figure 4 shows that 400 g E are able to extract essentially all of the conjugate Y from solution leaving only dimeric protein in solution. 300 ⁇ ig and 200 ig are able to extract the ma ority of Y from solution while 100 g was able to extract perhaps half of the Y.
  • the HAP showed high selectivity for binding the monomeric protein, consistent with selective binding via the bisphosphonate group, since the dimeric protein has no thiol groups and is expected to be mostly unmodified.
  • HAP nanoparticles (F) in partially aggregated form were labelled with s9a Tc-bisphosphonate ( [""Tc j - DPA-Ale) as described in Example 1.
  • the nanoparticles were washed with water (1 mh) three times, affording 102 MBq on 0.4 mg of HAP, Saline was added to the nanoparticle pellet, and the mixture was sonicated in order to resuspend the nanoparticles.
  • a BALES/c mouse was
  • HA nanoparticles in 1 mL water we e labelled with [ 18 Fj - fluoride .
  • the sample was centrifuged, the supernatant discarded and the pellet was washed with water (1 h) 3 times.
  • the washed pellet was then resuspended in saline for injection (100 ⁇ - ⁇ .
  • This suspension contains partially aggregated labelled nanoparticles.
  • the activity in the sample was 26 MBq . 50 ⁇ » of the suspension was then injected via the tail vein (i.v.) of a mouse that had been anaesthetised with isoflurane.
  • the mouse was transferred to the nano-SPET/CT scanner and scanned in the following sequence: CT (30 min) / PET (25 min) / PE (25 min) / PET (25 min) .
  • CT 30 min
  • PET 25 min
  • PE 25 min
  • PET 25 min
  • PET 25 min
  • the mouse was then rehydrated with saline and the above sequence was repeated.
  • the mouse was killed by C0 2 asphyxia.
  • PET images ( Figure 7) showed very high uptake in the lungs with some uptake in liver and spleen for the entire duration of the imaging protocol. There was no detectable uptake in sites of bone metabolism (e.g. ends of long bones). The biodistribution pattern is consistent with aggregated
  • Example 11 Uptake of hydr oxyapat i t e nanoparticles by macroptiagres This example demonstrates that macrophages take up HAP
  • nanoparticles by phagocytosis, and hence provides a mechanism for clearance of nanoparticles from circulation in vivo. This is essential to achieve high target-to-background ratio for targeted nanoparticles .
  • the murine macrophage cell line J7441DMEM was cultured in 10% FCS containing 5 mL Penicillin/St eptomycin (each 5000 ⁇ ig/mL) solution per 500 mL, L-glutamine 2 mM, sodium pyruvate 1 mM, HEPES 10 mM under 5% C0 2 S 37 °C . 500, 000 cells were placed in each well of 2 x 6-well plates in 2 mL cell medium, and incubated for 2 days. 100 ⁇ / mh of HAP samples E and F in PBS were added in triplicate for each type of nanoparticles, and 3 wells were left without nanoparticles .
  • the plates were incubated for 4 h, 5% C0 2 at 37°C, Cells were then washed x3 with PBS (2 mL) , then harvested and washed further with PBS, centrifuged ⁇ 1500 rpm, 5 mins) and resuspended in PIPES buffer 0.1 M (1 mL) . Fixative (1 mL 4% glutaraldehyde ) was added and incubated for 1 hr in a 15 mL plastic tube . The cells were then washed with 2 x PIPES buffer (0.1 M) and suspended in fresh PIPES (0.1 M, 1 mL) with 0.1% glutaraldehyde and then stained, fixed and sectioned for
  • macrophages float at the top of the Percoll layer while HAP nanoparticles form a pellet at the bottom.
  • the tubes were centrifuged at 1700 rpm for 15 min and the cell layer was removed carefully via pipette and washed x3 with PBS. These cells were then incubated with [ 1S F] - luoride, along wi h a macrophage-free sample containing only HAP and a sample of macrophages untreated with HAP. While the cell- free HAP samples showed high (>70%) binding of radioactivity, the macrophage sam les took up low levels of radioactivi y although significantly higher ( ⁇ 3.1%) than cells that had not been treated with HA
  • Macrophages free of HAP or Jl showed no measu able uptake of F-18 fluoride ( ⁇ 0.5%) whereas uptake in Jl-treated cells reached approx. 3% and uptake in HAP- treated cells reached 20%. Macrophages free of HAP or Jl showed low uptake ( ⁇ 4%) of [ 93 "Tc] -DPA-Ale whereas uptake in Jl-treated cells reached 87% and uptake in HAP-trated cells reached 80% . It is not clear from these experiments whether the tracer bound to internalised nanoparticles, or to incompletely ingested
  • this Example shows that the binding of the radiotracers to the nanoparticles can take place in the presence not only of serum ( Example 5 ) bu also of cells such as macrophages.
  • Example 12 alumiaitHn hydroxide nanoparticles (sample Jl)
  • Aluminium hydroxide suspension (0.1 mL - 0.9 mL) was added to Eppendorf tubes. Water was added to all the tubes so that the overall volume in the eppendorf tubes was 1.0 mL. [F-181 -fluoride (0.1 mL) was added to all the samples, which were then shaken at room temperature for 5 minutes. The samples were then centrifuged at 12000 rpm for 5 min . Afterwards, 0.1 mL of the supernatant from each sample was collected, and the activity was measured in a gamma counter. The standards consisted of 1 mL of water, and were subjected to the same treatment as the other samples (i.e.
  • % binding [1- (Activity in the sample) / (activity in the standard) ) *100
  • Aluminium hydroxide suspension (1 mL) was added to Eppendorf tubes . Different concentrations (1.0 M - 0.1 M) of sodium chloride (0.1 mL) was added to the samples . [F-18] -fluoride (0.1 mL) was added to all the samples, which were then shaken at room
  • Aluminium hydroxide suspension (1 mL) was added to Eppendorf tubes .
  • Differen concentrations (0.1 M - 10 "5 ) of sodium fluoride (0.1 mL) was added to the samples .
  • Aluminium hydroxide suspension (0.5 mL) was added to Eppendo tubes. Water was added to all the samples so that the overall volume in the tubes was 1.0 mL. Different concentrations (0.1M - 10 "5 M) of sodium fluoride (0,1 mL) was added to the samples . [F- 18] -fluoride (0,1 mL) was added to all the samples, which were then shaken at room temperature for 5 minutes. The samples were then centrifuged at 12000 rpm for 5 min. Afterwards, 0.1 mL of the supernatant from each sample was collected , and the activity was measured in a gamma counter .
  • % binding [1- (Activity in the sample) / (activity in the
  • Aluminium hydroxide suspension (0.1 mL) was added to Eppendorf tubes . Wa er was added to all the samples so that the overall volume in the tubes was 1.0 mL. Different concentrations (0.1M - 10 "5 M) of sodium fluoride (0.1 mL) were added to the samples . [F- 18] -fluoride (0.1 mL) was added to all the samples , which were then shaken at room temperature for 5 minutes . The samples were then centrifuged at 12000 rpm for 5 min. Afterwards, 0.1 mL of the supernatant from each sample was collected, and the activity was measured in a gamma counter .
  • % binding [1- (Activity in the sample) / (activity in the Std) ] *100.
  • Al (OH) 3 Img/mL was suspended in 50 mJM Tris-HCl buffer pH 7.4 (0.5 rtiL) and labelled with 99ra Tc ⁇ DPA-Ale, in triplicate. The particles were pelleted via centrifugation ( 5 in at 14000 rpm) and 150 juL of the supernatant was collected for labelling
  • the remaining supernatant was discarded.
  • the pellets were collected and washed in 50 mM Tris-HCl buffer pH 7.4 (1 mL) via centrifugation x4 ( 5 min at 14000 rpm) .
  • juL supernatant was removed and counted for activity.
  • the input activity was represented as triplicate pelleted standards as prepared in the initial Al (OH) 3 labelling.
  • the percentage activity associated with particles was calculated with respect to the standard input activity.
  • Al(OH) 3 labelled with ""Tc-DPA-Ale in serum was assessed.
  • Al(OH) 3 (lmg/mL) was suspended in 50 mM Tris- HCl buffer pH 7.4 (0.5 mL) and labelled with *'*Tc-DPA-Ale .
  • the particles were pelleted via centrifugation ( 5 min at 14000 rpm) and 100 ⁇ L ⁇ of the supernatant was collected for labelling

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Abstract

La présente invention concerne des nanoparticules et leurs utilisations en imagerie moléculaire qui sont formées à partir d'un matériau ionique inorganique capables de liaison à une sonde d'imagerie et éventuellement liée à un groupe de vectorisation primaire pour la liaison avec une cible dans un système biologique, par exemple, un site in vivo chez un sujet soumis à une imagerie moléculaire. Les nanoparticules peuvent être utilisées dans un mode de ciblage préalable dans lequel la nanoparticule est administrée à un patient pour atteindre la cible d'intérêt et est ultérieurement suivie par l'administration de la sonde d'imagerie. Selon d'autres aspects, l'invention concerne des nanoparticules dans lesquelles un radionucléide fluor est directement lié aux nanoparticules.
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Cited By (14)

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Publication number Priority date Publication date Assignee Title
EP2647389A1 (fr) * 2012-04-04 2013-10-09 Charité - Universitätsmedizin Berlin Dispersion de nanoparticules magnétiques, sa préparation et diagnostic et utilisation thérapeutique
WO2014141288A1 (fr) * 2013-03-12 2014-09-18 Amrita Vishwa Vidyapeetham University Art, procédé, manière, processus et systèmes d'un nano-biominéral pour imagerie de contraste multimodale et administration de médicament
WO2014163222A1 (fr) * 2013-04-05 2014-10-09 Intron Biotechnology, Inc. Agent de contraste d'imagerie à résonance magnétique à base de nanoparticules d'oxyde métallique, doté d'une cavité centrale
WO2014191569A1 (fr) * 2013-05-30 2014-12-04 Nanobiotix Composition pharmaceutique, préparation et utilisations de celle-ci
CN105999292A (zh) * 2016-05-06 2016-10-12 中南大学 一种多孔空心陶瓷微球的制备方法
WO2016209977A1 (fr) * 2015-06-22 2016-12-29 The Regents Of The University Of California Nanoparticules micellaires polymères
US20170168045A1 (en) * 2013-12-30 2017-06-15 The Curators Of The University Of Missouri Au multicomponent nanomaterials and synthesis methods
US10391058B2 (en) 2014-11-25 2019-08-27 Nanobiotix Pharmaceutical composition combining at least two distinct nanoparticles and a pharmaceutical compound, preparation and uses thereof
EP3458112A4 (fr) * 2016-05-19 2019-12-25 Amrita Vishwa Vidyapeetham Microbilles radio-opaques radio-marquées non iodées à contraste irm pour radio-embolisation
US10765632B2 (en) 2014-11-25 2020-09-08 Curadigm Sas Methods of improving delivery of compounds for therapy, prophylaxis or diagnosis
US10945965B2 (en) 2011-12-16 2021-03-16 Nanobiotix Nanoparticles comprising metallic and hafnium oxide materials, preparation and uses thereof
US11096962B2 (en) 2015-05-28 2021-08-24 Nanobiotix Nanoparticles for use as a therapeutic vaccine
US11304902B2 (en) 2014-11-25 2022-04-19 Curadigm Sas Pharmaceutical compositions, preparation and uses thereof
US11369681B2 (en) 2015-12-03 2022-06-28 Amrita Vishwa Vidyapeetham Radio-wave responsive doped nanoparticles for image-guided therapeutics

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0120694A2 (fr) 1983-03-25 1984-10-03 Celltech Limited Procédés pour la production des polypeptides ou protéines à chaînes multiples
EP0125023A1 (fr) 1983-04-08 1984-11-14 Genentech, Inc. Préparations d'immunoglobuline recombinante, méthodes pour leur préparation, séquences d'ADN, vecteurs d'expression et cellules d'hôtes recombinantes
EP0184187A2 (fr) 1984-12-04 1986-06-11 Teijin Limited Chaîne lourde d'immunoglobuline chimère souris-humaine et chimère de l'ADN codant celle-ci
EP0239400A2 (fr) 1986-03-27 1987-09-30 Medical Research Council Anticorps recombinants et leurs procédés de production
WO1994013804A1 (fr) 1992-12-04 1994-06-23 Medical Research Council Proteines de liaison multivalentes et multispecifiques, leur fabrication et leur utilisation
WO2005079867A2 (fr) 2004-02-20 2005-09-01 Algeta As Particules d'hydroxyapatite emettrices de rayons alpha
WO2008008917A2 (fr) 2006-07-12 2008-01-17 Mayo Foundation For Medical Education And Research Particules d'hydroxyapatite
WO2008125422A2 (fr) * 2007-03-22 2008-10-23 Guerbet Utilisation de nanoparticules metalliques dans le diagnostique de la maladie d'alzheimer

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0120694A2 (fr) 1983-03-25 1984-10-03 Celltech Limited Procédés pour la production des polypeptides ou protéines à chaînes multiples
EP0125023A1 (fr) 1983-04-08 1984-11-14 Genentech, Inc. Préparations d'immunoglobuline recombinante, méthodes pour leur préparation, séquences d'ADN, vecteurs d'expression et cellules d'hôtes recombinantes
EP0184187A2 (fr) 1984-12-04 1986-06-11 Teijin Limited Chaîne lourde d'immunoglobuline chimère souris-humaine et chimère de l'ADN codant celle-ci
EP0239400A2 (fr) 1986-03-27 1987-09-30 Medical Research Council Anticorps recombinants et leurs procédés de production
GB2188638A (en) 1986-03-27 1987-10-07 Gregory Paul Winter Chimeric antibodies
WO1994013804A1 (fr) 1992-12-04 1994-06-23 Medical Research Council Proteines de liaison multivalentes et multispecifiques, leur fabrication et leur utilisation
WO2005079867A2 (fr) 2004-02-20 2005-09-01 Algeta As Particules d'hydroxyapatite emettrices de rayons alpha
WO2008008917A2 (fr) 2006-07-12 2008-01-17 Mayo Foundation For Medical Education And Research Particules d'hydroxyapatite
WO2008125422A2 (fr) * 2007-03-22 2008-10-23 Guerbet Utilisation de nanoparticules metalliques dans le diagnostique de la maladie d'alzheimer

Non-Patent Citations (30)

* Cited by examiner, † Cited by third party
Title
ASCENCIO ET AL., J. PHYS. CHEM. B CONDENS. MATTER MATER. SURF. INTERFACES BIOPHYS., vol. 109, 2005, pages 8806
ASHOKAN A ET AL: "A molecular receptor targeted, hydroxyapatite nanocrystal based multi-modal contrast agent", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 31, no. 9, 1 March 2010 (2010-03-01), pages 2606 - 2616, XP026884285, ISSN: 0142-9612, [retrieved on 20100201], DOI: 10.1016/J.BIOMATERIALS.2009.11.113 *
ASHOKAN ET AL., BIOMATERIALS, vol. 31, 2010, pages 2606 - 2616
AXWORTHY ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 97, 2000, pages 1802
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
EMBERSON ET AL., J. IMMUNOL. METHODS., vol. 305, no. 2, 2005, pages 135 - 51
GIERSING ET AL., BIOCONJUG CHEM., vol. 12, no. 6, 2001, pages 964 - 71
GLAUS ET AL., BIOCONJUGATE CHEM., vol. 21, no. 4, 2010, pages 715 - 722
GORE ET AL., J. NUCL. MED., vol. 50, 2009, pages 999 - 1007
HNATOWICH ET AL., J. NUCL. MED., vol. 28, 1987, pages 1294
HOLLIGER ET AL., P.N.A.S. USA, vol. 90, 1993, pages 6444 - 6448
HU ET AL., CANCER RES., vol. 56, 1996, pages 3055 - 3061
HUSTON ET AL., PNAS USA, vol. 85, 1988, pages 5879 - 5883
JAUREGUI-OSORO MAITE ET AL: "Biocompatible inorganic nanoparticles for [18F]-fluoride binding with applications in PET imaging.", DALTON TRANSACTIONS (CAMBRIDGE, ENGLAND : 2003) 21 JUN 2011 LNKD- PUBMED:21394352, vol. 40, no. 23, 11 March 2011 (2011-03-11), pages 6226 - 6237, XP002656983, ISSN: 1477-9234 *
JUN ET AL., ANGEW. CHEM. INT. ED., vol. 47, 2008, pages 5122 - 5135
MATSON ET AL., J. AM. CHEM. SOC., vol. 130, 2008, pages 6731
MORAWSKI ET AL., MAGNETIC RESONANCE IN MEDICINE, vol. 52, 2004, pages 1255 - 1262
PAGANELLI ET AL., CANCER RES., vol. 51, 1991, pages 5960
PAGANELLI ET AL., NUCL. MED. COMMUN., vol. 12, 1991, pages 211
PULKKINEN ET AL., EUR. J. PHARM. BIOPHARM, vol. 70, 2008, pages 66 - 74
REITER ET AL., NATURE BIOTECH, vol. 14, 1996, pages 1239 - 1245
TAVARE ET AL., BIOCONJUGATE CHEM., vol. 20, 2009, pages 2071 - 2081
TORRES MARTIN DE ROSALES ET AL., CHEM. COMMUN., 2009, pages 4847 - 4849
TORRES MARTIN DE ROSALES ET AL.: "Bifunctional bisphosphonate imaging agents for the diagnosis and therapy of bone metastases", CHEM. COMMUN., 2009, pages 4847 - 4849
VITHA ET AL., LANGMUIR, vol. 24, 2008, pages 1952 - 1958
WARD, E.S. ET AL., NATURE, vol. 341, 1989, pages 544 - 546
WELCH ET AL., J. NUCL. MED., vol. 50, 2009, pages 1743 - 1746
YAO ET AL., J. NUCL. MED., vol. 26, 1995, pages 837
YOANN LALATONNE ET AL: "Bis-phosphonates-ultra small superparamagnetic iron oxide nanoparticles: a platform towards diagnosis and therapy", CHEMICAL COMMUNICATIONS, no. 22, 1 January 2008 (2008-01-01), pages 2553, XP055003463, ISSN: 1359-7345, DOI: 10.1039/b801911h *
ZHANG ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 100, 2003, pages 1891

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