WO2011045454A2 - Multifunctional nanostructures as spect/mri bimodal diagnosis agents - Google Patents

Multifunctional nanostructures as spect/mri bimodal diagnosis agents Download PDF

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WO2011045454A2
WO2011045454A2 PCT/ES2010/000424 ES2010000424W WO2011045454A2 WO 2011045454 A2 WO2011045454 A2 WO 2011045454A2 ES 2010000424 W ES2010000424 W ES 2010000424W WO 2011045454 A2 WO2011045454 A2 WO 2011045454A2
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magnetoferritin
superparamagnetic
peg
solution
pharmaceutical composition
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PCT/ES2010/000424
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Spanish (es)
French (fr)
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WO2011045454A3 (en
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José Manuel DOMÍNGUEZ VERA
Natividad GÁLVEZ RODRÍGUEZ
Belén FERNÁNDEZ LÓPEZ
Elsa Valero Romero
José Juan CALVINO GÁMEZ
Susana Trasobares Llorente
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Universidad De Granada
Universidad De Cádiz
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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
    • A61K49/0002—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
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • 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
    • A61K49/1851—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 having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
    • A61K49/1857—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 having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. PLGA
    • A61K49/186—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 having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. PLGA the organic macromolecular compound being polyethyleneglycol [PEG]
    • 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
    • A61K49/1866—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 the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
    • 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

  • Nanotechnology has made a new discipline flourish: Nanomedicine.
  • metal nanoparticles are designed and prepared to obtain bio-images through the simultaneous use of several techniques, effective distribution of drugs or techniques of promising therapies such as hyperthermia caused locally by magnetic nanoparticles. It is an area of tremendous potential subject to the development of new nanostructures for its advancement.
  • Magnetic nanoparticles have attracted attention primarily because of their potential use as contrast agents in MRI. This technique is based on the magnetic resonance of the protons of body tissues (water, membranes, lipids, proteins, etc.) and is currently the most powerful method of diagnosis.
  • the contrast in MRI can be improved with paramagnetic substances.
  • the ability of a compound to increase the relaxation rate of proton spins of surrounding water molecules is called relaxation and is defined as R1 -1 / T1 or R2-1 / T2.
  • Superparamagnetic nanoparticles are candidates to act as contrast agents in MRI. Like paramagnetic substances, they lose their magnetization when the external magnetic field is removed, but unlike them, their magnetization is significantly higher.
  • Nanomedicine One approach within Nanomedicine is the use of nanoparticles that can simultaneously combine different bioimaging techniques.
  • Each bioimaging modality has its own merits but also certain disadvantages and therefore multimodal imaging methods have a greater capacity to obtain a comprehensive and more detailed image.
  • MRI probably the most powerful imaging technique today, has a high spatial resolution but nevertheless does not show great sensitivity (10 5 M, with current technology) and requires accumulating significant concentrations of the contrast agent, which increases the risk of toxicity
  • OI has an extraordinary sensitivity (10 "12 M) but on the contrary it presents problems of spatial resolution and low tissue penetration.
  • the challenge is to combine the strengths of each imaging technique in a single contrast agent , which allows parallel images to be obtained using several techniques, although there are no multimodal contrast agents for routine clinical use today, a good number of them have been published, especially MRI-OI bimodal.
  • a key point for the in vivo biomedical application of metal nanoparticles is that they cannot be toxic and must remain in the bloodstream long enough to reach a biological target.
  • the immune system through the opsonin components, macrophages and antibodies (MPS, mononuclear phagocytic system) recognizes and removes particles from the bloodstream, with their simultaneous concentration in organs with high phagocytic activity (mainly the liver). Therefore, a sensible strategy is the use of nanoparticles that are able to evade the attack by the MPS and are not phagocytosed by macrophages, consequently increasing the half-life in plasma and allowing to reach a specific organ or tissue.
  • MPS mononuclear phagocytic system
  • An ideal bimodal agent for clinical application should have a high and specific accumulation in the appropriate cells, which would result in the possibility of diagnosing an even treatable disease increasingly early, in addition to having enough half-life in plasma sufficiently high and be able to reach a specific biological target.
  • the use of metal nanoparticles against classical metal compounds has the advantage of being able to more easily accumulate a greater amount of active metal material in a given tissue. This means that ultimately, the use of the drug is optimized and the necessary metal dose can be reduced, with the consequent decrease in the risk of toxicity.
  • Numerous physical and chemical methods have been used to prepare magnetic nanoparticles. Since the magnetic properties are very dependent on size, it is crucial that the method to be developed allows the obtaining of nanoparticles with uniform sizes.
  • a possible route to obtain metallic nanoparticles without aggregation and with controlled size is the use of a pre-organized molecular platform, with a cavity that can act as a nanoreactor for chemical and spatial control in the formation of nanoparticles.
  • a typical example of this type of molecules is the ferritin protein.
  • Apoferritin consists of a spherical protein formed by 24 subunits surrounding an aqueous cavity with a diameter of approximately 8 nm. The organization of multisubunities to form apoferritin generates the presence of channels. Eight hydrophilic channels of approximately 4A allow the entry of sufficiently small metal ions and molecules into the protein cavity.
  • an objective of the present invention is to provide a bimodal contracting agent, MRI-SPECT, based on a superparamagnetic magnetoferritin, characterized in that the magnetite core occludes 99m Tc, from now on magnetoferritin of the invention.
  • the superparamagnetic nanoparticle of the invention is constituted by a magnetite nanoparticle encapsulated in ferritin that has properties to behave as a contrast agent in MRI.
  • the magnetoferritin of the invention is biocompatible and has a biodistribution in different organs, significantly improving the properties of other contrast agents such as magnetite alone.
  • biocompatible polymer chains such as PEG (polyethylene glycol)
  • the preferred Te species for inclusion in the magnetoferritin of the invention is 99m TcO 4 " .
  • concentration of 99m Tc can be adjusted in function of the need, for example diagnosis or therapy but preferably is between 10 "9 -10 " 5 M.
  • the present inventors have managed to obtain magnetite particles of high Fe content in the ferritin cavity.
  • the Fe content can be modulated up to 3800 Fe atoms per unit of ferritin.
  • the amount of Fe from 3000 to 3800 Fe atoms per unit of ferritin is modulated, which makes it possible to improve its properties as a contrast agent in MRI.
  • the Te remains occluded within the magnetite, which is an advantage over the coordination complexes of Te since the concentration of Te per particle is much higher (up to 20 times higher ) and allows to accumulate a higher density of Te atoms. This greater accumulation results in an increase in gamma radiation and a higher resolution. Also, the accumulation optimizes the concentration of the radiolabel, which allows the use of lower doses.
  • the superparamagnetic magnetoferritin has chains of a biocompatible polymer covalently bonded to the surface of the magnetoferritin.
  • the presence of this polymer improves the properties of the magnetoferritin of the invention to be used as a contrast agent since it increases its overall stability, obtaining adequate average life times in the blood for use.
  • the preferred biocompatible polymer is polyethylene glycol (PEG), among other reasons for its industrial availability, its ease of incorporation to the surface of magnetoferritin and its high biocompatibility.
  • PEGylation Kohler, N .; Fryxell, GE; Zhang, MJ Am. Chem. Soc. 2004, 126, 7206. Paul, KG; Frigo, TB; Groman, JY; Groman, EV Bioconjugate Chem.
  • PEG is covalently linked to magnetoferritin.
  • This process can be carried out easily, by incubating a derivative of the reactive PEG with the nanoparticles.
  • the covalent union of PEG masks them of the immune system, increases hydrodynamic size (size in solution) which increases the half-life in blood and reduces their elimination by the immune system.
  • This process also increases the water solubility of said nanoparticles and generally gives it additional stability.
  • the number of PEG molecules that bind to the surface of the superparamagnetic nanoparticle can also be controlled.
  • PEG derivatives of the succinimidyl ester type allow the formation of an amide type covalent bond by reaction with the amino groups on the outer surface of the superparamagnetic nanoparticles.
  • the PEG must be derivatized with functional groups capable of joining by themselves or with the aid of a reagent to the surface of the magnetoferritin.
  • the PEG is preferably linked by amide bonds, therefore the preferred PEG derivatives, as discussed above, contain the succinimidyl ester functional group, which allows direct functionalization of the magnetoferritin, since this activated ester group It reacts with the amino groups surrounding the nanoparticles, forming the covalent bond type amide.
  • PEGs functionalized as succinimidyl ester are commercial and their covalent coupling to the superparamagnetic particle can be carried out after the introduction of 99m Tc. The number of PEG chains introduced can be controlled depending on the stoichiometry and PEG used.
  • the reaction is complete, such that the number of PEG covalently bonded to the surface of the nanoparticle coincides with the number of molecules of the PEG derivative / nanoparticles that is used in the reaction.
  • the number of chains introduced was checked through the use of an electrophoretic pattern, analyzing the different magnetoferritins derivatized with PEG and seeing their correspondence with a stepped and gradual increase in molecular weight and therefore a greater retention.
  • the PEG can be mono- or bifunctionalized with activated esters and PEG can be joined with different density and crystallinity.
  • the magnetoferritin stability of the invention is enhanced by the introduction of the biopolymer, and especially when this is PEG, but this greater increase in stability is not proportional to the number of chains introduced. Therefore, although between 1 and 72 chains can be introduced, free amino groups can be left if subsequent derivations are desired or simply to reduce manufacturing costs. Preferably on average between 3 and 10 PEG chains are incorporated per magnetoferritin and more preferably between 4 and 6.
  • PEG derivatives useful for the present invention are detailed below: i) MeO-PEG-COOH: PEG1 156 MeO-PEG (1 1) -COOH a-Methoxy-u) -propioic acid undecae (ethylene glycol) PEG -WM 588.7 g / mol, PEG1161 MeO-PEG- COOH a-Methoxy- o-carboxylic acid poly (ethylene glycol) PEG-WM 750 D, PEG1 158 MeO-PEG-COOH a-Methoxy-oü-carboxylic acid poly (ethylene glycol) PEG-WM 2,000 D, PEG1 160 MeO-PEG-COOH a-Methoxy- ⁇ - carboxylic acid poly (ethylene glycol) PEG-WM 5,000 D, PEG1 157 MeO- PEG-COOH a-Methoxy-oo-carboxylic acid poly (ethylene glycol) PEG-WM 10,000 D, PEG1 159 MeO- PEG
  • Another aspect is to provide a method for the synthesis of magnetoferritins of the present invention.
  • magnetite can be done through the process described by Douglas (Masaki Uchida, Masahiro Terashima, Charles H. Cunningham, Yoriyasu Suzuki, Deborah A. Willits, Ann F. Willis, Philip C. Yang, Philip S. Tsao, Michael V. McConnell, Mark J. Young and Trevor Douglas, Magnetic Resonance in Medicine 60: 1073-1081 (2008)), the present inventors have developed a variation, which provide some advantages.
  • the method of the invention comprises at least the steps of: a) preparing an apoferritin solution, preferably of a concentration between 0.1 and 100 mg / mL, and more preferably between 5 and 20 mg / mL, b) Prepare a solution comprising Fe (ll) and Fe (lll) in approximate stoichiometry of: 2, that is, between 1: 1, 8 a, 8: 1 c) prepare a solution of pertechnectate (Te) d) add the solution prepared in step (b) and the solution prepared in step (c) on the one prepared in step (a) are interleaved. f) Preferably, the superparamagnetic magnetoferritin doped with 99m Tc is isolated
  • step (b) is prepared by mixing a solution comprising iron sulfate (ll) ammonia hexahydrate with another comprising Fe (NO 3 ) 3 in HCI.
  • the spatial distribution can be determined, with resolution sub-nanometer, of the chemical elements present in the individual particles, using the electron energy loss technique (EELS).
  • EELS electron energy loss technique
  • This technique allows us to directly study the electronic transitions that occur in the atom when it is subjected to a beam of high-energy electrons, 200kV.
  • this technique measures the energy that the incident electron loses when it interacts with an atom.
  • the L2.3 transitions are directly studied where the 2p electrons of the atom are transferred to unoccupied states above the Fermi level. The energy required for this transition is a characteristic value for each atom and is equal to the energy lost by the incident electron.
  • the fine structure of the EELS spectrum of transition metals is characterized by the presence of two intense peaks (white lines) whose intensity and position in energy varies depending on the oxidation state of the material (Leapman et al Phys. Rev. Lett. 45, 397 (1980), Turquat et al. International Journal of Inorganic Materials 3 (2001) 1025-1032).
  • the detailed study of the fine structure of the absorption peak reflects information on the electronic state of the material and can study the possible variations in the oxidation state of iron or vanadium through the nanoparticles.
  • step (a) it is buffered with AMPSO at a pH between pH 7.5 and 9.5, preferably between 8.0 and 9.0.
  • the biocompatible polymer chains preferably PEG, are reacted with the magnetoferritin after the inclusion of the Tc magnetite.
  • Another aspect relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the magnetoferritins of the present invention and at least one pharmaceutically acceptable excipient, as well as the use of said pharmaceutical composition for the preparation of a medicament.
  • the magnetoferritin of the present invention as well as the pharmaceutical composition that includes them are useful for the preparation of a medicament for the diagnosis of different diseases, according to the use of molecules that confer specificity for a tissue or organ in question, but especially cancer , including cervical, head and neck, renal and ureter, colon, rectum and anus, endometrial, esophagus, stomach, liver, larynx, ovarian, pancreas, skin, prostate, lung, brain, testis, leukemia, melanoma, and lymphoma.
  • both the magnetoferritin of the present invention and the pharmaceutical composition comprising them as contrast agents in general and as a contrast agent in MRI or scintigraphy in particular are also useful.
  • Solution 1 A solution of apoferritin (Sigma-Aldrich Ref. A341-G, lot. 048K7004) with a concentration of 10 mg / ml in AMPSO buffer pH 8.6 (Sigma A6659) is prepared 10 ml. The solution is degassed with a strong stream of argon and under stirring for 10 min.
  • Solution 3 A solution of 0.1 M NaOH is degassed with a strong stream of argon and under stirring for 10 min.
  • Solution 4. A solution of belonging (Te) obtained from a commercial kit is degassed with a strong stream of argon and under stirring for 2 min.
  • Suspension 5. Slow addition of solution 2 to solution 1 is carried out. Additions of 0.25 ml are made every 2 min until 1 ml is completed. Before the addition of solution 2, 56 ⁇ of the 99m Tc solution (solution 4) is added. Solution 6. Suspension 5 is slowly added 1 ml of a 0.1 M sodium citrate solution to remove all metal compounds that have not been encapsulated in apoferritin.
  • the resulting solution is chromatographed (10 min) in size exclusion column (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17-0851-01), obtaining the final solution 6 containing doped mangnetite with 99m Tc encapsulated in the apoferritin cavity.

Abstract

The invention relates to multifunctional nanostructures consisting of superparamagnetic magnetoferritin which can also comprise chains of a biocompatible polymer covalently bound to the surface thereof. The invention also relates to a method for obtaining said nanostructures, as well as to the use thereof as a drug, preferably for the diagnosis of cancer or as a contrast agent.

Description

NANOESTRUCTURAS MULTIFUNCIONALES COMO AGENTES DE DIAGNOSIS BIMODAL MRI-SPECT  MULTIFUNCTIONAL Nanostructures AS MRI-SPECT BIMODAL DIAGNOSIS AGENTS
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
La integración de la Nanotecnología en la Biotecnología ha hecho florecer una nueva disciplina: la Nanomedicina. En este campo, se diseñan y preparan nanopartículas metálicas para obtener bioimágenes mediante el uso simultáneo de varias técnicas, distribución efectiva de fármacos o técnicas de terapias tan prometedoras como la hipertermia originada localmente por nanopartículas magnéticas. Es un área de tremendo potencial sujeta al desarrollo de nuevas nanoestructuras para su avance. The integration of Nanotechnology in Biotechnology has made a new discipline flourish: Nanomedicine. In this field, metal nanoparticles are designed and prepared to obtain bio-images through the simultaneous use of several techniques, effective distribution of drugs or techniques of promising therapies such as hyperthermia caused locally by magnetic nanoparticles. It is an area of tremendous potential subject to the development of new nanostructures for its advancement.
Las nanopartículas magnéticas han atraído atención principalmente por su uso potencial como agentes de contraste en MRI. Esta técnica se basa en la resonancia magnética de los protones de tejidos del cuerpo (agua, membranas, lípidos, proteínas, etc) y es actualmente el método más potente de diagnosis. El contraste en MRI puede mejorarse con sustancias paramagnéticas. La capacidad de un compuesto para incrementar la velocidad de relajación de los espines de protón de las moléculas de agua del entorno se llama relajación y se define como R1 -1/T1 o R2-1/T2. Las nanopartículas superparamagnéticas son candidatos para actuar como agentes de contraste en MRI. Al igual que las sustancias paramagnéticas, pierden su magnetización cuando se elimina el campo magnético externo, pero a diferencia de éstas, su magnetización es sensiblemente mayor. Por lo tanto, la relajación que producen es mucho más alta que las de los clásicos complejos paramagnéticos de Gd(lll). El efecto de las nanopartículas superparamagnéticas se puede describir en base a la heterogeneidad del intenso campo magnético que afecta a los protones del alrededor, induciendo un desfase del momento magnético y dando lugar a un acortamiento del tiempo de relajación T2. De este modo, las nanopartículas superparamagnéticas son unos buenos candidatos para el desarrollo de nuevos y elegantes agentes de contraste, permitiendo una detección temprana de patologías severas y de gran impacto social. Magnetic nanoparticles have attracted attention primarily because of their potential use as contrast agents in MRI. This technique is based on the magnetic resonance of the protons of body tissues (water, membranes, lipids, proteins, etc.) and is currently the most powerful method of diagnosis. The contrast in MRI can be improved with paramagnetic substances. The ability of a compound to increase the relaxation rate of proton spins of surrounding water molecules is called relaxation and is defined as R1 -1 / T1 or R2-1 / T2. Superparamagnetic nanoparticles are candidates to act as contrast agents in MRI. Like paramagnetic substances, they lose their magnetization when the external magnetic field is removed, but unlike them, their magnetization is significantly higher. Therefore, the relaxation they produce is much higher than those of the classic paramagnetic complexes of Gd (lll). The effect of superparamagnetic nanoparticles can be described based on the heterogeneity of the intense magnetic field that affects the surrounding protons, inducing a lag of the magnetic moment and resulting in a shortening of the relaxation time T2. Thus, superparamagnetic nanoparticles are good candidates for the development of new and elegant contrast agents, allowing early detection of severe pathologies and great social impact.
Un enfoque dentro de la Nanomedicina es el uso de nanopartículas que puedan combinar simultáneamente diferentes técnicas de bioimagen. Cada modalidad de bioimagen tiene sus propios méritos pero también ciertas desventajas y por lo tanto los métodos de imagen multimodales presentan mayor capacidad para obtener una imagen integral y más detallada. One approach within Nanomedicine is the use of nanoparticles that can simultaneously combine different bioimaging techniques. Each bioimaging modality has its own merits but also certain disadvantages and therefore multimodal imaging methods have a greater capacity to obtain a comprehensive and more detailed image.
MRI, probablemente la técnica de imagen más potente en la actualidad, tiene una alta resolución espacial pero sin embargo no presenta gran sensibilidad (105 M, con la tecnología actual) y requiere acumular concentraciones significativas del agente de contraste, lo que incrementa el riesgo de toxicidad. Sin embargo, OI tiene una extraordinaria sensibilidad (10"12 M) pero por el contrario presenta problemas de resolución espacial y baja penetración en tejidos. En definitiva, el reto es combinar los puntos fuertes de cada técnica de imagen en un único agente de contraste, que permita la obtención de imágenes paralelas mediante varias técnicas. Aunque al día de hoy no existen todavía agentes de contraste multimodales para uso clínico de rutina, han sido publicados un buen número de ellos, especialmente bimodales MRI-OI. MRI, probably the most powerful imaging technique today, has a high spatial resolution but nevertheless does not show great sensitivity (10 5 M, with current technology) and requires accumulating significant concentrations of the contrast agent, which increases the risk of toxicity However, OI has an extraordinary sensitivity (10 "12 M) but on the contrary it presents problems of spatial resolution and low tissue penetration. In short, the challenge is to combine the strengths of each imaging technique in a single contrast agent , which allows parallel images to be obtained using several techniques, although there are no multimodal contrast agents for routine clinical use today, a good number of them have been published, especially MRI-OI bimodal.
Un punto clave para la aplicación biomédica in vivo de las nanopartículas metálicas es que no pueden ser tóxicas y deben permanecer en el torrente sanguíneo el tiempo suficiente para alcanzar un blanco biológico. El sistema inmunológico a través de los componentes opsoninas, macrófagos y anticuerpos (MPS, sistema fagocítico mononuclear) reconoce y elimina las partículas del torrente sanguíneo, con su simultánea concentración en órganos con alta actividad fagocítica (principalmente el hígado). Por lo tanto, una estrategia sensata es el uso de nanopartículas que sean capaces de evadir el ataque por parte del MPS y no sean fagocitadas por los macrófagos, aumentando consecuentemente el tiempo de vida media en plasma y permitiendo alcanzar un órgano o tejido específico. A key point for the in vivo biomedical application of metal nanoparticles is that they cannot be toxic and must remain in the bloodstream long enough to reach a biological target. The immune system through the opsonin components, macrophages and antibodies (MPS, mononuclear phagocytic system) recognizes and removes particles from the bloodstream, with their simultaneous concentration in organs with high phagocytic activity (mainly the liver). Therefore, a sensible strategy is the use of nanoparticles that are able to evade the attack by the MPS and are not phagocytosed by macrophages, consequently increasing the half-life in plasma and allowing to reach a specific organ or tissue.
Un agente bimodal ideal para su aplicación clínica debería poseer una alta y específica acumulación en las células adecuadas, lo que tendría como consecuencia la posibilidad de diagnosticar de forma cada vez más precoz una enfermedad aún tratable, además de tener tiempos de vida media en plasma suficientemente altos y poder alcanzar un blanco biológico específico. En este sentido, el uso de nanopartículas metálicas frente a compuestos metálicos clásicos (sales o compuestos de coordinación) tiene la ventaja de poder acumular más fácilmente una mayor cantidad de material metálico activo en un tejido determinado. Esto hace que en definitiva, se optimice el uso del fármaco y que pueda ser disminuida la dosis metálica necesaria, con la consiguiente disminución en el riesgo de toxicidad. Numerosos métodos físicos y químicos han sido utilizados para preparar nanopartículas magnéticas. Puesto que las propiedades magnéticas son muy dependientes del tamaño, es crucial que el método a desarrollar permita la obtención de nanopartículas con tamaños uniformes. Una posible ruta para obtener nanopartículas metálicas sin agregación y con tamaño controlado es el uso de una plataforma molecular preorganizada, con una cavidad que pueda actuar como nanoreactor para el control químico y espacial en la formación de las nanopartículas. Un ejemplo típico de este tipo de moléculas es la proteína ferritina. La apoferritina consiste en una proteína esférica formada por 24 subunidades rodeando una cavidad acuosa con un diámetro de aproximadamente 8 nm. La organización de las multisubunidades para formar la apoferritina genera la presencia de canales. Ocho canales hidrofílicos de aproximadamente 4 Á permiten la entrada de iones metálicos y moléculas suficientemente pequeñas al interior de la cavidad de la proteína. Esto ha permitido la introducción de magnetita en el interior de la apoferritina produciendo magnetoferritina, la cual ha sido usada como método de diagnostico monomodal (MRI) (Journal of Magnetic Resonance Imaging. 4(3):497-505, 1994 May-Jun.). Por otro lado, el uso de 99mTcO4 " para diagnosis mediante gammagrafía SPECT (Single Photon Emission Computer Tomography) está ampliamente generalizado en Medicina Nuclear. El 99mTcO4 " tiene un periodo de vida media corto (6 h) y emite radiación gamma de 141 keV. Estos valores reducen su toxicidad y lo hacen un radionúclido idóneo para agente de contraste mediante SPECT. De hecho, en el mercado hay un buen número de fármacos de este radionúclido (Ceretec®, Tc-MAG®, Cardiolite®, etc), la mayoría de los cuales está en forma de complejo de Te de diferentes estados de oxidación y que son obtenidos in situ mediante reacción con diferentes ligandos (Chem. Rev. 1999, 99, 2205; Chem. Rev. 1999, 99, 2235). 1. La mayoría de los compuestos de Te, son compuestos de coordinación o sales siendo algunos nombres comercialesconocidos: cardiolite ® (para corazón), Tc-MAG ® (para riñon), etc. La US 20090035201 describe un ejemplo de un complejo 99mTcO4 '-Fe2O3, pero no trata el tema de la inclusión de este tipo de complejos en la cavidad de la magnetoferritina, y aun menos el uso del producto final como agente de contraste bimodal. An ideal bimodal agent for clinical application should have a high and specific accumulation in the appropriate cells, which would result in the possibility of diagnosing an even treatable disease increasingly early, in addition to having enough half-life in plasma sufficiently high and be able to reach a specific biological target. In this sense, the use of metal nanoparticles against classical metal compounds (salts or coordination compounds) has the advantage of being able to more easily accumulate a greater amount of active metal material in a given tissue. This means that ultimately, the use of the drug is optimized and the necessary metal dose can be reduced, with the consequent decrease in the risk of toxicity. Numerous physical and chemical methods have been used to prepare magnetic nanoparticles. Since the magnetic properties are very dependent on size, it is crucial that the method to be developed allows the obtaining of nanoparticles with uniform sizes. A possible route to obtain metallic nanoparticles without aggregation and with controlled size is the use of a pre-organized molecular platform, with a cavity that can act as a nanoreactor for chemical and spatial control in the formation of nanoparticles. A typical example of this type of molecules is the ferritin protein. Apoferritin consists of a spherical protein formed by 24 subunits surrounding an aqueous cavity with a diameter of approximately 8 nm. The organization of multisubunities to form apoferritin generates the presence of channels. Eight hydrophilic channels of approximately 4A allow the entry of sufficiently small metal ions and molecules into the protein cavity. This has allowed the introduction of magnetite into the apoferritin producing magnetoferritin, which has been used as a single-mode diagnostic method (MRI) (Journal of Magnetic Resonance Imaging. 4 (3): 497-505, 1994 May-Jun. ). On the other hand, the use of 99m TcO 4 " for diagnosis by SPECT scintigraphy (Single Photon Emission Computer Tomography) is widely used in Nuclear Medicine. The 99m TcO 4 " has a short half-life (6 h) and emits gamma radiation of 141 keV. These values reduce its toxicity and make it an ideal radionuclide for contrast agent using SPECT. In fact, in the market there are a good number of drugs of this radionuclide (Ceretec ® , Tc-MAG ® , Cardiolite ® , etc.), most of which is in the form of a complex of Te of different oxidation states and that are obtained in situ by reaction with different ligands (Chem. Rev. 1999, 99, 2205; Chem. Rev. 1999, 99, 2235). 1. Most of the compounds of Te, are coordination compounds or salts being some known trade names: cardiolite ® (for heart), Tc-MAG ® (for kidney), etc. US 20090035201 describes an example of a 99m TcO 4 ' -Fe 2 O3 complex, but does not address the issue of the inclusion of such complexes in the magnetoferritin cavity, let alone the use of the final product as a contrast agent. bimodal
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN En vista del estado de la técnica un objetivo de la presente invención es proporcionar un agente de contrate bimodal, MRI-SPECT, basado en una magnetoferritina superparamagnética, caracterizada porque el núcleo de magnetita ocluye 99mTc, a partir de ahora magnetoferritina de la invención. DETAILED DESCRIPTION OF THE INVENTION In view of the state of the art an objective of the present invention is to provide a bimodal contracting agent, MRI-SPECT, based on a superparamagnetic magnetoferritin, characterized in that the magnetite core occludes 99m Tc, from now on magnetoferritin of the invention.
El nanopartícula superparamagnética de la invención, es decir la magnetoferritina superparamagnética, está constituido por una nanopartícula de magnetita encapsulada en la ferritina que presenta propiedades para comportarse como agente de contraste en MRI. The superparamagnetic nanoparticle of the invention, that is to say superparamagnetic magnetoferritin, is constituted by a magnetite nanoparticle encapsulated in ferritin that has properties to behave as a contrast agent in MRI.
La magnetoferritina de la invención es biocompatible y presenta una biodistribución en distintos órganos, mejorando significativamente las propiedades de otros agentes de contraste como son la magnetita sola.The magnetoferritin of the invention is biocompatible and has a biodistribution in different organs, significantly improving the properties of other contrast agents such as magnetite alone.
Esta puede ser usada como agente de contraste bimodal en MRI (i), y gammagrafía-SPECT (iv) y muestra tiempos de vida medio en sangre, especialmente cuando tiene introducidas cadenas de polímero biocompatible como el (polietilenglicol) PEG, suficientemente extensos para distribuirse por el sistema circulatorio sin ser fagocitados en un tiempo menor de 3h. This can be used as a bimodal contrast agent in MRI (i), and SPECT-scintigraphy (iv) and shows half-life in blood, especially when biocompatible polymer chains such as PEG (polyethylene glycol) have been introduced, sufficiently large to distribute by the circulatory system without being phagocytosed in a time less than 3h.
Otras ventajas de la magnetoferritina de la presente invención es que la preparación de estas nanopartículas 99mTcO4 " se lleva a cabo a temperatura ambiente en un tiempo óptimo para su inyección en el cuerpo. Además no requiere reducción del Tc(VII) y su incorporación a la nanopartícula es prácticamente total, lo que permite una acumulación óptima del radionúclido. El hecho de que el pertecnectato vaya ocluido en la red del mineral de Fe, hace que pueda controlarse la cantidad de 99rnTcO4 " por partícula y por lo tanto, puedan prepararse fármacos de diferentes dosis de radionúclido, en función de las necesidades. Other advantages of the magnetoferritin of the present invention is that the preparation of these 99m TcO 4 " nanoparticles is carried out at room temperature at an optimal time for injection into the body. It also does not require reduction of Tc (VII) and its incorporation. the nanoparticle is practically total, which allows an optimal accumulation of the radionuclide. The fact that the pertecnectate is occluded in the Fe ore network makes it possible to control the amount of 99rn TcO 4 " per particle and therefore, drugs of different doses of radionuclide can be prepared, depending on the needs.
Las especies de Te preferidas para su inclusión en la magnetoferritina de la invención es 99mTcO4 ". La concentración de 99mTc se puede ajustar en función de la necesidad, por ejemplo diagnosis o terapia pero preferiblemente está comprendida entre 10"9-10"5M. The preferred Te species for inclusion in the magnetoferritin of the invention is 99m TcO 4 " . The concentration of 99m Tc can be adjusted in function of the need, for example diagnosis or therapy but preferably is between 10 "9 -10 " 5 M.
Los presentes inventores han logrado obtener partículas de magnetita de alto contenido en Fe en la cavidad de ferritina. El contenido en Fe puede ser modulado hasta 3800 átomos de Fe por unidad de ferritina. Preferiblemente se modula la cantidad de Fe de 3000 a 3800 átomos de Fe por unidad de ferritina, lo cual permite mejora sus propiedades como agente de contraste en MRI. The present inventors have managed to obtain magnetite particles of high Fe content in the ferritin cavity. The Fe content can be modulated up to 3800 Fe atoms per unit of ferritin. Preferably, the amount of Fe from 3000 to 3800 Fe atoms per unit of ferritin is modulated, which makes it possible to improve its properties as a contrast agent in MRI.
Durante la preparación de la nanopartícula de magnetita (i), el Te queda ocluido dentro de la magnetita, lo que es un ventaja frente a los complejos de coordinación de Te puesto que la concentración de Te por partícula es mucho mayor (hasta 20 veces mayor) y permite acumular una mayor densidad de átomos de Te. Esta mayor acumulación redunda en un incremento de la radiación gamma y en una mayor resolución. Asimismo, la acumulación optimiza la concentración del radiomarcador, lo que permite el uso de menores dosis. De acuerdo con una realización preferida de la presente invención la magnetoferrítína superparamagnética tiene cadenas de un polímero biocompatible covalentemente unidas a la superficie de la magnetoferritina. La presencia de este polímero mejora las propiedades de la magnetoferritina de la invención para ser usada como agente de contraste puesto que aumenta su estabilidad general obteniendo tiempos medios de vida en sangre adecuados para su uso. El polímero biocompatible preferido es polietilénglicol (PEG), entre otras razones por su disponibilidad industrial, su facilidad de incorporación a la superficie de la magnetoferritina y su alta biocompatibilidad. El proceso de unir covalentemente el polímero de PEG a otras moléculas, normalmente fármacos o proteínas terapéuticas es conocido como PEGylación (Kohler, N.; Fryxell, G. E.; Zhang, M. J. Am. Chem. Soc. 2004, 126, 7206. Paul, K. G.; Frigo, T. B.; Groman, J. Y.; Groman, E. V. Bioconjugate Chem. 2004, 15, 394.). En nuestro caso, el PEG va unido covalentemente a la magnetoferritina. Este proceso se puede llevar a cabo de forma sencilla, incubando un derivado del PEG reactivo con las nanopartículas. La unión covalente de PEG las enmascara del sistema inmune, aumenta la talla hidrodinámica (talla en solución) lo que aumenta el tiempo de vida media en sangre y reduce su eliminación por el sistema inmune. Este proceso además aumenta la solubilidad en agua de dichas nanopartículas y le confiere de forma general una estabilidad adicional. Se puede además controlar el número de moléculas de PEG que se unen a la superficie de la nanopartícula superparamagnética. Los derivados del PEG de tipo succinimidil ester permiten la formación de un enlace covalente de tipo amida por reacción con los grupos amino en la superficie externa de las nanopartículas superparamagnéticas. En resumen, para conseguir la unión de las cadenas de PEG y la magnetoferritina, el PEG debe de estar derivatizado con grupos funcionales capaces de unirse por si solos o con la ayuda de un reactivo a la superficie de la magnetoferritina. El PEG se une preferiblemente mediante enlaces amida, siendo por lo tanto los derivados de PEG preferidos, como ya se ha comentado antes, los que contienen el grupo funcional succinimidil ester, que permite una funcionalización directa de las magnetoferritina, ya que este grupo éster activado reacciona con los grupos aminos que rodean las nanopartículas, formando el enlace covalente tipo amida. Uno de los PEG preferidos es PEG1163 MeO-PEG- COO-Su a-Methoxy-oo-carboxylic acid succinimidyl ester poly(ethylene glycol) PEG-WM 2,000 Dalton. Los PEG funcionalizados como ester succinimidil son comerciales y su acoplamiento covalente a la partícula superparamagnético se puede llevar a cabo después de la introducción del 99mTc. El número de cadenas de PEG introducidas se pueden controlar dependiendo de la estequiometría y del PEG empleados. Por ejemplo, cuando se utilizan derivados de éster activados, dada la alta reactivad de dichos ésteres y de las aminas de la nanopartícula superparamagnética, la reacción es completa, de tal forma que el número de PEG enlazados covalentemente a la superficie de la nanopartícula coincide con el número de moléculas del derivado de PEG/nanopartículas que se utiliza en la reacción. Además el número de cadenas introducidas se comprobó a través del uso de un patrón electroforético, analizando las diferentes magnetoferritinas derivatizadas con PEG y viendo su correspondencia con un aumento escalonado y gradual del peso molecular y por ende de una mayor retención. El PEG puede ser mono- o bifuncionalizado con ésteres activados y se pueden unir PEG con diferente densidad y cristalinidad. La estabilidad de la magnetoferritina de la invención se ve aumentada por la introducción del biopolímero, y en especial cuando este es PEG, pero este mayor aumento de la estabilidad no es proporcional al número de cadenas introducidas. Por lo tanto, aunque se puede introducir entre 1 y 72 cadenas, se pueden dejar grupos amino libre por si se desea realizar subsiguientes derivarizaciones o simplemente para reducir los coste de fabricación. Preferiblemente se incorporan de media entre 3 y 10 cadenas de PEG por magnetoferritina y más preferiblemente entre 4 y 6. During the preparation of the magnetite nanoparticle (i), the Te remains occluded within the magnetite, which is an advantage over the coordination complexes of Te since the concentration of Te per particle is much higher (up to 20 times higher ) and allows to accumulate a higher density of Te atoms. This greater accumulation results in an increase in gamma radiation and a higher resolution. Also, the accumulation optimizes the concentration of the radiolabel, which allows the use of lower doses. In accordance with a preferred embodiment of the present invention the superparamagnetic magnetoferritin has chains of a biocompatible polymer covalently bonded to the surface of the magnetoferritin. The presence of this polymer improves the properties of the magnetoferritin of the invention to be used as a contrast agent since it increases its overall stability, obtaining adequate average life times in the blood for use. The preferred biocompatible polymer is polyethylene glycol (PEG), among other reasons for its industrial availability, its ease of incorporation to the surface of magnetoferritin and its high biocompatibility. The process of covalently bonding the PEG polymer to other molecules, usually drugs or therapeutic proteins, is known as PEGylation (Kohler, N .; Fryxell, GE; Zhang, MJ Am. Chem. Soc. 2004, 126, 7206. Paul, KG; Frigo, TB; Groman, JY; Groman, EV Bioconjugate Chem. 2004, 15, 394.). In our case, PEG is covalently linked to magnetoferritin. This process can be carried out easily, by incubating a derivative of the reactive PEG with the nanoparticles. The covalent union of PEG masks them of the immune system, increases hydrodynamic size (size in solution) which increases the half-life in blood and reduces their elimination by the immune system. This process also increases the water solubility of said nanoparticles and generally gives it additional stability. The number of PEG molecules that bind to the surface of the superparamagnetic nanoparticle can also be controlled. PEG derivatives of the succinimidyl ester type allow the formation of an amide type covalent bond by reaction with the amino groups on the outer surface of the superparamagnetic nanoparticles. In summary, to achieve the union of the PEG chains and the magnetoferritin, the PEG must be derivatized with functional groups capable of joining by themselves or with the aid of a reagent to the surface of the magnetoferritin. The PEG is preferably linked by amide bonds, therefore the preferred PEG derivatives, as discussed above, contain the succinimidyl ester functional group, which allows direct functionalization of the magnetoferritin, since this activated ester group It reacts with the amino groups surrounding the nanoparticles, forming the covalent bond type amide. One of the preferred PEGs is PEG1163 MeO-PEG-COO-Su a-Methoxy-oo-carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 Dalton. PEGs functionalized as succinimidyl ester are commercial and their covalent coupling to the superparamagnetic particle can be carried out after the introduction of 99m Tc. The number of PEG chains introduced can be controlled depending on the stoichiometry and PEG used. For example, when activated ester derivatives are used, given the high reactivity of said esters and the amines of the superparamagnetic nanoparticle, the reaction is complete, such that the number of PEG covalently bonded to the surface of the nanoparticle coincides with the number of molecules of the PEG derivative / nanoparticles that is used in the reaction. In addition, the number of chains introduced was checked through the use of an electrophoretic pattern, analyzing the different magnetoferritins derivatized with PEG and seeing their correspondence with a stepped and gradual increase in molecular weight and therefore a greater retention. The PEG can be mono- or bifunctionalized with activated esters and PEG can be joined with different density and crystallinity. The magnetoferritin stability of the invention is enhanced by the introduction of the biopolymer, and especially when this is PEG, but this greater increase in stability is not proportional to the number of chains introduced. Therefore, although between 1 and 72 chains can be introduced, free amino groups can be left if subsequent derivations are desired or simply to reduce manufacturing costs. Preferably on average between 3 and 10 PEG chains are incorporated per magnetoferritin and more preferably between 4 and 6.
A continuación se detallan algunos de los derivados de PEG útiles para la presente invención: i) MeO-PEG-COOH: PEG1 156 MeO-PEG(1 1 )-COOH a-Methoxy-u)-propioic acid undecae(ethylene glycol) PEG-WM 588,7 g/mol, PEG1161 MeO-PEG- COOH a-Methoxy- o-carboxylic acid poly(ethylene glycol) PEG-WM 750 D, PEG1 158 MeO-PEG-COOH a-Methoxy-oü-carboxylic acid poly(ethylene glycol) PEG-WM 2,000 D, PEG1 160 MeO-PEG-COOH a-Methoxy-ω- carboxylic acid poly(ethylene glycol) PEG-WM 5,000 D, PEG1 157 MeO- PEG-COOH a-Methoxy-oo-carboxylic acid poly(ethylene glycol) PEG-WM 10,000 D, PEG1 159 MeO-PEG-COOH a-Methoxy-oo-carboxylic acid poly(ethylene glycol) PEG-WM 20,000 D, PEG1 166 MeO-PEG-COO-Su a- Methoxy-ω-carboxylic acid succinimidyl ester poly(ethylene glycol) PEG- WM 750 D, PEG1163 MeO-PEG-COO-Su a-Methoxy-u)-carboxylic acid succinimidyl ester poly(ethylene glycol) PEG-WM 2,000 D, PEG1 165 MeO- PEG-COO-Su a-Methoxy-oo-carboxylic acid succinimidyl ester poly(ethylene glycol) PEG-WM 5,000 D, PEG1162 MeO-PEG-COO-Su a-Methoxy-ω- carboxylic acid succinimidyl ester poly(ethylene glycol) PEG-WM 10,000 D, PEG1164 MeO-PEG-COO-Su a-Methoxy-üú-carboxylic acid succinimidyl ester poly(ethylene glycol) PEG-WM 20,000 D, ii) HOOC-PEG-COOH:PEG1091 HOOC-PEG(12)-COOH a,u)-Bis(propionic acid) dodecae(ethylene glycol) PEG-WM 2,000 690,8 g/mol, PEG1083 HOOC-PEG-COOH α,ω-Bis-carboxy poly(ethylene glycol) PEG-WM 2,000 D, PEG1085 HOOC-PEG-COOH α,ω-Bis-carboxy poly(ethylene glycol) PEG-WM 3,000 DPEG 086 HOOC-PEG-COOH α,ω-Bis-carboxy poly(ethylene glycol) PEG-WM 6,000 D, PEG1082 HOOC-PEG-COOH α,ω- Bis-carboxy poly(ethylene glycol) PEG-WM 10,000 D PEG1084 HOOC- PEG-COOH α,ω-Bis-carboxy poly(ethylene glycol) PEG-WM 20,000 D, iii) NHS-PEG-NHS:PEG1 184 Su-OOC-PEG-COO-Su α,ω-Di-succinimidyl ester poly(ethylene glycol) PEG-WM 2,000 D, PEG1 186 Su-OOC-PEG- COO-Su α,ω-Di-succinimidyl ester poly(ethylene glycol) PEG-WM 3,000 D, PEG1 187 Su-OOC-PEG-COO-Su α,ω-Di-succinimidyl ester poly(ethylene glycol) PEG-WM 6,000 D, PEG1 183 Su-OOC-PEG-COO-Su α,ω-Di- succinimidyl ester poly(ethylene glycol) PEG-WM 10,000 D, PEG1 185 Su- OOC-PEG-COO-Su α,ω-Di-succinimidyl ester poly(ethylene glycol) PEG- WM 20,000 D, iv).H2N-PEG-COOH:PEG1096 H2N-PEG-COOH*HCI a- Amino-w-carboxy poly(ethylene glycol) hydrochloride PEG-WM 3,000 D, PEG1097 H2N-PEG-COOH*HCI a-Amino-oo-carboxy poly(ethylene glycol) hydrochioride PEG-WM 5,000 Dalton PEG1095 H2N-PEG-COOH*HCI a- Amino-io-carboxy poly(ethylene glycol) hydrochioride PEG-WM 10,000 Dalton Se elegió preferiblemente el PEG1 163 MeO-PEG-COO-Su a-Methoxy-ω- carboxylic acid succinimidyl ester poly(ethylene glycol) PEG-WM 2,000 Dalton. El derivado de PEG que contiene el grupo funcional succinimidil ester, permite una funcionalización directa de las magnetoferritinas ya que este grupo ester activado reacciona con los grupos aminos que rodean las nanopartículas, formando un enlace covalente tipo amida. Some of the PEG derivatives useful for the present invention are detailed below: i) MeO-PEG-COOH: PEG1 156 MeO-PEG (1 1) -COOH a-Methoxy-u) -propioic acid undecae (ethylene glycol) PEG -WM 588.7 g / mol, PEG1161 MeO-PEG- COOH a-Methoxy- o-carboxylic acid poly (ethylene glycol) PEG-WM 750 D, PEG1 158 MeO-PEG-COOH a-Methoxy-oü-carboxylic acid poly (ethylene glycol) PEG-WM 2,000 D, PEG1 160 MeO-PEG-COOH a-Methoxy-ω- carboxylic acid poly (ethylene glycol) PEG-WM 5,000 D, PEG1 157 MeO- PEG-COOH a-Methoxy-oo-carboxylic acid poly (ethylene glycol) PEG-WM 10,000 D, PEG1 159 MeO-PEG-COOH a-Methoxy-oo-carboxylic acid poly (ethylene glycol) PEG-WM 20,000 D, PEG1 166 MeO-PEG-COO-Su a- Methoxy-ω-carboxylic acid succinimidyl ester poly (ethylene glycol) PEG- WM 750 D, PEG1163 MeO-PEG-COO-Su a-Methoxy-u) -carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 D, PEG1 165 MeO- PEG-COO-Su a-Methoxy-oo-carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 5,000 D, PEG1162 MeO-PEG-COO-Su a-Methoxy- ω- carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 10,000 D, PEG1164 MeO-PEG-COO-Su a-Methoxy-üú-carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 20,000 D, ii) HOOC- PEG-COOH: PEG1091 HOOC-PEG (12) -COOH a, u) -Bis (propionic acid) dodecae (ethylene glycol) PEG-WM 2,000 690.8 g / mol, PEG1083 HOOC-PEG-COOH α, ω-Bis -carboxy poly (ethylene glycol) PEG-WM 2,000 D, PEG1085 HOOC-PEG-COOH α, ω-Bis-carboxy poly (ethylene glycol) PEG-WM 3,000 DPEG 08 6 HOOC-PEG-COOH α, ω-Bis-carboxy poly (ethylene glycol) PEG-WM 6,000 D, PEG1082 HOOC-PEG-COOH α, ω- Bis-carboxy poly (ethylene glycol) PEG-WM 10,000 D PEG1084 HOOC- PEG-COOH α, ω-Bis-carboxy poly (ethylene glycol) PEG-WM 20,000 D, iii) NHS-PEG-NHS: PEG1 184 Su-OOC-PEG-COO-Su α, ω-Di-succinimidyl ester poly ( ethylene glycol) PEG-WM 2,000 D, PEG1 186 Su-OOC-PEG- COO-Su α, ω-Di-succinimidyl ester poly (ethylene glycol) PEG-WM 3,000 D, PEG1 187 Su-OOC-PEG-COO-Su α, ω-Di-succinimidyl ester poly (ethylene glycol) PEG-WM 6,000 D, PEG1 183 Su-OOC-PEG-COO-Su α, ω-Di- succinimidyl ester poly (ethylene glycol) PEG-WM 10,000 D, PEG1 185 Su- OOC-PEG-COO-Su α, ω-Di-succinimidyl ester poly (ethylene glycol) PEG- WM 20,000 D, iv) .H2N-PEG-COOH: PEG1096 H2N-PEG-COOH * HCI a- Amino- w-carboxy poly (ethylene glycol) hydrochloride PEG-WM 3,000 D, PEG1097 H2N-PEG-COOH * HCI a-Amino-oo-carboxy poly (ethylene glycol) hydrochioride PEG-WM 5,000 Dalton PEG1095 H2N-PEG-COOH * HCI a- Amino-io-carboxy poly (ethylene glycol) hydrochioride PEG-WM 10,000 Dalton PEG1 163 MeO-PEG-COO-Su a-Methoxy-ω was preferably chosen - carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 Dalton. The PEG derivative containing the succinimidyl ester functional group allows direct functionalization of the magnetoferritins since this activated ester group reacts with the amino groups surrounding the nanoparticles, forming an amide-type covalent bond.
Los datos experimentales han demostrado que después de 3 h de inyección, las partículas se acumulan de forma significativa en pulmón. Por otra parte, puesto quee controlar la cantidad de Tc/partícula (en un rango 0- 20, con un 100% prácticamente de incorporación de Te) permite que se pueda alcanzar de sobra el rango óptimo de concentraciones de Te (10"9 M) y el de MRI (del orden de 10~5 M). Experimental data has shown that after 3 h of injection, the particles accumulate significantly in the lung. On the other hand, since controlling the amount of Tc / particle (in a range 0-20, with 100% practically incorporating Te) allows the optimum range of concentrations of Te (10 "9 M) to be exceeded. ) and that of MRI (of the order of 10 ~ 5 M).
Otro de los aspectos es proporcionar un método para la síntesis de las magnetoferritina de la presente invención. Aunque las introducción de la magnetita se puede hacer mediante el proceso descrito por Douglas (Masaki Uchida, Masahiro Terashima, Charles H. Cunningham, Yoriyasu Suzuki, Deborah A. Willits, Ann F. Willis, Philip C. Yang, Philip S. Tsao, Michael V. McConnell, Mark J. Young and Trevor Douglas, Magnetic Resonance in Medicine 60:1073-1081 (2008)), los presentes inventores han desarrollado una variación, que proporcionan algunas ventajas. El método de la invención comprende al menos las etapas de: a) preparar una disolución de apoferritina, preferiblemente de concentración entre 0,1 y 100 mg/mL, y más preferiblemente entre 5 y 20 mg/mL, b) Preparar una disolución que comprenda Fe(ll) y Fe(lll) en estequiometría aproximada de :2, es decir entre 1 :1 ,8 a ,8:1 c) preparar una disolución de pertecnectato ( Te) d) adicionar de forma intercalada la disolución preparada en el paso (b) y la disolución preparada en la etapa (c) sobre la preparada en el paso (a). f) Preferiblemente, se aisla la magnetoferritina superparamagnética dopada con 99mTc Another aspect is to provide a method for the synthesis of magnetoferritins of the present invention. Although the introduction of magnetite can be done through the process described by Douglas (Masaki Uchida, Masahiro Terashima, Charles H. Cunningham, Yoriyasu Suzuki, Deborah A. Willits, Ann F. Willis, Philip C. Yang, Philip S. Tsao, Michael V. McConnell, Mark J. Young and Trevor Douglas, Magnetic Resonance in Medicine 60: 1073-1081 (2008)), the present inventors have developed a variation, which provide some advantages. The method of the invention comprises at least the steps of: a) preparing an apoferritin solution, preferably of a concentration between 0.1 and 100 mg / mL, and more preferably between 5 and 20 mg / mL, b) Prepare a solution comprising Fe (ll) and Fe (lll) in approximate stoichiometry of: 2, that is, between 1: 1, 8 a, 8: 1 c) prepare a solution of pertechnectate (Te) d) add the solution prepared in step (b) and the solution prepared in step (c) on the one prepared in step (a) are interleaved. f) Preferably, the superparamagnetic magnetoferritin doped with 99m Tc is isolated
La estequiometría inicial de Fe(ll) y Fe(lll) es determinante para la preparación apropiada de magnetoferritina y por ende de sus propiedades magnéticas. A diferencia del método reportado por Douglas y colaboradores, dónde todo el Fe de partida está en su estado de oxidación Fe(ll) y se ha comprobado que con un riguroso control de la extensión de la oxidación, en nuestro método, el balance estequiométrico de partida confiere las condiciones óptimas y no requiere un control exhaustivo de la entrada de aire en el sistema. The initial stoichiometry of Fe (ll) and Fe (lll) is decisive for the proper preparation of magnetoferritin and hence its magnetic properties. Unlike the method reported by Douglas et al., Where all the initial Fe is in its oxidation state Fe (ll) and it has been proven that with a rigorous control of the extent of oxidation, in our method, the stoichiometric balance of departure confers optimal conditions and does not require exhaustive control of the air inlet in the system.
Las sales de hierro (II) y (III) útiles para la preparación de la magnetoferritina son conocidas por los expertos en la materia. En una realización preferida la etapa (b) se prepara mezclando una disolución que comprende sulfato de hierro(ll) amoníaco hexahidratado con otra que comprende Fe (NO3)3 en HCI. The iron (II) and (III) salts useful for the preparation of magnetoferritin are known to those skilled in the art. In a preferred embodiment step (b) is prepared by mixing a solution comprising iron sulfate (ll) ammonia hexahydrate with another comprising Fe (NO 3 ) 3 in HCI.
La inclusión de 99mTcO4" se lleva a cabo mediante pequeñas adiciones de 99mTc04" al mismo tiempo que se hace la red de magnetita. Se puede dializar la disolución resultante en bolsas de diálisis con tamaño de poro adecuado y separar las partículas de todo el material restante. En la disolución que no contiene las partículas se puede medir la concentración de Te y de esta forma se conoce el porcentaje de Te incorporado. En algunos casos se ha estudiado el comportamiento del MoO4 2", porque tiene una química muy similar a del 99mTc0 ", pero no es radioactivo. Se ha observado que para pequeños contenidos de Mo (0-20 átomos de Mo/magnetoferritina), la incorporación es prácticamente del 100%. The inclusion of 99m TcO 4 " is carried out by small additions of 99m Tc0 4" at the same time as the magnetite network is made. The resulting solution can be dialyzed into dialysis bags with adequate pore size and separate particles from all remaining material. In the solution that does not contain particles can measure the concentration of Te and in this way the percentage of Te incorporated is known. In some cases the behavior of MoO 4 2 " has been studied, because it has a chemistry very similar to 99m Tc0 " , but it is not radioactive. It has been observed that for small Mo contents (0-20 Mo / magnetoferritin atoms), the incorporation is practically 100%.
Con objeto de llevar una caracterización más pormenorizada que permita conocer la distribución de Te en la nanopartícula superparamagnética, se llevó a cabo un estudio mediante Microscopía Electrónica de Transmisión de muestras similares en las que en vez de usar el radiomarcador pertecnectato, se usaron otros aniones del tipo molibdato, vanadato, arseniato y fosfato. La química de estos aniones es similar a la del pertecnectato, especialmente si no hay cambios del estado de oxidación del metal. In order to carry out a more detailed characterization that allows to know the distribution of Te in the superparamagnetic nanoparticle, a study was carried out by means of Transmission Electron Microscopy of similar samples in which instead of using the radio-marker pertecnectate, other anions of the type molybdate, vanadate, arsenate and phosphate. The chemistry of these anions is similar to that of pertecnectate, especially if there are no changes in the oxidation state of the metal.
El estudio de Microscopía Electrónica de Transmisión es de especial utilidad en el caso de las nanopartículas dopadas con vanadato; esta técnica nos proporciona tanto información sobre la distribución del tamaño de las nanopartículas como sobre la composición química y distribución espacial de los estados de oxidación en nanopartículas individuales. Las imágenes de campo oscuro a alto ángulo son sensibles al número atómico del material, es decir, aquellas zonas en la que estén presentes los elementos más pesados se mostrarán en la imagen como puntos de mayor intensidad. En nuestro caso de estudio, las nanopartículas, al contener elementos de alto número atómico, aparecerán en la imagen con alto contraste, siendo visualizadas directamente y de forma individual. En estas condiciones de registro de la imagen se puede realizar medidas directas del tamaño de las nanopartículas presentes en la muestra, a partir de las cuales se puede establecer una distribución de tamaños de partícula. Adicionalmente se puede determinar la distribución espacial, con resolución sub-nanométrica, de los elementos químicos presentes en las partículas individuales, utilizando la técnica de pérdida de energía de los electrones (EELS). Esta técnica nos permite estudiar directamente las transiciones electrónicas que ocurren en el átomo cuando este es sometido a un haz de electrones de alta energía, 200kV. En particular esta técnica mide la energía que el electrón incidente pierde cuando interacciona con un átomo. Por ejemplo en el caso de los átomos de Vanadio, se estudian directamente las transiciones L2,3 donde los electrones 2p del átomo son transferidos a estados no ocupados sobre el nivel de Fermi. La energía requerida para esta transición es un valor característico para cada átomo y es igual a la energía pérdida por el electrón incidente. Así, midiendo la pérdida de energía de los electrones incidentes uno puede identificar los distintos elementos presentes en las nanopartículas, (V 513eV, O 532eV, Fe 708eV). Adicionalmente la estructura fina del espectro EELS de los metales de transición se caracteriza por la presencia de dos picos intensos (líneas blancas) cuya intensidad y posición en energía varía en función del estado de oxidación del material (Leapman et al Phys. Rev. Lett. 45, 397 (1980), Turquat et al. International Journal of Inorganic Materials 3 (2001 ) 1025-1032). El estudio detallado de la estructura fina del pico de absorción refleja información sobre el estado electrónico del material pudiendo estudiar las posibles variaciones del estado de oxidación del hierro o vanadio a través de las nanopartículas. The study of Transmission Electron Microscopy is especially useful in the case of nanoparticles doped with vanadate; This technique provides us with information about the nanoparticle size distribution as well as the chemical composition and spatial distribution of oxidation states in individual nanoparticles. Dark-field images at a high angle are sensitive to the atomic number of the material, that is, those areas where the heaviest elements are present will be shown in the image as points of greater intensity. In our case study, the nanoparticles, containing elements of high atomic number, will appear in the image with high contrast, being displayed directly and individually. Under these conditions of image registration, direct measurements of the size of the nanoparticles present in the sample can be made, from which a particle size distribution can be established. Additionally, the spatial distribution can be determined, with resolution sub-nanometer, of the chemical elements present in the individual particles, using the electron energy loss technique (EELS). This technique allows us to directly study the electronic transitions that occur in the atom when it is subjected to a beam of high-energy electrons, 200kV. In particular, this technique measures the energy that the incident electron loses when it interacts with an atom. For example in the case of Vanadium atoms, the L2.3 transitions are directly studied where the 2p electrons of the atom are transferred to unoccupied states above the Fermi level. The energy required for this transition is a characteristic value for each atom and is equal to the energy lost by the incident electron. Thus, by measuring the energy loss of the incident electrons one can identify the different elements present in the nanoparticles, (V 513eV, O 532eV, Fe 708eV). Additionally, the fine structure of the EELS spectrum of transition metals is characterized by the presence of two intense peaks (white lines) whose intensity and position in energy varies depending on the oxidation state of the material (Leapman et al Phys. Rev. Lett. 45, 397 (1980), Turquat et al. International Journal of Inorganic Materials 3 (2001) 1025-1032). The detailed study of the fine structure of the absorption peak reflects information on the electronic state of the material and can study the possible variations in the oxidation state of iron or vanadium through the nanoparticles.
El estudio de la composición química y los estados de oxidación en nanopartículas individuales fue llevado a cabo combinando las propiedades de las imágenes en campo oscuro a alto ángulo con la espectroscopia de pérdida de energía de los electrones, utilizando el método de adquisición conocido como esprectro-imagen (Tence, M. Quartuccio and C. Colliex, Ultramicroscopy 58 (1995) 42, Maigne et al Journal of Electron Microscopy 58(3): 99-109 (2009)). Este modo consiste en adquirir simultáneamente la señal de campo oscuro a alto ángulo y los espectros EELS mientras la sonda barre una zona predeterminada, imagen (1 D) o espectro línea. En particular utilizando un tiempo de adquisición de 2 segundos se adquirió un espectro EELS, con energía de dispersión de 0,5eV, cada 0,6 nm a lo largo de una línea de 36,7nm que pasa a través de las nanoparticulas. El análisis de cada uno de los espectros adquiridos (cuantificación y estudio del estado de oxidación) a lo largo de la nanopartíula nos proporciona la composición y estado de oxidación del metal caracterizado a la escala subnanométrica. Preferiblemente en el método en la disolución de la etapa (a) esta tamponada con AMPSO a un pH entre pH 7,5 y 9,5, preferiblemente entre 8,0 y 9,0. The study of the chemical composition and oxidation states in individual nanoparticles was carried out by combining the properties of the dark field images at high angle with the electron energy loss spectroscopy, using the acquisition method known as esprectro- image (Tence, M. Quartuccio and C. Colliex, Ultramicroscopy 58 (1995) 42, Maigne et al Journal of Electron Microscopy 58 (3): 99-109 (2009)). This mode consists of simultaneously acquiring the dark-field signal at high angle and the EELS spectra while the probe sweeps a predetermined area, image (1 D) or line spectrum. In particular, using an acquisition time of 2 seconds, an EELS spectrum was acquired, with dispersion energy of 0.5eV, every 0.6 nm along a 36.7nm line that passes through the nanoparticles. The analysis of each of the acquired spectra (quantification and study of the oxidation state) along the nanoparticle gives us the composition and oxidation state of the metal characterized to the sub-nanometer scale. Preferably in the method in the solution of step (a) it is buffered with AMPSO at a pH between pH 7.5 and 9.5, preferably between 8.0 and 9.0.
En una realización particular, las cadenas del polímero biocompatible, preferiblemente PEG, se hacen reaccionar con la magnetoferritina después de la inclusión de la magnetita Tc. In a particular embodiment, the biocompatible polymer chains, preferably PEG, are reacted with the magnetoferritin after the inclusion of the Tc magnetite.
Otro aspecto se refiere a una composición farmacéutica que comprende las magnetoferritina de la presente invención y al menos un excipiente farmacéuticamente aceptable, así como el uso de dicha composición farmacéutica para la preparación de un medicamento. Another aspect relates to a pharmaceutical composition comprising the magnetoferritins of the present invention and at least one pharmaceutically acceptable excipient, as well as the use of said pharmaceutical composition for the preparation of a medicament.
La magnetoferritina de la presente invención así como la composición farmacéutica que las incluye son útiles para la preparación de un medicamento para la diagnosis de diferentes enfermedades, según la utilización de moléculas que lo confieran especificidad por un tejido u órgano en cuestión, pero en especial cáncer, incluyendo cáncer cervical, de cabeza y cuello, renal y de uréter, de colon, recto y ano, de endometrio, de esófago, de estómago, de hígado, de laringe, de ovario, de páncreas, de piel, de próstata, de pulmón, de cerebro, de testículo, leucemia, melanoma, y linfoma. Además de para detectar enfermedades también son útiles tanto la magnetoferritina de la presente invención así como la composición farmacéutica que las comprende como agentes de contraste en general y como agente de contraste en MRI o gammagrafía en particular. The magnetoferritin of the present invention as well as the pharmaceutical composition that includes them are useful for the preparation of a medicament for the diagnosis of different diseases, according to the use of molecules that confer specificity for a tissue or organ in question, but especially cancer , including cervical, head and neck, renal and ureter, colon, rectum and anus, endometrial, esophagus, stomach, liver, larynx, ovarian, pancreas, skin, prostate, lung, brain, testis, leukemia, melanoma, and lymphoma. In addition to detecting diseases, both the magnetoferritin of the present invention and the pharmaceutical composition comprising them as contrast agents in general and as a contrast agent in MRI or scintigraphy in particular are also useful.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. EJEMPLOS Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention. EXAMPLES
Síntesis de magnetoferritina-99mTc: Synthesis of magnetoferritin- 99m Tc:
Disolución 1. Se preparan 10 mi una disolución de apoferritina (Sigma- Aldrich Ref. A341- G, lot. 048K7004) de concentración 10mg/ml en tampón AMPSO pH 8,6 (Sigma A6659). La disolución se desgasifica con una corriente fuerte de argón y en agitación durante 10 min. Solution 1. A solution of apoferritin (Sigma-Aldrich Ref. A341-G, lot. 048K7004) with a concentration of 10 mg / ml in AMPSO buffer pH 8.6 (Sigma A6659) is prepared 10 ml. The solution is degassed with a strong stream of argon and under stirring for 10 min.
Disolución 2. Se preparan dos disoluciones: 5 mi de Sal de Mohr (Amonium Iron (II) sulfate hexahydrate, Aldrich Chem. 20,350-5) 0,05 M en HCI 0,01 M y 5 mi de Fe (NO3)3 0,1 M en HCI 0,01 M, se mezclan y se desgasifica con una corriente fuerte de argón y en agitación durante 10 min. Solution 2. Two solutions are prepared: 5 ml of Mohr Salt (Amonium Iron (II) sulfate hexahydrate, Aldrich Chem. 20,350-5) 0.05 M in 0.01 M HCI and 5 ml of Fe (NO 3 ) 3 0.1 M in 0.01 M HCI, mixed and degassed with a strong argon stream and stirred for 10 min.
Disolución 3. Una disolución de NaOH 0,1 M se desgasifica con una corriente fuerte de argón y en agitación durante 10 min. Disolución 4. Una disolución de pertenectato ( Te) obtenida a partir de kit comercial se desgasifica con una corriente fuerte de argón y en agitación durante 2 min. Suspensión 5. Se lleva a cabo la adición lenta de la disolución 2 sobre la disolución 1. Se llevan a cabo adiciones de 0,25 mi cada 2 min hasta completar 1 mi. Antes de la 5 adición de la disolución 2, se adicionan 56 μΙ de la disolución de 99mTc (disolución 4). Disolución 6. A la suspensión 5 se le adiciona lentamente 1 mi de una disolución de citrato sódico 0,1 M para eliminar todo los compuestos metálicos que no hayan quedado encapsulados en la apoferritina. La disolución resultante se cromatografía (10 min) en columna de exclusión por tamaño (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17-0851-01 ), obteniendo la disolución final 6 que contiene mangnetita dopada con 99mTc encapsulada en la cavidad de la apoferritina. Solution 3. A solution of 0.1 M NaOH is degassed with a strong stream of argon and under stirring for 10 min. Solution 4. A solution of belonging (Te) obtained from a commercial kit is degassed with a strong stream of argon and under stirring for 2 min. Suspension 5. Slow addition of solution 2 to solution 1 is carried out. Additions of 0.25 ml are made every 2 min until 1 ml is completed. Before the addition of solution 2, 56 μΙ of the 99m Tc solution (solution 4) is added. Solution 6. Suspension 5 is slowly added 1 ml of a 0.1 M sodium citrate solution to remove all metal compounds that have not been encapsulated in apoferritin. The resulting solution is chromatographed (10 min) in size exclusion column (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17-0851-01), obtaining the final solution 6 containing doped mangnetite with 99m Tc encapsulated in the apoferritin cavity.
Adición del radioisótopo de Te a la magnetoferritina. Disolución 4. Se usa un generador de 99Mo/99mTc de 12 GBq de actividad calibrada. La elución realizada es analizada en términos de actividad de 99mTc. Conocida la relación específica mCi/pg de 99mTc, es posible controlar la cantidad de Te utilizada y por ende su actividad radioquímica. Suspensión 5. Se lleva a cabo la adición lenta de la disolución 2 sobre la disolución 1. Se llevan a cabo adiciones de 0,25 mi cada 2 min hasta completar 1 mi. Antes de la 5 adición de la disolución 2, se adicionan 0,5 mi de la disolución de 99rT1Tc (disolución 4). Para la realización del control de calidad usamos tiras de papel Whatman 3 MM de 10 cm de longitud y 0,5 cm de ancho, en las que depositamos una alícuota (150 μΙ) del radiofármaco marcado, y para su desarrollo las introducimos en tanques cromatográficos con acetona hasta unos 0,5 mm de la base. Cuando la cromatografía se ha desarrollado la medimos en el Radio Cromatógrafo Minigita Raytest. En el origen de la tira cromatográfica se quedarán los coloides (Rf=0), en este caso la magnetoferritina dopada con Te, y en el frente el pertecnetato libre (Rf=1 ) Addition of the radioisotope of Te to the magnetoferritin. Solution 4. A 99 Mo / 99m Tc generator with 12 GBq of calibrated activity is used. The elution performed is analyzed in terms of 99m Tc activity. Once the specific mCi / pg ratio of 99m Tc is known, it is possible to control the amount of Te used and therefore its radiochemical activity. Suspension 5. Slow addition of solution 2 to solution 1 is carried out. Additions of 0.25 ml are made every 2 min until 1 ml is completed. Before the addition of solution 2, 0.5 ml of the 99rT1 Tc solution (solution 4) is added. To perform the quality control we use Whatman 3 MM paper strips 10 cm long and 0.5 cm wide, on which we deposit a aliquot (150 μΙ) of the labeled radiopharmaceutical, and for its development we introduce them in chromatographic tanks with acetone up to about 0.5 mm from the base. When the chromatography has developed we measure it on the Raytest Minigit Chromatograph Radio. In the origin of the chromatographic strip the colloids will remain (Rf = 0), in this case the magnetoferritin doped with Te, and in the front the free pertechnetate (Rf = 1)
Acoplamiento de PEG El derivado de polietilenglicol MeO-PEG-NHS a-Methoxy- -carboxylic acíd succinimidyl ester poly(ethylene glycol) (PEG-MW 2.000 Dalton)/ MW 2.000 g/mol) se adquirió en Iris Biotech GmbH (PEG1164, lot. 125447). PEG Coupling The polyethylene glycol derivative MeO-PEG-NHS a-Methoxy- -carboxylic acid succinimidyl ester poly (ethylene glycol) (PEG-MW 2,000 Dalton) / MW 2,000 g / mol) was purchased from Iris Biotech GmbH (PEG1164, lot . 125447).
Disolución 7. 1000 moles de PEG (0,0058 g en 0,5 mi de agua bidestilada) fueron añadidos a la disolución 8 y se dejó 30 min en agitación suave y a temperatura ambiente. Se cromatografió (10 min) en una columna de exclusión por tamaño (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17-0851 -01 ) hasta obtener una disolución pura de nanopartículas de magnetita dopadas con 99mTc, acopladas covalentemente con un biopolímero de PEG. Solution 7. 1000 moles of PEG (0.0058 g in 0.5 ml of double distilled water) were added to solution 8 and left 30 min under gentle stirring and at room temperature. Chromatograph (10 min) on a size exclusion column (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17-0851 -01) until a pure solution of magnetite nanoparticles doped with 99m is obtained Tc, covalently coupled with a PEG biopolymer.

Claims

REIVINDICACIONES
1. - Una magnetoferritina superparamagnética, caracterizada porque el núcleo de magnetita ocluye 99mTc. 1. - A superparamagnetic magnetoferritin, characterized in that the magnetite core occludes 99m Tc.
2. - La magnetoferritina superparamagnética según la reivindicación anterior, caracterizada porque la especie de Te es 99mTc04 ~. 2. - The superparamagnetic magnetoferritin according to the preceding claim, characterized in that the Te species is 99m Tc0 4 ~ .
3.- La magnetoferritina superparamagnética según cualquiera de las reivindicaciones anteriores, caracterizada porque la concentración de la especie 99mTc está comprendida entre 10"9-10"5M. 3. The superparamagnetic magnetoferritin according to any of the preceding claims, characterized in that the concentration of the 99m Tc species is between 10 "9 -10 " 5 M.
4. - La magnetoferritina superparamagnética según cualquiera de las reivindicaciones anteriores, caracterizada porque tiene cadenas de un polímero biocompatible covalentemente unidas a la superficie de la magnetoferritina. 4. - The superparamagnetic magnetoferritin according to any of the preceding claims, characterized in that it has chains of a biocompatible polymer covalently bonded to the surface of the magnetoferritin.
5. - La magnetoferritina superparamagnética según la reivindicación anterior, caracterizada porque el polímero biocompatible es polietilén glicol. 5. - The superparamagnetic magnetoferritin according to the preceding claim, characterized in that the biocompatible polymer is polyethylene glycol.
6. - La magnetoferritina superparamagnética según la reivindicación anterior, caracterizada porque el polietilenoglicol es PEG1163 MeO-PEG- COO-Su a-Methoxy- -carboxylic acid succinimidyl ester poly(ethylene glycol) PEG-WM 2,000 Dalton. 6. - The superparamagnetic magnetoferritin according to the preceding claim, characterized in that the polyethylene glycol is PEG1163 MeO-PEG-COO-Su a-Methoxy- -carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 Dalton.
7. - La magnetoferritina superparamagnética según cualquiera de las dos reivindicaciones anteriores, caracterizada porque el PEG está unido mediante enlaces covalentes de tipo amida. 7. - The superparamagnetic magnetoferritin according to any of the two preceding claims, characterized in that the PEG is linked by covalent bonds of the amide type.
8. - La magnetoferritina superparamagnética según cualquiera de las tres reivindicaciones anteriores, caracterizada porque tiene entre 3 y 10 cadenas de PEG unidas covalentemente a la superficie de la magnetoferritina. 8. - The superparamagnetic magnetoferritin according to any of the three preceding claims, characterized in that it has between 3 and 10 PEG chains covalently bonded to the surface of the magnetoferritin.
9. - Un método para la síntesis de cualquiera de las magnetoferritina superparamagnética como se definen en las reivindicaciones anteriores que comprende: a) preparar una disolución de apoferritina, preferiblemente de concentración entre 0,1 y 100 mg/mL, y más preferiblemente entre 5 y 20 mg/mL, b) Preparar una disolución que comprenda Fe (II) y Fe(lll) en estequiometría aproximada de 1 :2. c) preparar una disolución de pertenectato (99mTc) d) adicionar de forma intercalada la disolución preparada en el paso (b) y la disolución preparada en la etapa (c) sobre la preparada en el paso (a). f) Aislar la magnetoferritina superparamagnética dopada con 99mTc 9. - A method for the synthesis of any of the superparamagnetic magnetoferritins as defined in the preceding claims comprising: a) preparing an apoferritin solution, preferably of a concentration between 0.1 and 100 mg / mL, and more preferably between and 20 mg / mL, b) Prepare a solution comprising Fe (II) and Fe (lll) in stoichiometry of approximately 1: 2. c) to prepare a solution of pentactate ( 99m Tc) d) to intercalate the solution prepared in step (b) and the solution prepared in step (c) over that prepared in step (a). f) Isolate the superparamagnetic magnetoferritin doped with 99m Tc
10. - El método según la reivindicación anterior en la que la disolución de la etapa (b) se prepara mezclando una disolución que comprende sulfato de hierro (II) amoníaco hexahidratado con otra que comprende Fe (N03)3 en HCI. 10. - The method according to the preceding claim wherein the solution of step (b) is prepared by mixing a solution comprising iron (II) sulfate ammonia hexahydrate with another comprising Fe (N0 3 ) 3 in HCI.
1 1. - El método según cualquiera de las dos reivindicaciones anteriores, donde la disolución de la etapa (a) esta tamponada con AMPSO a un pH entre pH 7,5 y 9,5, preferiblemente entre 8,0 y 9,0. 1 1. The method according to any of the two preceding claims, wherein the solution of step (a) is buffered with AMPSO at a pH between pH 7.5 and 9.5, preferably between 8.0 and 9.0.
12. - El método según cualquiera de las tres reivindicaciones anteriores para la síntesis de cualquiera de las magnetoferritina superparamagnética como se definen en las reivindicaciones 6 a 10, que comprende hacer reaccionar las magnetoferritina superparamagnética con el polímero biocompatible. 12. - The method according to any of the three preceding claims for the synthesis of any of the superparamagnetic magnetoferritins as defined in claims 6 to 10, which comprises reacting the superparamagnetic magnetoferritins with the biocompatible polymer.
13. - Una composición farmacéutica que comprende la magnetoferritina superparamagnética como se en cualquiera de las reivindicaciones 1 a 8 y al menos un excipiente farmacéuticamente aceptable. 13. - A pharmaceutical composition comprising the superparamagnetic magnetoferritin as in any one of claims 1 to 8 and at least one pharmaceutically acceptable excipient.
14. - Uso de la composición farmacéutica según la reivindicación anterior para la preparación de un medicamento. 14. - Use of the pharmaceutical composition according to the preceding claim for the preparation of a medicament.
15. - El uso de la composición según la reivindicación anterior para la preparación de un medicamento para la diagnosis de cáncer. 15. - The use of the composition according to the preceding claim for the preparation of a medicament for the diagnosis of cancer.
16. - La magnetoferritina superparamagnética según cualquiera de las reivindicaciones 1 a 8 o de la composición farmacéutica según la reivindicación 13 para la preparación de un medicamento para su uso como agente de contraste. 16. - The superparamagnetic magnetoferritin according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 13 for the preparation of a medicament for use as a contrast agent.
17. - La magnetoferritina superparamagnética según cualquiera de las reivindicaciones 1 a 10 o de la composición farmacéutica según la reivindicación 17 para la preparación de un medicamento para su uso como agente de contraste en MRI o SPECT. 17. - The superparamagnetic magnetoferritin according to any one of claims 1 to 10 or of the pharmaceutical composition according to claim 17 for the preparation of a medicament for use as a contrast agent in MRI or SPECT.
PCT/ES2010/000424 2009-10-14 2010-10-13 Multifunctional nanostructures as spect/mri bimodal diagnosis agents WO2011045454A2 (en)

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