WO2011095661A1 - Magnetic nanoparticles for use in a pharmaceutical composition - Google Patents

Abstract

The invention relates to a pharmaceutical composition that includes a magnetic nanoparticle and at least one active ingredient, preferably an antineoplastic or immunomodulator, for treating illnesses, in particular cancer. The nucleus of the magnetic nanoparticle includes a magnetic iron oxide. The surface of the nanoparticle includes ligands bonded thereto by means of an ionic bond, a covalent bond, a hydrogen bridge, a hydrophobic bond, an electrostatic bond and/or a metal-ligand coordination bond, wherein the ligand includes an active region having at least one group chosen from - CO₂H, -PO₃H, -PO₄H, -SO₃H and -SO₄H. Moreover, the active ingredient is bonded to the active region of the ligands by means of ionic bonds.

Description

 MAGNETIC NANOPARTICLES FOR USE IN A COMPOSITION

 PHARMACEUTICS

The present invention relates to the use of a pharmaceutical composition comprising a magnetic nanoparticle and at least one active ingredient, preferably an antineoplastic or immunomodulator, for the treatment of diseases, especially cancer. Therefore it belongs to the field of medicine technique. STATE OF THE PREVIOUS TECHNIQUE

One of the physiological functions of the immune system is to detect and eliminate the transformed cells that continually arise in the body before they manifest clinically. This process is called immunovigilance and is based on the involvement of effector cells of the innate and acquired systems, and certain immune molecules.

In the literature we can find numerous experimental evidences that support the idea of immunovigilance. The use of models of mice deficient in any of the components of the immune system, such as T cells, natural killer or specific molecules such as interferon-gamma, perforin, interleukin-12 or TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), show unequivocally greater incidences of tumor development. On the other hand, the administration of interleukin-12 or immune system stimulants such as α-galactosylceramide reduces the incidence of primary tumors in normal mice. In humans there is also evidence to suggest that the hypothesis of immune surveillance is fulfilled, such as the fact that immunosuppressed patients have higher incidence of tumors than immunocompetent patients, that the presence of lymphocytes infiltrated in a tumor is a sign of good prognosis, and that cancer patients are capable of generating innate immune responses and acquired against the type of tumor they suffer (Ikeda et al, Cancer Sci, 65: 697-703, (2004)).

However, immunovigilance represents only part of the complex relationship between the immune system and cancer. The presence of a tumor indicates that the cells that form it have been able to prevent recognition by the immune system, or escape the effects of it. The process between immunovigilance and evasion, called immunoedition, consists of three phases: elimination, balance and escape. The elimination phase represents the classic concept of immunovigilance. In this phase the immune system is able to detect and destroy the transformed cells that are generated. The equilibrium phase represents the latency period that would exist between the end of the elimination phase and the appearance of a clinically detectable tumor. In this phase, what is known as immunoselection is carried out, that is, those cells that, thanks to the great genomic plasticity of the tumor cells, that usually have altered the mechanisms of DNA repair and accumulate mutations more frequently than the cells normal, they acquire a phenotype that makes them less immunogenic and more resistant in their environment, which allows them to survive and divide, so that the developing tumor is enriched in this cell type that cannot be eliminated by the immune system. In the escape phase, tumor cells have accumulated a series of changes that allow them to escape both innate and acquired immunity, so that they can develop freely. There is also a process of active suppression of the immune response by the tumor, which is known as immunosubversion. This process includes the overproduction of immunosuppressive cytokines or the release of soluble forms of ligands that block certain immune cell receptors. The immune response against tumors begins with the participation of innate immune system cells, such as Natural Killer (NK), Natural Killer T (NKT) and Τγδ cells, which recognize transformed cells and stimulate to produce interferon-gamma. This initial interferon-gamma triggers a cascade of reactions such as the induction of angiostatic chemokines that block neovascularization and the recruitment of effector cells from the immune system to the area where the tumor is located. Interferon-gamma also acts directly on tumor cells, on which they have antiproliferative and proapoptotic effects. These effects cause the death of part of the tumor cells, which are to be processed by dendritic cells, which migrate to nearby lymph nodes and present tumor antigens to CD4 + or CD8 + T cells specific to them. These T cells are activated, proliferate and migrate to the immediate vicinity of the tumor following gradients of cytokines and chemokines. Once they reach their destination, these cells will recognize and destroy the tumor cells that express the corresponding antigens. Interferon-gamma, therefore, plays a fundamental role in the antitumor immune response. Interferon-gamma is a member of a family of proteins that were originally identified by their ability to protect cells from viral infections (Wheelock EF, Science, 149: 310-31 1 (1965)). These proteins are divided into two classes following structural and functional criteria, and taking into account the stimuli that induce their expression. Type I interferons are induced in response to a viral infection. Type II interferon or immune interferon corresponds to interferon-gamma, and is predominantly produced by T lymphocytes, natural killer cells (NK) and NKT cells after activation by immune and inflammatory stimuli (Boehm et al, Annu Rev Immunol, 15: 749-95 (1997)). Human and murine interferon-gamma are formed by glycosylated polypeptide chains that homodimerize by binding non-covalently and giving rise to a 50 kDa protein. The two chains are associated antiparallel and form a dimer that has two identical binding sites to its receptor (Bach et al, Annu Rev Immunol, 15: 563-91 (1997)). There are numerous studies that point to interferon-gamma as the most effective cytokine against tumors. Injection of monoclonal antibodies capable of blocking interferon-gamma in experimental animals suppresses the ability to reject transplanted tumors. The incidence of primary tumors is higher and they also grow faster and at a lower dose of carcinogens in 129 / SvEv mice deficient in molecules involved in the interferon-gamma receptor signaling pathway than in normal mice. Also in mice where tumor formation is induced genetically {mice deficient in p53), the lack of molecules of the interferon-gamma receptor signaling pathway or of interferon-gamma itself induces greater tumor formation.

In the past, different cytokines have been used as possible therapeutic agents against cancer, with varying results depending on the mode of administration. The appearance of important side effects is the main limitation of the systhemic administration, in addition to the fact that the concentration of cytokine that is achieved is well below what is required at the site of action, and the increase in the level of cytokine is transitory (Margolin et al, J Immunother Emphasis Tumor immunol, 14: 70-6 (1993)). The injection of genetic vectors into the tumor mass or the implantation of genetically modified cells to produce cytokines has a variable efficiency depending on the vector or the type of cell used (Miller et al., Blood. 82: 3686-94 (1993)). Injection of tumor cells modified to produce cytokines and irradiated as vaccines has side effects similar to systemic administration. The most promising results have been obtained with the use of microspheres of different cytokine-loaded materials (Arara et al, J Surq Oncol, 94: 403-12 (2006)). , although there is still a need for methods that allow the controlled and specific release of active substance, and especially those potentially toxic if administered systematically. DESCRIPTION OF THE INVENTION

The present invention provides a pharmaceutical composition comprising conjugates formed by magnetic nanoparticles and an active ingredient, said composition being also useful for the treatment of cancer diseases.

As can be seen in the examples, the authors of the present invention tested the electrostatic interaction between murine interferon-gamma and different types of magnetic nanoparticles. The result was positive when magnetic nanoparticles with negative surface charge at physiological pH were used and it was also found that interferon-gamma was still functional after the interaction, and that the release of interferon occurred especially in the place where the particles accumulated by The influence of a magnetic field.

The antitumor composition of the present invention can be administered in the form of a preparation containing conjugates formed by magnetic nanoparticles to which molecules of the active ingredient that can be a cytokine such as interferon-gamma are adsorbed, in order to be able to direct and concentrate said cytokine in the region of interest by using external magnetic fields.

According to the present invention, immobilization or attraction to the area of interest of the composition allows obtaining a therapeutically relevant concentration in the area of interest, but nevertheless the concentration of active ingredient in the rest of the tissue and / or fluids, for example in blood, is lower than if compared with the administration of said active substance by other routes of administration. These advantages consequently lead to a reduction in doses and a reduction in the risk levels of various side effects, especially highly aggressive active ingredients such as interferon-gamma. Therefore, a first aspect of the present invention relates to the use of a pharmaceutical composition comprising a conjugate, wherein said conjugate comprises a magnetic nanoparticle and at least one active principle for the preparation of a medicament, characterized in that:

 - the core of the magnetic nanoparticle comprises a magnetic iron oxide;

- The nanoparticle comprises on its surface ligands bound thereto by ionic bond, covalent bond, hydrogen bridge, hydrophobic bond, metal ligand coordination bond or combinations thereof, where the ligand comprises an active region comprising at least one selected group between -CO ₂ H, -PO ₃ H, -P0 ₄ H, -SO ₃ H and -S0 ₄ H; Y

- where the active substance is linked to the active region of the ligands by ionic bonds. By "an active region" is meant the region of the ligand that binds to the active ingredient to form the conjugate described in the present invention. Said region comprises at least one group selected from -C0 ₂ H, -PO ₃ H, -P0 ₄ H, -SO ₃ H and -S0 ₄ H. More preferably this active region of the ligand comprises at least one group selected from -CO ₂ H, -P0 ₄ H - and -S0 ₄ H. More preferably the active region of the ligand comprises -CO ₂ H.

By "ligand" is meant in the present invention a compound comprising an active region, described above, where it binds to the active ingredient and comprising an adhesive region which is bonded to the surface of the nanoparticle through ionic bonding, bonding covalent, hydrogen bridge, hydrophobic bond or metal ligand coordination bond. Preferably this adhesive region comprises at least one group selected from -C0 ₂ H, -NH ₂ , -SH, -CONH ₂ , -OH, -PO ₃ H, -PO ₄ H, -SO ₃ H and - SO ₄ H. More preferably it comprises at least one group selected from -CO ₂ H, -OH, -PO ₄ H and -SO ₄ H. Even more preferably, the adhesive region comprises -CO ₂ H. In addition, the ligand between the adhesive region and the active region may comprise a hydrocarbon chain of between 1 and 20 carbon atoms. More preferably the hydrocarbon chain comprises between 1 and 10 carbon atoms. Even more preferably, the hydrocarbon chain is of the formula -CnH ₂ n-, where n is an integer between 1 and 10. This hydrocarbon chain may be optionally substituted and / or have one or more unsaturations. The objective of this chain is to unite the active region with the surface of the particle. More preferably the hydrocarbon chain comprises at least one group selected from ether, amido, amino, thioether, alkene, alkyne, C3-C-6 cycloalkane and / or phenyl. And said hydrocarbon chain may be substituted with at least one group selected from -SH, -OH, -CO ₂ H, -NH ₂ , -SH, -CONH2, -OH, -PO3H, -PO ₄ H, -SO ₃ H , -SO ₄ H, F, Cl, Br and I. More preferably the hydrocarbon chain is substituted with at least one -SH group.

In a preferred embodiment, the magnetic nanoparticles described in the present invention comprise at least one ligand selected from dimethylmercaptosuccinic acid, dimercaptomaleic acid and dimercaptopentadionic acid. More preferably it comprises at least dimethylmercaptosuccinic acid.

In another preferred embodiment of the present invention, the magnetic nanoparticle is obtainable by the decomposition of ferric acetylacetonate in solution at a temperature between 235 and 290 ^Q C in ether in the presence of a fatty acid, preferably the fatty acid is oleic acid. For example, a synthesis of nanoparticles is described in this article (Park J, Joo J, Soon GK, Jang Y and Hyeon T 2007 Angew.Chem. Int. Edn 46 4630, "Synthesis of monodisperse spherical nanocrystals"). More preferably the magnetic nanoparticle is obtainable by the decomposition of ferric acetylacetonate in solution at a temperature between 250 and 270 ^S C.

The decomposition of ferric acetylacetonate can be carried out in presence of an organic solvent, or mixtures thereof, of boiling temperature greater than 235 ^S C.

In a more preferred embodiment, the decomposition of ferric acetylacetonate to obtain the magnetic nanoparticles is carried out in the presence of biphenyl ether, benzyl ether, octyl ether, octadecene, trioctylamine or ethylene glycol.

In another preferred embodiment of the present invention, the binding between the ligand and the magnetic nanoparticle is performed by a ligand exchange process (a process known to any person skilled in the art and described in "Nanotechnologies for the Life Sciences. Volume 7 Biofunctionalization of Nanomaterials. Edited by Challa SSR Kumar ").

In a preferred embodiment of the present invention, the magnetic nanoparticle has at least an average particle size greater than 6 nm. Preferably the magnetic nanoparticle has at least an average particle size between 8 and 1 nm. The particle size is the average size that results from adjusting to a logarithmic distribution the data obtained from TEM photographs, measuring at least 500 particles (Partióle size analysis in ferrofluids, Journal of Magnetism and Magnetíc Materials, K. O ^' Grady and A. Bradbury, 39 (1983) 91-94).

In another preferred embodiment of the present invention, the conjugate has an average hydrodynamic particle size of less than 200 nm. More preferably the conjugate has an average hydrodynamic particle size between 40 and 150 nm. Even more preferably, the conjugate has an average hydrodynamic particle size between 85 and 1 nm. The measurement or method of measurement of hydrodynamic radii is known to any person skilled in the art and is described in: "Effect of Nanoparticle and Aggregate Size on the Relaxometric Properties of MR Contrast Agents Based on High Quality Magnetite Nanoparticles Alejandro G. Roca, Sabino Veintemíllas- Verdaguer, Marc Port, Caroline Robic, Carlos J. Serna, and Maria P. Morales, J. Phys. Chem. B 2009, 1 13, 7033-7039 ").

The term "active substance", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different effect on the diagnosis, cure, mitigation, treatment , or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect. In a particular embodiment the active ingredient is a cytokine. This can also be an antineoplastic or immunomodulatory agent. Proteins with biological activity are also preferred, and especially interferon. Interferon can be selected from interferon alpha, beta, omega, epsilon, kappa, gamma, lambda and any combination thereof. More preferably the active ingredient is interferon gamma. Since the union between the active region and the active principle is preferably carried out by ionic interactions, it is important, and therefore preferred that the active principle has at least a positive charge at a pH between 2 and 8, and even more preferably it has at least a positive charge at a pH between 5.5 and 7.5. This charge may be that it has an active ingredient by its very nature, such as interferon gamma, or that it is introduced by chemical derivatization of the active substance, for example by the introduction of amino groups.

By "antineoplastic" refers to substances that prevent the development, growth, and / or proliferation of malignant tumor cells. These substances can be of natural, synthetic or semi-synthetic origin. Many of the antineoplastic drugs are prodrugs, that is, a drug is administered that is less toxic, or has better pharmacodynamic characteristics, and once In the body it becomes another more effective, safe and selective drug against its therapeutic target.

By "immunomodulator" is meant a substance a substance that alters the immune response through the increase or reduction of the ability of the immune system to produce antibodies or sensitized cells to recognize and react with the antigen that began its production.

The interferon-gamma for use in the pharmaceutical composition of the present invention may be any of the natural interferon-gamma, recombinant interferon-gamma and derivatives thereof provided that they exhibit an interferon-gamma activity. The interferon-gamma described in the examples is murine recombinant interferon-gamma, since the experiments performed were based on the use of a mouse tumor model.

Other pharmacologically active substances could be added to the pharmaceutical composition of the invention if necessary, in addition to those already mentioned. In another preferred embodiment of the present invention, the conjugate comprises at least 1 x 10 ^"3 mg of active ingredient, preferably antineoplastic or immunomodulator, per mg of conjugate. More preferably, the conjugate comprises at least 10x10 ^{" 3} mg of active ingredient, preferably cytokine, per mg of conjugate. Even more preferably between 2x10 ^"3 mg and 6x10 ^{" 3} mg of active ingredient, preferably cytokine, per mg of conjugate.

Another preferred embodiment of the present invention comprises a pharmaceutical composition with at least one pharmaceutically acceptable excipient or carrier. The term "pharmaceutically acceptable excipient" means any ingredient that has no therapeutic activity and that is non-toxic and therefore suitable as an excipient. Suitable excipients include the excipients commonly used in pharmaceutical products, such as microcrystalline cellulose, lactose, starch, magnesium stearate, crosspovidone, povidone and talc. The administration of the compounds of this invention can be carried out by any method that releases the compound, preferably to the desired tissue. These procedures include oral, intravenous, intramuscular, subcutaneous or intramedullary, intraduodenal, etc. Preferably, the pharmaceutical composition of the present invention can be administered locally by injection in an area near the region of interest (intramuscularly, subcutaneously, intradically), injection into an area near the region of interest and subsequent immobilization by magnetic fields or, preferably, intravenous injection and attraction to the area of interest through magnetic fields. Therefore, another preferred embodiment of the present invention comprises the use of any of the pharmaceutical compositions described for the preparation of an intravenous composition.

For the purpose of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be used, as well as sterile aqueous solutions of the corresponding water-soluble salts. If necessary, such aqueous solutions can be adequately buffered, and the liquid diluent first became isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this regard, all sterile aqueous media employed can be easily obtained by standard techniques well known to those skilled in the art. In the parental formulations for intravenous use, preferably the conjugate is dissolved or dispersed in an aqueous solution. This solution is not especially limited, being able to use a wide variety of solutions as long as their pH is in a range in which the cytokine, or in general the active substance, is natively and has a positive charge (at pH 2-8 ). Generally, the compounds of the present invention are administered in single doses (for example once a day) or multiple doses.

The compounds of the present invention are administered in a therapeutically effective dose. The term "therapeutically effective" refers to an amount of a drug or a therapeutic agent that elicits the desired biological or medical response of a tissue, a system or an animal {including man) that is being sought by a researcher or clinician. In any case, the amount and rate of the compound administered will, of course, depend on the subject being treated, the severity of the condition, the manner of administration and the opinion of the prescribing physician. Therefore, because of the variability between patients, the doses given below are a guide and the doctor can adjust the doses of the compounds to achieve the treatment that the doctor considers to be appropriate for the patient. When considering the degree of treatment desired, the doctor must balance a number of factors such as the patient's age, the presence of a pre-existing disease as well as the presence of other diseases. The term "treatment" means the management and care of a patient for the purpose of combating the disease or disorder.

Another aspect of the present invention relates to the use of any of the pharmaceutical compositions described above for the preparation of a composition for the treatment of cancer.

The term "cancer" or "cancerous", as used herein, refers to any malignant tumor. The terms "tumor" or "tumor", as used herein, refers to transformed cells that exhibit uncontrolled growth. Depending on its possible evolution it may be a benign tumor, which remains in its starting place and does not produce metastasis; or malignant, invasive or metastatic tumor.

Tumors or cancers of the head and neck are, for example, but not limited to, a tumor or cancer of the lip, a tumor or cancer of the oral cavity, a tumor or cancer of the salivary gland, a tumor or a cancer of the nasal cavity and paranasal sinus, a tumor or an oropharyngeal cancer, a tumor or a hypopharyngeal cancer, a tumor or a larynx cancer, a tumor or a nasopharyngeal cancer, or a metastatic squamous neck cancer with a hidden primary tumor. Tumors or cancers of the digestive system are, for example, but not limited to, a tumor or cancer of the esophagus, a tumor or stomach cancer, a tumor or cancer of the small intestine, a tumor or cancer of the duodenum, a tumor or a colon cancer, a tumor or rectal cancer, a tumor or anus cancer, a tumor or liver cancer, a tumor or a gallbladder cancer, a tumor or an extrahepatic bile duct cancer, or a tumor or pancreatic cancer. Tumors or cancers of the nervous system are, for example, but not limited to, an astrocytoma, a glioblastoma, a xanthoastrocytoma, an oligodendroglioma, an oligoastrocytoma, an ependymoma, a subependymoma, an astroblastoma, a glioma, a gangliocytoma, a gangliocytoma, a gangliocytoma, a gangliocytoma, a gangliocytoma, a gangliocytoma, a gangliocytoma, a gangliocytoma, a gangliocytoma , a liponeurocytoma, a paraganglioma, an ependimoblastoma, a medulloblastoma, a supratentorial primitive neuroectodermal tumor, a pineoblastoma, a pynealocitoma, a papilloma or a carcinoma of the choroid plexus, a meningioma, a hemangiopericytoma, a saratoma, a germ tumor vitelline, an embryonic carcinoma, a choriocarcinoma, a mixed germ cell tumor, an adenoma or a pituitary carcinoma, a craniopharyngioma, a hemangioblastoma, a schwannoma. Tumors or cancers of the urinary system are, for example, but not limited to, a tumor or a renal cell cancer, a tumor or a bladder cancer, a tumor or a transitional cell cancer of the renal pelvis and ureter, or a tumor or a urethral cancer. Gynecological tumors or cancers are, for example, but not limited to, a tumor or an ovarian cancer, a tumor or a cervical cancer, a tumor or a vaginal cancer or a tumor or vulvar cancer. Examples of tumors or cancers of the male genital tract are, for example, a tumor or prostate cancer, a tumor or a testicular cancer or a tumor or a penile cancer. Examples of tumors or cancers of the respiratory system are, for example, a tumor or a non-small cell lung cancer, a tumor or a small cell lung cancer or a malignant mesothelioma. Tumors or cancers of the endocrine system are, for example, a tumor or a thyroid cancer, a tumor or a parathyroid cancer, a tumor or a cancer of the pituitary gland, a tumor or a cancer of the adrenal cortex, a tumor or an islet cell cancer of the endocrine pancreas or a pheochromocytoma. They are sarcomas, for example, but not limited to, a fibrosarcoma, a histiocytoma, a dermatofibrosarcoma, a liposarcoma, a rhabdomyosarcoma, a leiomyosarcoma, a fusocellular sarcoma, a hemangiosarcoma, a Kaposi sarcoma, a lymphangiosarcoma, a sionovial sarcoma, a neurofibrosis a chondrosarcoma or osteosarcoma. They are non-melanoma skin tumors or cancers, for example, but not limited to, a tumor or a basal cell cancer, a tumor or a squamous cell cancer or a tumor or a Merkel cell cancer. They are hemopoietic or lymphoid tissue neoplasms, for example, but not limited to, a lymphoma or a lymphoblastic precursor cell B leukemia, a lymphoma or a peripheral cell B leukemia, a lymphoma or a lymphoblastic T cell precursor leukemia, a lymphoma or a peripheral T-cell leukemia, a lymphoma or an NK cell leukemia, a Hodgkin lymphoma, a hystocyte neoplasm, a dendritic cell neoplasm, an acute myeloid leukemia, an acute lymphoblastic leukemia, an acute biphenotypic leukemia or a myelodysplastic syndrome .

In a preferred embodiment, the cancer would be breast, colon, lung, uterus, prostate, pancreas, melanoma and / or bladder. A further aspect of the present invention relates to an assembly comprising a pharmaceutical composition and a magnet, characterized in that the pharmaceutical composition comprises a conjugate comprising a Magnetic nanoparticle and at least one active ingredient for the preparation of a medicine, characterized in that:

 - the core of the magnetic nanoparticle comprises a magnetic iron oxide;

- The nanoparticle comprises on its surface ligands attached thereto by ionic bond, covalent bond, hydrogen bridge, hydrophobic bond, metal ligand coordination bond or any combination thereof, where the ligand comprises an active region comprising at least one selected group between -CO ₂ H, -PO ₃ H, -PO ₄ H, -SO ₃ H and -SO ₄ H; and - where the active ingredient is linked to the active region of ligands through ionic bonds.

The pharmaceutical composition that is part of the system of the present invention can be any of the compositions described above.

In a preferred embodiment of the assembly of the present invention, the magnet has at least a power of 0.05 Teslas. More preferably the magnet has at least a power of 0.1 Teslas, even more preferably it has at least a power of 0.2 to 0.45 Teslas.

In another preferred embodiment of the assembly of the present invention, the magnet is selected from a ferrite, neodynium magnet and / or an electromagnet. Said magnet can be a neodymium disc with a diameter between 3 and 7 mm and a thickness between 1, 5 and 5 mm.

Another aspect of the present invention relates to the use of the assembly of the present invention for use in medicine.

Another aspect of the invention relates to a pharmaceutical composition comprising: a conjugate comprising a magnetic nanoparticle and at least one active ingredient; characterized in that: the core of the magnetic nanoparticle comprises a magnetic iron oxide; the nanoparticle comprises on its surface ligands bound thereto by ionic bond, covalent bond, hydrogen bridge, hydrophobic bond, electrostatic bond and / or metal ligand coordination bond, where the ligand comprises an active region with at least one group selected from - CO ₂ H, -PO ₃ H, -P0 ₄ H, -SO ₃ H and -S0 ₄ H; and where the active ingredient is linked to the active region of the ligands by ionic bonds. The embodiments described for the first aspect of the invention are also preferred for the present aspect. 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 and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES Figure 1 shows the mean tumor volume (mm ³ ) after one week of intravenous treatment. The magnetic field was applied by subcutaneous implantation of magnets on the right flank of the mice.

Figure 2 shows the variation of the mean tumor volume (mm ³ ) over time during the application of different intravenous treatments. Treatment with nanoparticles (np) and interferon-gamma (ifn) together with the external application of a magnetic field causes a decrease in tumor size. Figure 3 shows the average levels of interferon gamma in the serum of mice that have received different intravenous treatments for two weeks The level of interferon-gamma in blood is lower when administered together with nanoparticles and a magnetic field is applied.

Figure 4 shows the average levels of interferon-gamma in the tumor environment in mice that received different intravenous treatments for two weeks. The application of the magnetic field produced the accumulation of interferon-gamma in the tumor area.

Figure 5 represents the mass fraction that is a measure of the nanoparticles accumulated in each organ with respect to the result obtained in control organs from mice that received injections of PBS. The nanoparticle solution was administered intravenously (i.v.) or subcutaneously (s.c.) It is observed that administration subcutaneously does not provide an adequate distribution of nanoparticles in the organs where they usually accumulate {spleen, liver and kidney).

Figure 6 shows the quantitative analysis of the amount in grams of iron oxide nanoparticles accumulated in the different organs obtained from the saturation magnetization values of the organs with respect to the magnetization value of the isolated magnetic particles.

Figure 7 shows the tumors extracted from mice to which different amounts of nanoparticles were administered intravenously, and on which an external magnetic field of 0.2 Tesla was stained with Prussian blue, marking the presence of iron in the tissue. The presence of nanoparticles is greater when the largest amount of nanoparticles is administered.

Figure 8 shows the tumors extracted from mice to which 300 µg of nanoparticles were administered intravenously, and on which an external magnetic field of 0.2 or 0.6 Teslas was stained with Prussian blue, which marks the presence of iron in the tissue. The presence of nanoparticles is greater at greater intensity of applied magnetic field.

EXAMPLES

A. - Preparation of conjugates with interferon-gamma.

The synthesis of nanoparticles and the formation of conjugates composed of magnetic and interferon-gamma nanoparticles was carried out as described in Mejías et al, J Control Relay, 130: 168-74 (2008).

B. - Cell cultures.

The murine Pan02 cell line was cultured in DMEM medium supplemented with 10% FCS (fetal bovine serum), L-Glutamine and non-essential amino acids, and to which a mixture of penicillin and streptomycin was added, under standard culture conditions (37 ^Q C, 5% C0 ₂ and 90% relative humidity). Pan02 cells were washed with PBS and resuspended in the same buffer at a concentration of approximately 45x10 ⁶ cells / ml.

Pan02 cells prepared in this way were subcutaneously transplanted into the right flank of 12-week-old C57BL76 mice, both female and male, so that each of them received 100 µΙ of the cellular solution. The animals were raised regularly for a week, at which time the developing tumor becomes visible.

C- Treatment.

The treatment was carried out twice a week for two weeks. The animals were divided into 5 treatment groups: treatment with the composition with and without application of magnetic field (dose between 0.9x10 ^"3 and 1, 2x10 ^{" 3} mg of conjugated interferon-gamma per animal), treatment with interferon-gamma (0.9x10 ^"3 and 1, 2x10 ^{" 3} mg of interferon-gamma per animal), treatment with nanoparticles (300 μg per animal) and control treatment with PBS. All treatments are applied in a final volume of 100 μΙ_ per animal. In each treatment session the animals were anesthetized by intraperitoneal administration of a mixture of ketamine and xylazine (2.1 μΙ_ Imalgene 500 + 0.7μΙ_ Xilagesic / g weight). After the application of anesthesia, the tumor size of each animal was measured with a caliber and the corresponding treatment was administered intravenously. After administration of the treatment, a magnetic field of between 0.2 and 0.6 Tesla was applied over the tumor region for one hour. After two weeks the animals were sacrificed and the tumors were removed for analysis.

D. - Calculation of tumor volume. In each treatment session the major (y) and minor (x) diameters of the tumors were measured, and the tumor volume was calculated by the formula:

Vol = yπx ²

 E. - Specific iron staining.

After tumor extraction, they were fixed in 4% paraformaldehyde in PBS for 16 hours, and included in paraffin blocks. Serial cuts of 7μηι were made that were stained with prussian blue (potassium hexacyanoferrate (II)) (Sigma) following the manufacturer's instructions. This staining marks in blue the areas of the tissue where there is iron, so that the presence of nanoparticles can be determined.

F. - Measurement of interferon-gamma levels in the tumor environment. Some of the tumors removed were cut into small fragments and plated on multi-cycle plates (24 well). 1 mL of RPMI-1640 medium supplemented with 10% FCS per well was added and incubated for 24 hours under standard culture conditions. After this period the medium was collected and the amount of interferon-gamma present was measured in triplicate by ELISA (Mouse IFN-γ ELISA Set, BD biosciences). G.- Effect of treatment on tumor development in vivo.

The treatment with the antitumor composition developed in the present invention proved to produce a decrease in tumor volume when an external magnetic field was applied over the area of interest. This effect is not observed when the control treatment is applied, the treatment with the composition without a magnetic field, or when a treatment with interferon-gamma or nanoparticles is applied separately. The following table shows the average tumor volumes at the beginning and at the end of the different treatments:

H.- Presence of nanoparticles in the area of interest.

Prussian blue staining of the sections obtained from tumors undergoing the different treatments indicated that the nanoparticles reached the area of interest when a magnetic field was applied over the area. In the case of treatment with conjugates without the application of a magnetic field, the presence of nanoparticles is clearly lower, while tumors undergoing control and interferon-gamma treatments separately did not present nanoparticles. These results are compatible with those obtained in other previous studies of nanoparticle biodistribution using magnetic fields [Wu et al, J Magn Magn Mat, 31 1: 372-5 (2007)]. F.- Interferon-gamma levels in the tumor environment.

The level of interferon-gamma after treatment with conjugates and application of a magnetic field turned out to be much higher than after application of conjugates without magnetic field or interferon separately, whereas after control treatment or treatment with nanoparticles separately the levels Interferon-gamma are virtually undetectable.

Interferon-gamma level (pg / mL)

Treatment

 SD measure

 Control (PBS) 0 0

Nanoparticles (300 ₍ ug) 15.44 2.1

 Interferon-gamma (1 μg) 176.57 56.86

 - field 203.99 66.98

 Conjugate

 + field 620.62 189.87

Claims

1. - The use of a pharmaceutical composition comprising: a conjugate comprising a magnetic nanoparticle and at least one active ingredient for the preparation of a medicament; characterized in that: the core of the magnetic nanoparticle comprises a magnetic iron oxide; The nanoparticle comprises on its surface ligands bound thereto by ionic bond, covalent bond, hydrogen bridge, hydrophobic bond, electrostatic bond and / or metal ligand coordination bond, where the ligand comprises an active region with at least one group selected from - CO ₂ H, -PO ₃ H, -PO ₄ H, -SO ₃ H and -SO ₄ H; and where the active ingredient is linked to the active region of the ligands by ionic bonds. 2. - The use according to the preceding claim, wherein the active region of the ligand comprises at least one group selected from -CO ₂ H, -P0 ₄ H - and -S0 ₄ H. 3. - The use according to the preceding claim, wherein the active region of the ligand comprises -CO ₂ H. 4. The use according to any of the preceding claims, characterized in that the ligand comprises an adhesive region which is linked to the surface of the nanoparticle. 5. - The use according to the preceding claim, characterized in that the adhesive region comprises at least one group selected from -C0 ₂ H, -NH ₂ , -SH, -CONH ₂ , -OH, -PO ₃ H, -PO ₄ H, -SO ₃ H and -SO ₄ H. 6. - The use according to the preceding claim, characterized in that the adhesive region comprises at least one group selected from -CO ₂ H, -OH, - PO ₄ H and -SO ₄ H. 7. - The use according to the preceding claim, characterized in that the region adhesive comprises -CO ₂ H. 8. - The use according to any of the preceding claims, characterized in that the ligand, between the adhesive region and the active region, comprises a hydrocarbon chain of between 1 and 20 carbon atoms. 9. - The use according to the preceding claim, characterized in that the hydrocarbon chain comprises between 1 and 10 carbon atoms. 10. The use according to the preceding claim, characterized in that the hydrocarbon chain comprises is of the formula -C _n H _2n -, n being an integer between 1 and 10, the chain being optionally substituted. The use according to any of the three preceding claims, characterized in that the hydrocarbon chain comprises at least one group selected from ether, amido, amino, thioether, alkene, alkyne, C3-C6 cycloalkane and / or phenyl. 12. - The use according to any of the four preceding claims, characterized in that the hydrocarbon chain is substituted with at least one group selected from -SH, -OH, -CO ₂ H, -NH ₂ , -SH, -CONH ₂ , - OH, - PO ₃ H, -PO ₄ H, -SO ₃ H, -SO ₄ H, F, Cl, Br and I. 13. - The use according to the preceding claim, characterized in that the hydrocarbon chain is substituted with at least one -SH group. 14. - The use according to any of the preceding claims, characterized in that the nanoparticle comprises at least one ligand selected from dimethylmercaptosuccinic acid, dimercaptomaleic acid and dimercaptopentadionic acid. 15. - The use according to any of the preceding claims, characterized because it comprises at least dimethylmercaptosuccinic acid. 16. - The use according to any of the preceding claims, characterized in that the magnetic nanoparticle is obtainable by the decomposition of ferric acetylacetonate in solution at a temperature between 235 and 290 ^Q C in ether in the presence of a fatty acid. 17. - The use according to the preceding claim, characterized in that the magnetic nanoparticle is obtainable by the decomposition of ferric acetylacetonate in solution at a temperature between 250 and 270 ^e C. 18. The use according to any of the two preceding claims, characterized in that the decomposition of ferric acetylacetonate is carried out in the presence of an organic solvent, or mixtures thereof, of boiling temperature greater than 235-C. 19. The use according to any of the three preceding claims, characterized in that the decomposition of ferric acetylacetonate is carried out in the presence of biphenyl ether, benzyl ether, octyl ether, octadecene, trioctylamine or ethylene glycol. 20. - The use according to any of the four preceding claims, characterized in that the fatty acid is oleic acid. 21. The use according to any of the five preceding claims, characterized in that the union between the ligand and the magnetic nanoparticle is carried out by a process of ligand exchange. 22. The use according to any of the preceding claims, characterized in that the magnetic nanoparticle has at least an average particle size greater than 6 nm. 23. The use according to the preceding claim, characterized in that the magnetic nanoparticle has at least an average particle size between 8 and 11 nm. 24. The use according to any of the preceding claims, characterized in that the conjugate has an average hydrodynamic particle size of less than 200 nm. 25. - The use according to the preceding claim, characterized in that the conjugate has an average hydrodynamic particle size between 40 and 150 nm. 26. - The use according to the preceding claim, characterized in that the conjugate has an average hydrodynamic particle size between 85 and 115 nm. 27. The use according to any of the preceding claims, characterized in that the active ingredient has at least a positive charge at a pH between 2 and 8. 28. - The use according to any of the preceding claims, characterized in that the active ingredient has at least a positive charge at a pH between 5.5 and 7.5. 29. - The use according to any of the preceding claims, characterized in that the active ingredient is antineoplastic or immunomodulator. 30. The use according to any of the preceding claims, characterized in that the active ingredient is a protein. 31. - The use according to any of the preceding claims, characterized in that the active ingredient is interferon. 32. - The use according to the preceding claim, characterized in that the interferon is selected from interferon alpha, beta, omega, epsilon, kappa, gamma, lambda and any of its combinations. 33. - The use according to the preceding claim, characterized in that the interferon is gamma interferon. 34. - The use according to any of the preceding claims, characterized in that the conjugate comprises at least 1x10 ³ mg of active ingredient per mg of conjugate. 35. The use according to any of the preceding claims, characterized in that the conjugate comprises at least 10x10 ^"3 mg of active ingredient per mg of conjugate. 36. The use according to any of the preceding claims, characterized in that the conjugate comprises between 2x10 ^"3 mg and 6x10 ^{" 3} mg of active ingredient per mg of conjugate. 37. - The use according to any of the preceding claims, characterized in that the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient or vehicle. 38. - The use according to any of the preceding claims, characterized in that the conjugate is dissolved or dispersed in an aqueous solution. 39. The use according to any of the preceding claims for the preparation of an intravenous composition. 40. - The use according to any of the preceding claims for the preparation of a composition for the treatment of cancer. 41. - The use according to any of the preceding claims for the preparation of a composition for the treatment of breast cancer, colon, lung, uterus, prostate, pancreas, melanoma and / or bladder. 42.- An assembly comprising a pharmaceutical composition and a magnet, characterized in that the pharmaceutical composition comprises a conjugate comprising a magnetic nanoparticle and at least one active principle for the preparation of a medicament, characterized in that: the core of the magnetic nanoparticle comprises a magnetic iron oxide; The nanoparticle comprises on its surface ligands bound thereto by ionic bond, covalent bond, hydrogen bridge, hydrophobic bond and / or metal ligand coordination bond, where the ligand comprises an active region comprising at least one group selected from -CO ₂ H, -PO ₃ H, - P0 ₄ H, -SO ₃ H and -SO ₄ H; and where the active ingredient is linked to the active region of ligands through ionic bonds. 43.- The assembly according to the preceding claim, characterized in that the magnet has at least a power of 0.05 Teslas. 44. - The assembly according to the preceding claim, characterized in that the magnet has at least a power of 0.1 Teslas. 45. - The assembly according to the preceding claim, characterized in that the magnet has at least a power of 0.2 to 0.45 Teslas. 46. - The assembly according to any of the previous four claims, characterized in that the magnet is selected from a ferrite, neodynium and / or an electromagnet magnet. 47. - The assembly according to the preceding claim, characterized in that the magnet is a neodymium disc with a diameter between 3 and 7 mm and a thickness between 1, 5 and 5 mm. 48.- The assembly according to any of the previous six claims for Its use in medicine. 49.- A pharmaceutical composition comprising: a conjugate comprising a magnetic nanoparticle and at least one active ingredient; characterized in that: the core of the magnetic nanoparticle comprises a magnetic iron oxide; The nanoparticle comprises on its surface ligands bound thereto by ionic bond, covalent bond, hydrogen bridge, hydrophobic bond, electrostatic bond and / or metal ligand coordination bond, where the ligand comprises an active region with at least one group selected from - C0 ₂ H, -P0 ₃ H, -P0 ₄ H, -S0 ₃ H and -S0 ₄ H; and where the active ingredient is linked to the active region of ligands through ionic bonds.