FR2930890A1 - Submicron particles, useful for the fabrication of a medicament for the treatment and/or prevention of tumor diseases such as cancer, and proliferative diseases such as arthritis, comprise an oxide and/or oxohydroxide of a rare earth - Google Patents

Abstract

Submicron particles having a size of 1-800 nm, comprise at least 30%, preferably at least 90 mass% of an oxide and/or oxohydroxide of a rare earth, where total rare earths are responsible for a specific radioactive activity of at least 0.5 MBq per gram of particles and preferably at least 5, due to disintegration of the rare earth element, at least 10% of the disintegrations producing an emission of radiations. Independent claims are included for: (1) a pharmaceutical composition in an injectable form comprising particles in combination with excipient; and (2) a process for preparing particles or a composition comprising preparing non-radioactive particles of which the composition and the structure are defined and activating the particles by a neutron flux to obtain the desired radioactivity. ACTIVITY : Cytostatic; Antiarthritic. MECHANISM OF ACTION : None given.

Description

The invention relates to novel targeted radiotherapy or brachytherapy agents. More precisely, the invention relates to submicron particles with a high concentration of rare earth oxide or oxohydroxide, the so-called rare earths having a radioactive activity. These particles behave like radionuclides and are used in metabolic radiotherapy or brachytherapy, or more generally in the field of therapy and diagnosis. Radionuclides are commonly used in various fields in biology, but also as markers or tracers for the purpose of characterization of media or as therapeutic agents. The therapeutic effect of radionuclides or radioelements used in medicine is based on the effect strongly ionizing particulate radiation 13- and emitted by radionuclides, resulting in a cascade of cytotoxic reactions that ultimately lead to cell death (radiobiological effects). This therapeutic effect has been used for many decades in adjuvant treatment of malignant tumors, but also in the treatment of non-cancerous diseases. We distinguish between targeted radiotherapy defined as the use of radioactive molecules with the double property of being ionizing and being able to target, after parenteral injection, a target tissue, and brachytherapy. Brachytherapy is an irradiation technique that involves introducing radioactive sources into or within the tumor. These source devices are usually in the form of rods a few millimeters long and a millimeter in diameter deposited within the target to be treated. They can also consist of nano or micrometric structures. The elements are deposited within the target tissue, and the therapeutic efficacy is related to the homogeneous distribution within the entire target area, difficult to obtain. The use of molecular elements makes it possible to obtain a very good dispersion of these, but with diffusion also outside the area to be treated, too fast, with a risk of reduced therapeutic efficacy in the area of interest and systemic or locoregional side effects (radiobiological effects in healthy tissues). Targeted radiotherapy relies on concentrating the radioactive molecules essentially in the target tissue to limit the ionizing effect to target cells in order to achieve a therapeutic effect with limited side effects (no radiobiological effect on healthy cells). It is therefore necessary that the radiotoxic element is targeted towards the target tissue. This targeting can be achieved: - by a particular tropism of certain elements, such as strontium chloride 89, a calcium analogue, or analogs of samarium 153-labeled bi-phosphonates for the bone matrix used for the palliative treatment of bone metastases; or the specific internalization of iodine by the thyroid cells allowing the treatment of hyperthyroidism and the adjuvant treatment of thyroid remains by iodine-131 in differentiated thyroid cancers or hyperthyroidism - by coupling the ionizing therapeutic element to a tumor cell recognition molecule, such as anti-bodies (treatment of lung or medullary thyroid cancers with anti-ACE antibodies labeled with iodine-131, treatment of lymphomas with radiolabelled anti-bodies); [90] Y-labeled CD.) Coupling can also be done with peptides (example of the use of [90] Y or [177] Lu-labeled octreotide / lanreotide, used in the treatment of gastroenteropancreatic neuroendocrine tumors (GEP) and paragangliomas) or with polysaccharides or other complex molecules (example of the use of a radiolabelled guanidine analogue, the [131] I-MIBG for the treatment of metastatic pheochromocytomas, paragangliomas and neuroblastomas). The encapsulation of the radio-element can allow, after injection by arterial catheterization of a seat tissue, a tumor, to concentrate within it the therapeutic element by limiting the extra-tumoral diffusion and the radiobiological effect on the healthy tissues. (example of treatment of hepatic metastases by injection of [131] I-lipiodol).

Apart from radioactive lipiodol treatment of hepatic metastases and thyroid cancers with radioactive iodine, metabolic radiotherapy is currently limited to palliative treatments, either because of their low efficacy or because of insufficient targeting of tumor cells ( which generates significant side effects). We have already described in some documents of the literature charged particles in rare earth and in particular in holmium. - "Characterization of holmium loaded alginate microspheres for multimodality imaging and therapeutic." J. Biomedical Materials Research A 10 (2007) 82, 892. - "Removal of chloroform from biodegradable therapeutic microspheres by radiolysis" International Journal of Pharmaceutics (2006) 315, 67. which discloses polylactates loaded with Ho166. The present invention relates to microspheres containing microspheres as a reservoir of holmium-loaded fluids. 2004) 26, 925. - "Radioapharmaceuticals and radioactive microspheres for locoregional ablation of abnormal tissues" US2004131543 which describes particles of diameter greater than 1 micron .- "Supramolecular aggregates containing chelating agents and bioactive peptides for delivery of antitumor drugs and contrast agents in MRI or nuclear medicine "WO 2006/128643 which discloses aggregates of polymers -" Distribution of radiation in synovectomy of the knee with 166Ho-FHMA using image fusion "Cancer Biotherapy & Radiopharmaceuticals (2005) 20, 333 which discloses aggregates of All the particles described in these documents are both lightly loaded with holmium and size around the micron. They therefore have a non-optimized radioactive effect (at least in metabolic radiotherapy or brachytherapy) since either the charge in radioactive element is too low or the size of the micrometric particle causes the absorption within the particle of the radiation. 13- issued. In this context, the subject of the present invention is submicron particles that can be used in therapeutic or diagnostic procedures, characterized in that: they have a size of between 1 and 800 nm, they comprise at least 30%, preferably at least 50%, more preferably at least 90%, by weight of at least one oxide and / or an oxohydroxide of at least one rare earth, all the rare earths are responsible for a specific radioactive activity of at least 0.5 MBq per gram of particles, due to a disintegration of the rare earth element (s), at least 10% of these decays producing an R-type emission. The particles according to the invention have certain advantages in relation to to molecular radionuclides used in therapy. In brachytherapy, the inertia of the particles should limit the extra-tumoral diffusion of the radioactive elements that could be assimilated to intra-medical devices. In metabolic radiotherapy, the number of radioactivated atoms (and not radioactivatable given that the activation is done before the injection) per vector is high and the particle can have contrast properties (magnetic, optical) that make it non only detectable in scintigraphy but also in MRI and optical imaging. The present invention also relates to injectable pharmaceutical compositions of these particles, their use for the manufacture of a medicament for the treatment and / or prevention of tumor diseases, proliferative diseases, as well as their method of preparation. In this regard, two types of particles are of particular interest: particles of size less than 20 nm consisting of a holmium oxide core, in particular of between 1 and 10 nm in size, and coated with a layer of polysiloxane functionalized, in particular with a thickness of 0.1 to 5 nm, ensuring their colloidal stability. Even if the proportion by weight of holmium is reduced by the presence of the polysiloxane, this layer is sufficiently fine to stop only slightly ionizing radiation. particles of submicron size between 50 and 800 nm composed almost exclusively of holmium oxide. Compared with the developed microparticles described in the literature, these particles have a higher Ho load, in particular of the order of 90% by mass.

Various other characteristics appear from the description given below with reference to the appended figures which show, by way of non-limiting examples, embodiments of the object of the invention. Figure 1. represents the particle size distribution obtained by PCS (Photon Correlation Spectroscopy) of the holmium oxide particles obtained in Example 1 below after functionalization with DTPABA, at the different stages of their preparation. Figure 2 demonstrates the radioactive character of the particles of Figure 1 after neutron activation. FIGS. 3 and 4 show in-vivo scintigraphy images of the particles of FIG. 1. FIG. 5a shows a scanning electron microscope view of the particles obtained in example 3. FIG. 5b represents a view by electron microscopy of FIG. scanning the particles obtained in Example 4.

Figure 6 demonstrates the radioactive character of the particles of Figure 5a after neutron activation and their in-vivo detectability by gamma scintigraphy. It presents the gamma scintigraphy image obtained, after injection, into the thigh of a rat, of particles presented in Figure 5a.

Figures 7, 8 and 9 show images obtained by in vivo optical imaging acquired by a CDD camera.

FIG. 9 represents the in vivo optical images acquired successively for 4 days in the rat presented in FIG. 8. FIG. 10 shows the MRI images (echo sequence of spin TR = 1000 msec / TE = 50 msec, 7 T) of aqueous samples containing increasing concentrations of colloidal particles of the core / coating type. Figure 11 is an in vivo MRI image obtained after intravenous injection. Figure 12A shows the image acquired in optics by superimposing the image in fluorescence with excitation: 495 nm / emission: 518 nm to the bright field image and Figure 12B represents the image in MRI (echo sequence of spin TR = 1000 msec / TE = 50 msec, 7T), of the two xenografts ex-vivo taken from the same rat. Figure 13 shows the in vivo and ex vivo images acquired by a CDD camera with an excitation: 495 nm / emission: 518 nm and highlights the biodistribution after intratumoral injection of nanoscale colloids. The particles used according to the invention have a size of between 1 and 800 nm. They may consist exclusively of a core comprising at least one oxide or oxohydroxide of at least one rare earth, or. a mixture of oxides, a mixture of oxohydroxides or a mixture of oxide and oxohydroxide of at least one rare earth or may be in the form of a core surrounded by a coating. In this case, the core comprises at least one oxide or oxohydroxide of at least one rare earth, or a mixture of oxides, oxohydroxides or oxide and oxohydroxide of at least one rare earth. According to an alternative embodiment, the particles consist exclusively of a rare earth oxide, a mixed rare earth oxide (that is to say at least two rare earths), an oxohydroxide a rare earth, a rare earth oxohydroxide (that is to say at least two rare earths) or a mixture thereof. Similarly, in the case where the particles according to the invention are in a coated form, according to an alternative embodiment, the core of the particles consists exclusively of an oxide of a rare earth, a mixed oxide of earth rare (ie at least two rare earths), rare earth oxohydroxide, mixed rare earth oxohydroxide (ie at least two rare) or one of their mixtures. Of course, in the particles according to the invention, rare earth elements are in radioactive form, since they have a specific radioactive activity. The specific radioactive activity is defined as the number of decays per second, it is expressed as Becquerel (Bq) or MBq and per unit mass of particles. To distinguish the activity due to rare earths or lanthanides from the activity due to other possible elements, it is sufficient to evaluate the type and energy of ionizing radiation from colloids. From these data, it is possible to go back to the type of decays and thus clearly identify the part resulting from the disintegration of the rare earths or lanthanides. By way of example, the rare earth or rare earths are chosen from holmium 166, cerium 141, samarium 153, europium 155, erbium 169, thulium 170, ytterbium 169, dysprosium 165, lutetium 177, gadolinium 159. The advantage of using rare earths is that after activation, elements become emitters of highly energetic and highly ionizing radiation. The strongly ionizing character is due to the emission by the radioactive element of an E3 radiation which is responsible for the direct therapeutic effect (the ionizing effect occurring within the tumor cells) or indirect effect (the effect occurring at the level of the supporting cells such as the endothelial cells of the neovessels). Some elements (strontium 89, yttrium 90) are already used in targeted radiotherapy or brachytherapy. In the context of the invention, the rare earth or rare earths are preferably chosen from lanthanides. Some lanthanides, and particularly the holmium, have the advantage, compared to strontium 89 and yttrium 90, that once activated, they have both a favorable radioactive half-life, a very high emission energy and a gamma emission allowing a detection of sites of accumulation by scintigraphy. It is therefore possible to verify by scintigraphy the location of the particles.

More generally, the choice of the radioelement is made on several criteria: it must have an optimal radioactive period (neither too long to limit the problems of radioprotection, nor too short not to limit the therapeutic effect), and a emission energy 13 - high (a few hundred keV minimum) and if there is a related emission, it must be low energy, to limit the problems of radiation protection. For example, an element having a period of 1 to a few days, a 13- emission of the order of 500 to 1500 keV and an emission y less than 150 keV is a good candidate. The other important element is that the radioactive element which would diffuse out of the volume to be targeted after local injection, or would not be targeted after intratumoral injection, is rapidly eliminated, urinary, without any non-specific sequestration, especially in the system. reticuloendothelial. The rare earth family, as shown by the following examples made with particles according to the invention, made it possible to achieve such an objective. In the context of the invention, all the rare earths present within the particles have a specific radioactive activity of at least 0.5 MBq per gram of particles, due to a disintegration of the rare earth elements, at least 10% of these decays producing an emission of R- radiation. Advantageously, all the rare earths have a specific radioactive activity of at least 5 MBq per gram of particles. The decays produce, for at least 10%, an emission of radiation R The radioactivity of the rare earths may correspond to the global radioactivity of the particle, unless this radioactivity is weakly absorbed by a coating surrounding the core of the particle-based oxide or oxohydroxide rare earth. Also, advantageously, when the particles according to the invention comprise a core containing the oxide or oxides and / or oxohydroxides of rare earth and a coating consisting of at least one organic and / or inorganic component, this coating stops at most 25. % of radiation 13 "emitted by the heart.

The coating, when present, consists of at least one organic and / or inorganic constituent. As will come out of the. description and examples which follow, this coating, when present, is advantageously constituted by a polysiloxane (which constitutes an inorganic constituent) but may also comprise organic molecules either directly attached to the surface of the core, or grafted onto a inorganic coating, even being included within this inorganic coating. The particles used according to the invention can be solely, as previously stated, constituted by oxides and / or oxohydroxides of rare earth, and in particular lanthanide. The rare earth group consists of the group of lanthanides (atomic elements between 57 and 71, lanthanum or lutetium) to which are added yttrium and scandium. They can also be either in purely inorganic form and include in this case a core consisting of rare earth oxides and / or oxohydroxides, and in particular of lanthanide and coated with an inorganic coating. They may also be in the form of inorganic and organic hybrid particles and consist of a core of oxides and / or oxohydroxides of at least one rare earth, and in particular a lanthanide and an organic and / or inorganic coating. In addition to oxides and / or oxohydroxides of rare earth, the core may comprise other compounds: the core may be in the form of a ceramic composite, metal, or include halides, phosphates, vanadates, silicates ... or organic compounds, fluorescent compound type, magnetic, opacifier, drug liberator, targeting molecules. Such compounds may in particular be present in the core when there is no coating. The hydrodynamic size of the particles is measured in laser granulometry by photon correlation spectroscopy (Photon Correlation Spectroscopy). In the case where the particle is composite, the size of each successive layer is measured by this technique after each step of the development. The geometric size of each particle and its various constituents is measured by transmission electron microscopy. It corresponds to an average diameter in the case where the particles are not spherical. A particle population is characterized by a particle size distribution, which distribution is itself characterized by a mean size and a standard deviation. In the context of the invention, when it is indicated that the size of the particles is between 1 and 800 nm, it means that at least one of these two sizes, average hydrodynamic size or geometric mean size is included in this range. The particles are preferably of core-embedding shape (also called core-shell) and, preferably, substantially spherical. More generally, the rare earth oxide or oxohydroxide core may be spherical, faceted (the dense planes then forming the interfaces) or elongated. In the same way, the final particle is spherical, faceted or elongated. The core, or the particle itself when there is no coating, is advantageously between 1 and 800 nm.

The submicron particles according to the invention are doubly advantageous alternatives to the particles currently used, since they contain a mass percentage of rare earths very high and they are architected for a maximum therapeutic effect, the rare earth may be place in the immediate vicinity of the area to be treated. In particular, the particles below are particularly preferred: submicron particles comprising a core based on at least one rare earth oxide and / or oxohydroxide, in particular lanthanide, preferably of holmium or lutetium, and which may have an inorganic or organic coating not exceeding 5% by weight of the particle, of total size (with the coating) in the range from 50 to 800 nm, nanoscale particles comprising a core based on at least an oxide and / or oxohydroxide of rare earth, especially of lanthanide, preferably of holmium or lutetium and which can have an inorganic or organic coating not exceeding 50% by mass of the particle, of total size (with the coating ) in the range of 1 to 50 nm. According to one embodiment of the invention, it is advantageous to use particles of small dimensions, and in particular in colloidal form, preferably with a size in the range from 1 to 20 nm, preferably in the range from 1 to 10. nm. Indeed, small particles generally have better colloidal stability and are more suitable for injections.

Their small size and high stability allow better diffusion: from the vascular compartment to the target cells, through an unrestricted capillary barrier (due to their small size) and high diffusion through the interstitial tissues to cells, after intravascular injection. the tissue diffusion of small particles is high, responsible for a greater tissue retention after passage of the capillary barrier. As tumors are most often hypervascularized (compared to healthy tissues), even in the absence of specific functionalization of the particles, tissue uptake is higher in tumors as evidenced by the MRI studies performed. Compared with molecules for which elimination can also be done by the kidneys, the small particles have a longer residence time inside the body and allow a preferential accumulation in the areas of interest.

The small size of the particles allows urinary elimination of the particles (unlike larger particles), allowing the rapid elimination of the fraction of particles not captured at the level of the tumor tissues. It limits non-specific uptake by cells of the reticuloendothelial system. This very favorable biodistribution has been confirmed by MRI studies. The small particle size allows: - a long residence time and increased permeation in the damaged areas (unlike molecules) - facilitated renal elimination (compared to larger particles) - increased ionizing treatment efficiency (by compared to larger particles) - less nonspecific uptake by cells of the reticuloendothelial system (compared to larger particles) to reduce the occurrence of side effects by ionization of healthy tissues such as the liver. For all the reasons explained above, it is preferable to use, according to a variant of the present invention, particles having a size of less than or equal to 20 nm and preferably less than or equal to 10 nm, in the case of particles exclusively consisting of at least one oxide and / or an oxohydroxide of at least one rare earth or having a core smaller than 20 nm, and preferably less than 10 nm, when it is a particle in the form of heart and coating.

According to a particularly advantageous variant of the invention, the part of the particle containing the oxides and / or oxohydroxides will comprise at least two different rare earths, each representing more than 10% by weight of all the rare earths and, preferably, more than 20%. This makes it possible to combine detectability by MRI and metabolic therapy or to include two radionuclides with different radioactive properties (half-lives, energies of particulate and complementary gamma rays).

In such a case where several different rare earths will be used, the oxides and / or oxohydroxides of these different rare earths may be either in successive layers or in the form of a solid solution. The solid solution is easier to achieve from the point of view of synthesis. The choice of rare earths, all of which have very similar chemistries, makes it easy to produce adaptable solid solutions. Moreover, a multilayer structure can be interesting if one wants to increase the contrast in magnetic resonance imaging (by putting lanthanides with high contrast on the surface). Indeed, as is well known, a number of lanthanides give signals in magnetic resonance imaging (MRI), which allows, provided you choose the proportions and nature of the lanthanides to cause during therapy a signal in MRI, allowing in vivo detection of the presence of particles and / or monitoring of therapy. Thus, the rare earth (s) will contain, advantageously, at least 50% by mass of lanthanide causing an MRI signal, allowing an in vivo detection of the presence of said particles, and preferably at least 50% by weight of gadolinium ( Gd), dysprosium (Dy), holmium (Ho) or mixtures of these lanthanides. The combination of a therapeutic effect and detectability properties by in vivo imaging is a major asset of such therapeutic agents. In fact, the in vivo imaging will make it possible to quantify tumor tissue uptake of the particles, to establish the kinetics of uptake to know the delay after injection where it is maximal in the tumor. This knowledge makes it possible to optimally determine the delay in which the external irradiation must be carried out, in order to obtain maximum effect (maximum radio-sensitizing effect). The external detection by imaging of these particles makes it possible to follow and adapt the therapy. These particles are responsible for a positive or negative modification of the MRI contrast that can possibly be quantified. Magnetic Resonance Imaging provides high resolution images.

According to one variant of the invention which is of particular interest when the radiotherapy process is coupled with MRI monitoring, particles are used in which the part containing oxides and / or oxohydroxides of rare earth contains at its periphery lanthanides causing a MRI signal, preferably gadolinium, and at least one other rare earth in its central part. These particles may, in particular, be: - either in the form of particles consisting exclusively of at least one oxide and / or one oxohydroxide at least one rare earth, which contains on its periphery lanthanides which give rise to an MRI signal, preferably gadolinium, and at least one other rare earth in its central part - either in the form of a heart consisting of at least an oxide and / or an oxohydroxide of at least one rare earth, and surrounded by a coating consisting of at least one organic and / or inorganic constituent, the core containing at least one oxide and / or one ox The at least one rare earth hydroxide contains at its periphery lanthanides causing an MRI signal, preferably gadolinium, and at least one other rare earth in its central portion. Indeed, the contrast in MRI being done by an action of the paramagnetic ion in the perimeter of its first coordination layers, it is preferable to put the lanthanides which will act as contrast agents on the surface of the heart. The rare earths of high atomic number emitting strong ionizing radiation will then preferentially be located in the center of this heart. As explained above, the particles used according to the invention are advantageously in the form of a core and a coating. This coating may be either inorganic, organic and inorganic, or purely organic. According to a variant of the invention, in the case where the particles have a coating, it is advantageous for said rare earth oxide (s) and / or oxohydroxides to represent at least 30% by weight of all the inorganic constituents of said particle, the organic constituents being covalently bound to the hybrid particle and representing less than 30% by weight of the final particle. Note that when we speak of "all inorganic constituents", we include in this set oxides and oxohydroxides of rare earth. In general, the function of the coating is multiple. This is on the one hand to protect the heart of an untimely dissolution and on the other hand to easily adapt the stability of the particles to the injection system (avoid uncontrolled agglomeration) because of the numerous surfactants it is possible to fix on its surface. Finally, another function of the coating is to adapt fillers and surface chemistry to improve biodistribution and promote local overconcentrations in tumor areas. For this purpose, the polysiloxane constitutes a particularly preferred coating according to the invention. According to a variant of the invention, the particles comprise a core in the form of at least one oxide and / or an oxohydroxide of at least one rare earth and a polysiloxane coating representing 1 to 70% by weight of the inorganic constituents of the particles. . Preferably, the ratio number of silicon atoms of the coating with polysiloxane / number of rare earth atoms is between 0.1 and 10. The choice of this ratio makes it possible to effectively protect the core of the particle and / or adapt its colloidal stabilization and / or adapt its magnetic interaction with water and therefore its characteristics of contrast agents.

According to a variant of the invention, a coating consisting of polysiloxane, on which the molecules are fixed, particularly preferably, hydrophilic organic molecules, will be chosen. Advantageously, the particles furthermore comprise hydrophilic organic molecules with a molar mass of less than 5000 g / mol and preferably less than 450 g / mol, preferably chosen from organic molecules comprising alcohols and carboxylic acids functions. amines, amides, esters, ether-oxides, sulfonates, phosphonates or phosphinates, covalently bound to at least 5% of the silicon atoms of the polysiloxane coating. These organic molecules advantageously comprise the following units given below with their preferred molar mass (Mw): poly (ethylene glycol) bis (carboxymethyl) ether (PEG), 250 <Mw <5000 gmol-1 - Polyoxyethylene bis (amine ), 250 <Mw <5000 gmol-1-O-Methyl-O-succinylpolyethyleneglycol, Mw of the order of 2000 g.mol -1 Methoxypolyethyleneglycolamine (Mw of the order of 750 g mol -1) - Diethylenetriaminepentaacetic acid (DTPA) and its derivatives, in particular DTPA dithiole (DTDTPA) - 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (DOTA) and derivatives - succinic and mercaptosuccinic acid - Glucose and derivatives, for example dextran - amino acids or hydrophilic peptides (aspartic acid, glutamic acid, lysine, cysteine, serine, threonine, glycine, etc.) More generally, the organic molecules will advantageously comprise alcohol or carboxylic acid functions or amines or amides or esters or ether-oxides or sulfonates or phosphonates or phosphinates and will preferably be covalently bound to at least 10% of the silicon atoms of said polysiloxane. Organic molecules comprising a polyethylene glycol, DTPA, DTDTPA (dithiole DTPA) or succinic acid unit will preferably be selected. Among the above organic molecules, those which have rare earth complexing properties, in particular those whose complexation constant is greater than 1015, will advantageously be chosen. For this purpose, DTPA or DOTA will preferably be chosen. Furthermore, according to an alternative embodiment, the particles according to the invention carry targeting molecules, in particular molecules which have at least one recognition site enabling them to react with a ligand of biological interest. According to a particular embodiment, the particles according to the invention comprise a core and a surface coating, in which one or more targeting molecules are coupled. Examples of target molecules include proteins, peptides, antibodies, lipids, carbohydrates and nucleic acids. As particularly preferred target molecules, mention may be made of anti-bodies targeting at least one target molecule involved in the angiogenesis of a VEGF receptor, the integrin av (33, the endoglin CD 105 or annexin Al According to another variant embodiment, the particles according to the invention carry fluorescent or luminescent molecules, chosen in particular from the derivatives of rhodamine or fluorescein, cyanine 5. The presence of fluorescent or luminescent molecules makes it possible, in particular Optical detection of particles according to the invention The particles according to the invention may carry various molecules mentioned above, these molecules may in particular be grafted onto the surface and / or within the coating. the particles according to the invention may carry both target molecules and fluorescent molecules, the particles according to the invention may be used es for the manufacture of a medicament for the treatment and / or prevention of tumor diseases such as cancer, or proliferative diseases such as polyarthritis.

The particles according to the invention can be used in all the current fields of brachytherapy and targeted radiotherapy in oncology or in inflammatory proliferative pathologies (for example synoviortheses in destructive articular pathologies such as rheumatoid arthritis). They can also be used in adjuvant therapies of all types of cancers by direct intratumoral injection: for example superficial tumors, such as skin cancers or breast cancers. They can also be used in adjuvant therapy by injection of nanoparticles coupled to bioeffectors specific to all types of tumors with metastatic development, which allows the treatment of primary and secondary sites and non-solid tumors.

The subject of the invention is also the compositions in injectable form, comprising particles according to the invention, in association with at least one pharmaceutically acceptable excipient. It will be readily understood that, depending on the intended application, particularly depending on the nature of the tumor to be treated and the injection technique, the proportion of rare earth oxide and / or oxohydroxide may vary in large proportions in the injected suspension. However, in general, the compositions used according to the invention will advantageously contain between 0.5 and 200 g / l of rare earth oxide and / or oxohydroxide.

The composition will be injected into the body either directly into the tumor to be treated, or parenterally, in particular intravenously. The presence of a layer of polysiloxane gives, after injection in vivo, to the particle a behavior in adequacy with the desired objective of irradiation. Moreover, the very small size of the core and the coating makes it possible, depending on the functionalization, to obtain sufficiently stable and controllable particles for injections in vivo, intravenously or directly intratumorally. Generally speaking, the particles according to the invention, and more particularly the particles of small size, make it possible to obtain, locally at the level of the tumor, an overconcentration. These particles can be designed to locally present at the level of the tumor an overconcentration (either directly related to the increase of the blood irradiation zone, or linked to a passive diffusion within the tumor, or by functionalization with active biomolecules ).

A great interest of the method comes from the preferential use of oxide or oxohydroxide lanthanide particles having interesting magnetic properties and can be followed by MRI imaging. The submicron particles according to the invention have many advantages: first of all, it is possible to concentrate in a single particle a large number of rare earth elements having important therapeutic potentialities. to obtain drainage towards the lymph nodes after intratumoral injection Moreover, it is possible, as previously mentioned, to add to the particle other active elements, in particular: targeting molecules (biological effectors); organic fluorophores allowing optical diagnosis - other lanthanide elements allowing MRI imaging - other lanthanide elements allowing other therapies For example, it is possible to carry out an initial treatment using the rare earth radioactivity, in particular the holmium or lutetium, and then perform X-ray resuscitation treatments, since these elements s are also radiosensitizers As has been said previously, the particles according to the invention are of small size and are optionally surface-coated to have a high stability in biological media and can be injected parenterally into a living being. The surface and the size of the particles can be optimized to allow the best possible diffusion to obtain: a) after direct intra-tumor injection a homogeneous diffusion in the whole of the tumor and possibly a lymphatic drainage with accumulation of the particles in the ganglia of tumor drainage (allowing concomitant treatment of the tumor and loco-regional lymph node metastases); the rapid elimination of particles that would undergo extra-tumoral diffusion, limiting the risk of side effects b) after parenteral injection especially intravenous, a favorable biodistribution with little non-specific uptake by the cells of the reticuloendothelial system, long-lasting blood retention, high tumor uptake (by passive diffusion in hypervascularized tumors and / or by breaking the blood-brain barrier in brain tumor sites, and specific recognition by tumor cells of a biological effector grafted onto the particles), and renal elimination allowing rapid elimination of uncaptured particles (limitations of side effects). Thus, it is possible to effectively target all tumor sites (primary and secondary and limit the radiobiological effects of healthy tissue). A combination of the two types of injection (local direct at some of the tumor sites and parenteral) will allow treatment of all primary and secondary tumor sites, identified and known, or not. Iterative injections in more or less rapid time intervals can optimize intra-tumor particle concentration and increase the therapeutic effect without increasing the side effects. The possibility of detecting particles according to the invention in vivo by imaging makes it possible: a) in a pre-clinical phase to optimize the various parameters of synthesis and injection of the particles, b) to the phase. Clinical to verify and quantify in each individual the rate of capture of particles in the tumor sites, to detect other tumor sites, and thus have elements of analysis of the overall therapeutic efficacy (improvement of patient survival. .) and by tumor sites (reduction of the size of the tumor, etc.). In the pre-clinical phase, non-activated particles may be used, and will be detected by optics (pre-clinical studies in small animals, superficial structures in humans). In the clinical phase, the activated particles will be detected by gamma-scintigraphy, and the pre-injection of non-activated particles will make it possible to check by MRI (and optics for superficial areas) the good quality of particle capture (high tumor uptake, capture extra-tumoral weak). Indeed, the activation does not change the structural properties of the particle, biodistribution and tumor uptake properties studied with unactivated particles will be comparable to those of activated particles. This method will make it possible to combine local and remote processing. For example, direct intra-tumor injections into superficial tumors, such as mammary tumors, will allow neo-adjuvant treatment of advanced tumors, before surgery, with imaging treatment and imaging of the tumor drainage ganglia (sentinel lymph nodes), which can be collected and analyzed specifically. Arterial catheterization injection into hepatic tumors will provide optimal intra-tumor diffusion, with limited irradiation of healthy peri-tumoral tissues. For example, in the case of the use of holmium particles 166 (emission y max = 80 keV, period of the order of 26.7 hours) or lutetium 177 (emission y max = 208 keV, period 6, 71 days with a p-max emission = 497 keV), several publications describe a very high energy of beta particles less than holmium 166 [85% of p-energy particles between 1.76 and 1.84 MeV (US Patent No. 5061475); maximum energy of 13- to 1.84 MeV particles (Mumper RJ et al., JNM 1991; 32: 2139-2143)]. Precautions to be taken with the current treatment of liver tumors which uses lipid micelles labeled with iodine-131 (emission y max = 723 keV, period 8.02 days with a p-max emission = 606 keV) and requires significant radiation protection measures, such as isolation of the patient for several days.

In a general manner, the skilled person can easily manufacture the particles according to the invention, based on various literature documents. More precisely, with regard to the synthesis of the various particles used according to the invention, the following elements will be noted: rare earth oxides and oxohydroxides are preferably manufactured according to processes using a polyol as a solvent, for example according to the approach described in the publication by R. Bazzi et al. (Bazzi, R., Flores, MA, Louis, C., Lebbou, K., Zhang, W., Dujardin, C., Roux, S., Mercier, B, Ledoux, G., Bernstein, E .; Perriat, P. Tillement, 0. Synthesis and properties of europium-based phosphor on the nanometer scale: Eu2O3, Gd2O3: Eu, and Y2O3: Eu. Journal of Colloid and Interface Science (2004), 273 (1), 191- 197.). In this gun obtained particles of a size between 1 and 20 nm. They can also be obtained according to the methods described in the publication M. Flores et al. (Flores-Gonzalez, M., Ledoux, G., Roux, S., Lebbou, K., Perriat, P., Tillement, O., Preparing nanometer sac, Tb-doped Y203, luminescent powders by the polyol, Journal of Solid. State Chemistry (2005), 178, 989-997.). In this case, particles having a size of between 100 and 800 nm are obtained. They can also be made by lyophilization synthesis and partial thermal decomposition treatment, as described in the publication of C. Louis et al. (Louis, C., Bazzi, R., Flores, Mo A., Zheng, W., Lebbou, K, Tillement, W. Mercier, B. Dujardin, C. Perriat, P. Synthesis and characterization of Gd203: Eu3 + phosphor nanoparticles by sollyophilization technique, Journal of Solid State Chemistry (2003), 173 (2), 335-341. In this case, particles having a size of between 50 and 500 nm are obtained. for coating with polysiloxane layers, a number of techniques may be employed, derived from those initiated by Stoeber (Stober, J. Colloid Interf Sci 1968, 26, 62). The method employed for the coating of particles can also be used as presented in the publication of C. Louis et al. (Louis, C., Bazzi, R., Marquette, C. and Bridot, J.

L. Roux, S; Ledoux, G.; Mercier, B.; Blum, L.; Perriat, P.; Tillement, 0.; Nanosized hybrid particles with double luminescence for biological labeling, Chemistry of Marerials (2005), 17, 1673-1682.) And International Application WO 2005/088314. for organic surface grafting, it is advisable to directly use silanes containing hydrophilic organic functions or to go through two steps by grafting onto the amine surface compounds, for example when using APTES. We can, for example, refer to the application WO 2005/088314 and the examples below. the surface functionalization of the coated rare earth oxide particles aims, inter alia, at introducing hydrophilic molecules in order to promote the free circulation of the particles in the vascular system and thus to avoid undesirable non-specific accumulation. One of the possible strategies consists in inserting into the polysiloxane layer (during its formation) organoalkoxysilanes whose organic part is hydrophilic, for example PEG chains terminated by an alkoxysilyl group, a glucose derivative or a phosphonate group linked to a grouping. alkoxysilyl. Another way to make the particles perfectly circulating is to post-functionalize the polysiloxane layer by hydrophilic molecules having a reactive group capable of ensuring the attachment of the molecule on the polysiloxane layer which must have graft sites. It is therefore necessary to use organoalkoxysilanes having reactive groups such as derivatives of succinic anhydride, amines, thiols, maleimides, carboxylic acids, isocyanates, isothiocyanates and epoxides on which these polysiloxane layers are made. The hydrophilic molecules will be covalently bonded (as a result of a condensation reaction). For the most part, the hydrophilic entities comprise a hydrocarbon chain with at least one ionizable function (carboxylic acid, amine, phosphate, phosphinate, phosphonate, sulphonate) and / or several alcohol and / or ether-oxide and / or thiol functions. The process for the preparation of particles according to the invention comprises, for example: the preparation of non-radioactive particles whose composition and structure have been defined previously, the activation by a neutron flux of said particles to obtain the desired radioactivity . Activation can be accomplished by positioning said particles within a neutron stream from a neutron reactor or a source from a cyclotron. In particular, it is possible to use a neutron flux produced by a small-medium-sized cyclotron-driven system, preferably with a transmission energy of at most 70 MeV, where the above-mentioned system comprises a solid target. of Be or Ta or W, cooled by a stream of water which also acts as the first stage of neutron moderation, and a neutron reflection-moderation system of lead-graphite or graphite. The activation may be, for example, carried out with an activator coupled with a cyclotron operating under the following conditions: proton energy of 30 to 80 MeV, proton beam current of 10 to 400 pA, irradiation time of 0, 5 to 20 h, total neutron flux from 6 X 101 ° to 5 X 1012 n.cm-2.s-1. This makes it possible to obtain specific activities of between 0.5 and about 100 GBq / g of holmium, in the case of particles of holmium oxide. The activation can also be carried out in a nuclear reactor. In this case the activation is obtained by irradiation under a neutron flux of the order of 1012 n.cm-2.s-1 for a period that may vary from a few minutes to a few hours. This makes it possible to obtain specific activities of between 0.5 and 100 GBq / g of holmium, in the case of particles of holmium oxide.

Such a system has the following advantages: the non-radioactive injectable suspension can be prepared in large quantities and diffused as a pharmaceutical product, this suspension being able to be irradiated just before injection, the distribution of the non-radioactive injectable suspension is easier and cheaper, than a radioactive suspension, - isotopes with different energies can be used with the suspension for injection, - the method allows irradiation just before injection and allows the use of short-lived isotopes.

Also, according to an advantageous variant, an injectable pharmaceutical composition of non-radioactive particles will be prepared and then subjected to activation by a neutron flux. The examples below have no limiting character but allow to illustrate the invention.

High resolution transmission electron microscopy is performed on a JEOL-2010 microscope with an acceleration voltage of 200kV. A drop of diluted colloidal solution (1: 30 in diethylene glycol) is placed on a carbon grid for analysis. Measurement of the particle size in colloidal suspension is carried out by photon correlation spectroscopy on a Malvern Zetasizer nano-S. The measurement of the cores is at the synthesis exit directly in the diethylene glycol. After coating, the measurement is also done in diethylene glycol. After functionalization and reconcentration, the measurement is carried out in water. The device used to make the gamma spectra is a model 1480 WIZARD 3 "gamma counter of the brand Wallac, the current name of the company: Perkin Elmer.The system allows the automation of measurement with a door device The results are printed on paper Example 1. Synthesis of Particles Comprising a Functionalized Polysiloxane Coated Holmium Oxide Core A colloid is prepared by dissolving a amount of 56 gL -1 of salts of holmium chloride in a volume of 200 mL of diethylene glycol To the solution obtained, an addition of 7.5 mL of sodium hydroxide at a concentration of 3M is carried out at room temperature in 1 h 30. The final size of the particles, measured by photon correlation spectroscopy (PCS), is about 1.5 nm, around which a layer of functionalized polysiloxane with a thickness of 0.5n m is synthesized by sol-gel route. In a solution containing 200 ml of colloid and 800 ml of diethylene glycol, 3.153 ml of aminopropyltriethoxysilane (APTES), 2.008 ml of tetraethylorthosilicate (TEOS) and 7.650 ml of a solution of catalysis at a concentration of 10 m in water and 0 ml are added. 1M triethylamine The reaction is carried out at 40 ° C in an oil bath and stirring in several stages. At t = Oh addition of 3% of the volume of APTES and TEOS at t = 1h addition of 3% of the volume of catalysis solution at t = 2h addition of 7% of the volume of APTES and TEOS at t = 1h 3h addition of 7% of the volume of catalysis solution at t = 4h addition of 10% of the volume of APTES and TEOS at t = 5h addition of 10% of the volume of catalyst solution at t = 6h addition of 15% of the volume of APTES and TEOS at t = 7h addition of 15% of the volume of catalysis solution at t = 23h addition of 15% of the volume of APTES and TEOS at t = 24h addition of 15% of the volume of solution of catalysis at t = 25h addition of 25% of the volume of APTES and TEOS at t = 26h addition of 25% of the volume of catalysis solution at t = 27h addition of the remaining 25% of the volume of APTES and TEOS at t = 28h addition of the remaining 25% of the volume of catalysis solution at t = 76h end of the synthesis

The particles thus coated are then functionalized: either by poly (ethylene glycol) bis (carboxymethyl) ether (Mn = 250) (PEG250): 0.3612 g of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide, 0, 3489 g of pentafluorophenol and 181.5 μl of PEG 250 are dissolved in 5 ml of isopropanol with stirring for 1 h 30 min. The solution obtained is then mixed with 1 mmL of solution of coated particles. The mixture is then stirred for 15 hours. Either diethylenetriaminepentaacetic acid dianhydride (DTPABA): 0.616 g of DTPA are dissolved in 17.3 ml of anhydrous dimethylsulfoxide (DMSO) with stirring for 1 h 30 min. The solution obtained is then mixed with 100 ml of solution of coated particles. The mixture is then stirred for 15 hours. After one or other of these functionalizations, the solution of particles obtained is dialyzed against ethanol. The dialysis bath is changed 3 times every 24 hours. At the end of the dialysis, 100 ml of water are added to the solution. The mixture thus obtained is placed on a rotary evaporator under reduced pressure in order to evaporate the liquid and obtain 75mL of solution in the end. The aqueous nanoparticle solution can be stored at -18 ° C or lyophilized under dynamic vacuum at 0.15mbar and -52 ° C in a CHRIST Alpha 1-2 freeze-dryer. The oxide mass% measured using a plasma source quadrupole mass spectrometer ("ICP-MS" "Inductively Coupled Plasma-Mass Spectrometer"). After coating, there is a value of 35.6% of holmium, corresponding to an equivalent of 41% of lanthanide oxide.

The hydrodynamic sizes (measured by PCS) are: -After synthesis: 1.5 nm -After coating: 3.1 nm -After functionalization with DTPABA: 3.6 nm -After lyophilization: 4.0 nm Figure 1 represents the particle size distribution obtained in PCS (Photon Correlation Spectroscopy) of holmium oxide particles obtained at different stages of their elaboration.

Example 2. Synthesis of Particles Comprising a Functionalized Polysiloxane Coated Gd / Ho (50/50) Oxide Core A colloid is prepared by dissolving an amount of 56 gL-1 of salts of holmium chloride and of gadolinium in a volume of 200mL of diethylene glycol. To the solution obtained, an addition of 7.5mL of sodium hydroxide at a concentration of 3M is carried out at room temperature in 1h30. The mixture is then heated at 180 ° C for 4h. The final size of the particles, measured by photon correlation spectroscopy (PCS) is about 1.3 nm. In a solution containing 40 ml of colloid and 160 ml of diethylene glycol, 0.421 ml of aminopropyltriethoxysilane (APTES), 0.267 ml of tetraethylorthosilicate (TEOS) and 1.020 ml of a solution of catalysis at a concentration of 10 M in water and 0.1 M are added. triethylamine.

The reaction is carried out at 40 ° C. in an oil bath and with stirring in several stages. at t = Oh addition of 10% of the volume of APTES and TEOS at t = 1h addition of 10% of the volume of catalysis solution at t = 2h addition of 25 ° / o of the volume of APTES and TEOS at t = 3h addition of 25% of the volume of catalyst solution at t = 4h addition of the remaining 65% of the volume of APTES and TEOS at t = 5h addition of the remaining 65% of the volume of catalyst solution at t = 53h end of synthesis.

The functionalization is then carried out as in Example 1: - Either with poly (ethylene glycol) bis (carboxymethyl) ether (Mn = 250) (PEG250) - or with diethylenetriaminepentaacetic acid dianhydride (DTPABA) The hydrodynamic sizes (measured by PCS) are: -After synthesis: 1.3 nm -After coating: 2 nm -After functionalization with DTPABA: 4.5 nm The oxide mass% measured using a plasma source quadrupole mass spectrometer ("ICP-MS" "Inductively Coupled Plasma-Mass Spectrometer"). After coating, there is a value of 39.2% of holmium, corresponding to an equivalent of 45% of lanthanide oxide.

EXAMPLE 3 Synthesis of a Holmium Oxide Powder by Nitrate Method The powder is obtained by mixing 54.3 ml of an acid solution of holmium nitrate of concentration [Ho] = 0.264 mol.l -1 in volume of 100 mL of diethylene glycol. The solution is then heated at 110 ° C. under reduced pressure for 1 hour in order to evaporate the water. The temperature is then raised to 170 ° C under atmospheric pressure until precipitation of the particles. After precipitation, the solution is left stirring for 30 minutes before stopping the heating. 100 ml of ethanol are then added to the solution, when the latter has returned to ambient temperature, with vigorous stirring. The solution is then centrifuged at 7000 rpm for 5 minutes.

The powder is then washed with ethanol and then centrifuged again under the same conditions and that 3 times in succession. After washing, the powder is dried in an oven at 100 ° C. for 12 hours and then slowly raised to 700 ° C. according to the following thermal program: 1 hour at 150 ° C. 1 hour at 200 ° C. 1 hour at 225 ° C. 1 hour at 250 ° C. C 1 hour at 275 ° C. 1 hour at 300 ° C. 1 hour at 325 ° C. 15 hours at 350 ° C. 1 hour at 400 ° C. 1 hour at 450 ° C. 1 hour at 500 ° C. 1 hour at 600 ° C. 1 hour at 700 ° C. The particles thus obtained have an average hydrodynamic size of 310 nm (average standard deviation of 70 nm) by measurement in photon correlation spectroscopy (PCS).

The particles are 99% pure holmium oxide.

Example 4. Synthesis of a chloride powder of holmium oxide The powder is obtained by dissolving 37.54 g of HoCl3; 6H20 in 20mL of water. 100 ml of diethylene glycol are then added and the solution is heated to 110 ° C. At this temperature, 36 mL of 6M NaOH is added.

The solution is stirred for 30 min at 110 ° C before raising the heating temperature to 180 ° C for 3h. The solution obtained is then washed 3 times with ethanol and centrifuged. The solid obtained is dried for 12 hours at 100 ° C. and then heated to a temperature of up to 900 ° C., according to the following thermal program: 12 hours at 250 ° C. 1 hour at 300 ° C. 5 hours at 500 ° C. 3 hours at 700 ° C. 10 2 h at 900 ° C. Once heated to 900 ° C., the particles are washed with water and then centrifuged before being heated again at 500 ° C. for 1 hour. The particles thus obtained have a mean hydrodynamic size of about 300 nm by measurement in photon correlation spectroscopy (PCS).

Example 5. Preparation of suspensions for injection. Containers containing the dry radioactive particles are placed under a protective hood. Solutions were prepared under this hood from (a) the particles of Example 1, (b) powders of Example 3. (a) A 250mg of powder of holmium oxide particles functionalized with of DTPABA and lyophilized as obtained in Example 1, 3,690 ml of physiological injectable liquid composed of sterile water and sodium chloride (NaCl) diluted to 9 per 1000 were added in order to obtain a colloidal solution at a concentration of concentration of 100mmol.L-1 in Ho element. The solution obtained is vortexed in order to obtain a homogeneous solution. (b) To 250 mg of holmium oxide powder as obtained in Example 3, 1.220 ml of physiological injectable liquid composed of sterile water and sodium chloride (NaCl) diluted to 9 per 1000 are added. to obtain a solution at a concentration of 20% by weight of holmium oxide. The solution obtained is vortexed to obtain a homogeneous solution. "

Example 6 Synchrotron activation of colloids and holmium oxide powders Activation of the particles is carried out using a compact activator (λ 1 x 1 m of graphite) coupled with a medium size cyclotron. The principle of this activator is based on an evolution of the so-called Adiabatic Resonance Crossing technique validated at CERN in 1997. It is used here in coupling with the Scanditronix 40 MeV-50 pA cyclotron of the Joint Research Center at Ispra. Activation of Ho particles of medium hydrodynamic size after lyophilization of 4 nm, functionalized with DTPABA and then lyophilized as obtained in Example 1 and 310 nm, as obtained in Example 3 having respective contents of holmium element of 26% by mass and 86% by weight, was carried out. The particles in both cases in the form of powder were irradiated with the neutrons produced by a proton beam of 34 MeV and 38 pA for 4.7 hours. A specific activity of 9.0 × 10 and 1.6 × 108 Bq per gram of particles was obtained, respectively, at the end of the irradiation. These particles were then prepared according to Example 5 to obtain ready-to-inject particle suspensions.

Example 7 Injection into Rats and In Vivo Detection Suspensions of the radioactive particles obtained in Example 6 were injected 72 h after the end of intravenous irradiation or in subcutaneously grafted tumors in Fisher rats. Figure 2 demonstrates the radioactive character of the coated and functionalized particles of Figure 1 after neutron activation. This spectrum is consistent with data from the gamma emission literature, with a peak emission at 77 keV and another at 38 keV. The particles have therefore been activated by a neutron flux and the element obtained is indeed holmium 166.

The coated and functionalized particles of FIG. 1 which have undergone neutron activation are detected by gammascintigraphy in vivo. FIG. 3 shows a gamma scintigraphy image acquired 1 hour after intra-tumor direct injection in a rat carrying two tumor xenografts, from 200 μl to 100 mmol.L-1 in Ho element per tumor of a suspension of colloidal particles. polysiloxane-coated holmium oxide, functionalized with DTPABA and having a mean hydrodynamic size measured in PCS of 4 nm, as obtained in Examples 1 and 5, after neutron activation, with an activity of the order of 2 pCi by tumors. This experiment demonstrates that particles with a holmium oxide core, after activation by a neutron flux, are radioactive and can be detected in vivo by gamma-scintigraphy in tumors. FIG. 4 shows a gamma scintigraphy image of a rat carrying an acquired tumor xenograft after intravenous injection of a suspension of polysiloxane-coated holmium oxide colloidal particles having a total size of 5 nm, as obtained in Example 1, after neutron activation, with an activity of the order of 1 pCi. This experiment demonstrates that these particles in the form of a colloidal suspension can also be injected by syringe, locally or parenterally, and that they exhibit excellent intratumoral diffusion and urinary elimination after intravenous injection.

Particles of Figure 5a with a hydrodynamic size of about 300 nm, neutronally excited are detected by in vivo gammascintigraphy. Figure 6 is the gamma scintigraphy image obtained after injection of these particles into the thigh of a rat with a high-pressure syringe (3000 bar). 0.8 ml of a suspension of particles, with an activity of 0.056 MBecquerel, were injected. This scintigraphic image demonstrates that holmium particles of this size can be efficiently elaborated, activated and injected.

Experiments were also carried out with suspensions of particles identical to the particles of Example 1 encapsulating within them fluorescein (FITC) or cyanine molecules incorporated according to the method described in Chemistry of Materials (2005), 17 (7). ), 1673-1682 and Journal of the American Chemical Society (2007), 129 (16), 5076-5084. After in vivo injection, the particles were detected by in vivo optical imaging acquired by a CDD camera. A first experiment was carried out, by injecting by intratumoral injection, into a rat carrying 2 tumor xenografts: 500 μl of a 109 mM suspension of colloidal particles of core / coating type [heart: holmium oxide, coating: polysiloxane total size of 5 nm], non-activated FITC carriers in the left tumor, 500 μl NaCl at 9 ° / ° 0 in the tumor on the right. Figure 7 shows the image obtained by in vivo optical imaging acquired by a CDD camera (HamamatsuTM) with an excitation: 495 nm / at emission: 518 nm, 15 minutes after intratumoral injection. The brigt field image is the image acquired in visible light. A second experiment was carried out, by injecting by intratumoral injection, in a rat carrying 2 tumor xenografts, 500 μl of a suspension at 100 mM of colloidal particles of the caeur / coating type [heart: holmium oxide, coating: polysiloxane total size of 5 nm], non-activated cyanine carriers in each tumor. Figure 8 shows the image obtained by in vivo optical imaging acquired by a CDD camera with an excitation: 650 nm / at emission: 672 nm, 15 minutes after intratumoral injection. The fate of the particles after intratumoral injection, in the 2 tumors of 250 μl of colloidal suspension defined in FIG. 9 by tumor, was followed in the long term by light microscopy. FIG. 9 represents the in vivo optical images acquired in the same rat and under the conditions defined in FIG. 9 successively for 4 days (OJ corresponds to the day of the injection and 33 to the third day post-injection and therefore to the 4th day examination) after the injection against both tumors. These images demonstrate that these particles can be detected in vivo by optical imaging, and that the fluorescence emitted by these particles is very stable. This means, in particular, that the integrity of the particles is preserved in the tumor. Experiments were also performed to demonstrate particle detection by MRI. MRI images are obtained with a device dedicated to small animal imaging (BruckerTM, 7T, horizontal field). FIG. 10 shows the MRI images (echo sequence of spin TR = 1000 msec / TE = 50 msec, 7T) of aqueous samples containing increasing concentrations of colloidal particles in accordance with example 1 of the core / coating type [core: d holmium, coating: polysiloxane total size 5 nm] (1: conc = 0/2 = conc = 0.05 mM / 3 = 0.1 mM / 4 = 1 mM / 5 = 5 mM) at pH = 7, 4, at room temperature and at 7T. The contrast increase is calculated according to the following formula: EHC Sample X = [Signal (Sample X) -Signal Sample 1] / 15 Sample Signal 1 The calculation confirms the visual analysis, with negative contrast at low concentration, visible between 0.1 mM and 1 mM (EHC sample 2 = -8%, sample 3 = -21%, sample 4 = -24%). At high concentration, a signal enhancement is observed (positive contrast, EHC sample 5 = + 36%). This inversion of the effect on the contrast of the image (first negative then positive) must occur for higher values but close to 1 mM. FIG. 11 is an in vivo MRI image obtained after intravenous injection (spin multi-echo sequence TR = 1000 msec / TE = 50 msec, 7 T) in a rat, of 50 μl of a colloidal particle suspension conforming to FIG. Example 1 at 100 mM heart / coating type [core: holmium oxide, coating: 5 nm total polysiloxane] non-activated cyanine carriers. The images A and B correspond respectively to coroanal sections centered on the two kidneys and on the bladder; the indexes 30 correspond to the images before injection; image A2 was acquired between 3.5 and 5.6 minutes after injection, and image B2 between 51 and 53 minutes after injection.

There is a decrease in the signal at the level of the pyelocalicielles cavities (white arrow) of the kidneys, and at the level of the bladder an increase of the signal (except at the level of the bladder neck, where a decrease of the signal is noted). These signal variations observed in vivo are superimposable on what is observed in vitro: at the level of the kidney where the particles transit but do not accumulate, the local concentration of particles remains low (negative contrast), whereas in the bladder, the particles accumulate over time, without elimination (the anesthesia causing a blockage mictionnel), ending up giving a positive contrast.

A study coupling multi-modal MRI imaging and optics has been performed. Figure 12A shows the optically acquired image by superimposing the fluorescence image with excitation: 495 nm / emission: 518 nm to the bright field image and Figure 12B represents the MRI image. (spin echo sequence TR = 1000msec / TE = 50 msec, 7T), of the two ex-vivo xenografts taken from the same rat 15 minutes after injection into the right tumor of 500 μl of a suspension of 100 mm colloidal particles core / coating type [core: holmium oxide, coating: polysiloxane 5 nm total size] FITC carriers, non-activated.

The detectable fluorescence is noted within the tumor, showing a fairly homogeneous distribution of the particles within the tumor. In MRI, we note the sharp difference in contrast of the 2 tumors, the particles being responsible for an intense negative contrast.

All these results demonstrate that these particles are detectable in vivo by multi-modal imaging, which makes it possible to follow the scattering of particles in situ within the tumor after intratumoral injection (brachytherapy) by imaging, check the site migration after parenteral injection (metabolic radiotherapy) functionalized particles or not. Imaging will then be a tool for monitoring and evaluating therapy.

This in vivo detection can be carried out with non-activated MRI and optical particles, making it possible to have evaluation and optimization tools in the pre-clinical phases, or by scintigraphy after activation, allowing control in the patient and the adaptation of the therapy. These results also demonstrate the very good diffusion within the tumors of particles after direct intratumoral injection (brachytherapy) allowing a homogeneous treatment of the whole tumor volume. These results also show that the particles remain permanently within the tumor (high intra-tumor residence time), and that those that diffuse outside the tumor are eliminated rapidly urinary, making the hypothesis of a the radiotoxic effect on healthy tissue is very unlikely. This aspect is confirmed by Figure 13 which shows the biodistribution after intratumoral injection of nanoscale colloids. FIG. 13 shows the in vivo images acquired at dorsal (A) and ventral (B) incidence by a CDD camera with an excitation: 495 nm / at emission: 518 nm, 90 minutes after left intra-tumor injection (Tg 100 μl of a 109 mM suspension of colloidal particles of heart-type / coating type [heart: holmium oxide, coating: 5 nm total-size polysiloxane] carrying FITC, non-activated, in a carrier rat of 2 tumor xenografts. In the tumor on the right (Td), 500 μl of NaCl at 9 ° / ° O were injected. The images presented on the right, were obtained ex vivo and acquired with the same parameters, on organs taken postmortem 120 minutes. after injection, right kidneys (Rd) and left (Rg), liver (F) and urine (U). The images Rt, Ft and Ut correspond to kidneys, liver and urine taken from a control rat. These images show that after a direct intratumoral injection, a small fraction of the particles exhibit extra-tumoral diffusion, but are rapidly eliminated urinary, without retention in the reticuloendothelial system (absence of significant fluorescence in the liver). .

Claims (26)

CLAIMS1 - Submicronic particles that can be used in therapeutic or diagnostic procedures, characterized in that: they have a size of between 1 and 800 nm, they comprise at least 30%, preferably at least 50%, and more preferably at least 50%; less than 90%, by mass of at least one oxide and / or oxohydroxide of at least one rare earth, - all the rare earths are responsible for a specific radioactive activity of at least 0.5 MBq per gram of particles and preferably at least 5, due to a disintegration of the rare earth element (s), at least 10% of these decays producing an emission of R- radiation. 2 - Particles according to claim 1, characterized in that they are in colloidal form and have a size in the range of 1 to 20 nm, preferably in the range of 1 to 10 nm. 3 - Particles according to claim 1 or 2, characterized in that the rare earth or rare earths are chosen from holmium 166, cerium 141, samarium 153, europium 155, erbium 169, thulium 170, Ytterbium 169, dysprosium 165, lutetium 177, gadolinium 159. 4 - Particles according to any one of claims 1 to 3, characterized in that said particles contain oxides and / or oxohydroxides of at least two different rare earths, each representing more than 10% by weight of the totality rare earths and preferably more than 20%. 5 - Particles according to claim 4, characterized in that the oxides and / or oxohydroxides of different rare earths are in successive layers. 6 - Particles according to claim 4, characterized in that the oxides and / or oxohydroxides of different rare earths are in the form of a solid solution. 7 - Particles according to one of claims 1 to 6, characterized in that the rare earth or rare earths comprise at least 50% by weight of lanthanide causing a signal in MRI, allowing in vivo detection of the presence of said particles. 8 - Particles according to claim 7, characterized in that the rare earth or rare earths contain at least 50% by weight of gadolinium (Gd), dysprosium (Dy), holmium (Ho) or mixtures of these lanthanides. 9 - Particles according to one of claims 1 to 8, characterized in that they are - either in the form of particles consisting exclusively of at least one oxide and / or an oxohydroxide of at least one rare earth, - either in the form of a core consisting of at least one oxide and / or an oxohydroxide of at least one rare earth, and surrounded by a coating consisting of at least one organic and / or inorganic constituent, characterized in that the particle or the constituent core of the particles containing at least one oxide and / or an oxohydroxide of at least one rare earth contains on its periphery lanthanides causing an MRI signal, preferably gadolinium, and at least one other rare earth in its central part. 10 - Particles according to one of claims 1 to 9, characterized in that they comprise a core containing the oxide or oxides and / or oxohydroxides of rare earth and a coating consisting of at least one organic constituent and / or inorganic which stops at most 25% of the radiation [3- emitted by the heart. 11 - Particles according to claim 10, characterized in that the one or more rare earth oxides and / or oxohydroxides represent at least 30% by weight of all the inorganic constituents of said particle, the organic constituents being covalently bound to the hybrid particle and representing less than 30% by weight of the final particle. 12 - Particles according to one of claims 1 to 11, characterized in that said particles comprise a core in the form of at least one oxide and / or an oxohydroxide of at least one rare earth and a polysiloxane coating representing 1 to 70 % by mass of the inorganic constituents of said particle. 13 - Particles according to claim 12, characterized in that the ratio number of silicon atoms of the polysiloxane coating / number of rare earth atoms is between 0.1 and 10 14 - Particles according to one of claims 9 to 13, characterized in that said coating is a polysiloxane coating and in that said particle further comprises hydrophilic organic molecules with a molar mass of less than 5000 g / mol and preferably of less than 450 g / mol, preferably chosen from organic molecules containing alcohols, carboxylic acids, amines, amides, esters, ether-oxides, sulphonates, phosphonates or phosphinates, covalently bound at least 5% silicon atoms of said polysiloxane. 15 15 - Particles according to claim 14, characterized in that said hydrophilic organic molecules contain a polyethylene glycol unit, DTPA, DTDTPA (dithiole DTPA) or a succinic acid. 16 - Particles according to claim 14 or 15, characterized in that said hydrophilic organic molecules are rare earth complexing agents whose complexing constant is greater than 1015, preferably DTPA or DOTA. 17 - Particles according to one of claims 1 to 16, characterized in that they comprise a core and a surface coating in which one or more target molecules are coupled. 25 18 - Particles according to claim 17, characterized in that the target molecule or molecules are chosen from proteins, peptides, antibodies, lipids, carbohydrates and nucleic acids. 19 - Particles according to claim 18, characterized in that the target molecule or molecules are an antibody targeting at least one target molecule involved in the angiogenesis of a VEGF receptor, the integrin av 83, the endoglin CD 105 or annexin Al. 20 - Use of particles according to one of claims 1 to 19 for the manufacture of a medicament for the treatment and / or prevention of tumor diseases such as cancers. 21 - Use of particles according to one of claims 1 to 19 for the manufacture of a medicament for the treatment and / or prevention of proliferative diseases such as polyarthritis. 22 - Injectable pharmaceutical compositions, comprising particles according to one of claims 1 to 19, in combination with at least one pharmaceutically acceptable excipient. 23 - Process for preparing particles according to one of claims 1 to 19 or a composition according to claim 22, characterized in that it comprises - the preparation of non-radioactive particles whose composition and structure are defined in the following: one of claims 1 to 19 and - the activation by a neutron flux of said particles to obtain the desired radioactivity. 24 - Method according to claim 23, characterized in that said activation is performed by positioning said particles in a neutron flux from a neutron reactor or a source from a cyclotron. 25 - Method according to one of claims 23 or 24, characterized in that it comprises a step of preparation of an injectable pharmaceutical composition, upstream of the activation by a neutron flux. 26 - Method according to one of claims 23 to 25, characterized in that the activation is carried out in a neutron flux produced by a system driven by a cyclotron of emission energy of at most 70 MeV, where the aforesaid system consists of a solid target of Be or Ta or W, cooled by a stream of water which also acts as a first stage of neutron moderation, and a neutron reflection-moderation system of lead-graphite or graphite .30