RU2540510C2 - Method of obtaining biocompatible gallium(iii)-containing highly disperse particles for manufacturing diagnostic preparations for positron-emission tomography - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to method of obtaining biocompatible highly disperse polylactide particles for in situ manufacturing of diagnostic means for positron-emission tomography by combination of said particles with solution, containing cations of gallium 68 (III). Claimed method includes combination of solution of bidentate aromatic ligand, namely quercetin, quinalizarin, alizarin or 8-hydroxyquinoline in polar solvent with solution, containing lactic acid copolymers in low-polar solvent and intensive mixing of obtained mixture with obtaining highly homogenous mixture. After that, at least three volumes of water solution of polyvinyl alcohol with molecular weight 9.0÷80 kDa are added and intensively mixed with obtaining highly homogenous mixture. Obtained mixture is evaporated with obtaining suspension, which is filtered by filter with pore size less than 30 mcm with obtaining filtrate, with further addition of lyoprotector, freezing and lyophilisation. Invention also relates to biocompatible highly disperse particles for manufacturing radiopharmaceutical preparation for positron-emission tomography, their application for carrying out positron-emission tomography and to diagnostic composition, containing effective amount of gallium-68 (III), bound with said particles.

EFFECT: obtaining biocompatible highly dispersive polylactide particles for in situ manufacturing of diagnostic means for positron-emission tomography.

33 cl, 8 tbl, 6 dwg, 2 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to the field of nuclear medicine, and more specifically to methods for the manufacture of radiopharmaceutical diagnostic tools for positron emission tomography.

BACKGROUND

Today, the success of the treatment of many diseases depends on how early and successfully the diagnosis of the disease was carried out. Modern medicine has a large number of diagnostic methods. These are imaging methods (radiography, magnetic resonance imaging (MRI), positron emission tomography, computed tomography, etc.), and biochemical analyzes, and biopsy (for the diagnosis of neoplasms).

Modern methods of positron emission tomography (two-photon emission tomography) are based on the detection of a pair of gamma quanta resulting from the annihilation of positrons after the decay of unstable radioisotopes in tissues and cells, while the location of the radiation sources is made taking into account the fact that as a result of annihilation two quantum, and the directions in which they propagate are strictly opposite to each other.

One of the main advantages of positron emission tomography is that, unlike other diagnostic methods, the inclusion of radionuclides in the composition of biologically active compounds allows us to study their distribution and metabolism in the human body in dynamics. In many cases, this circumstance makes PET an indispensable method of research, despite the relatively low spatial resolution of modern PET scanners.

As radioisotopes in positron emission tomography, radionuclides are used which are subject to positron beta decay. The most common are carbon-11 (T _^1/2 20.4 min), nitrogen-13 (T _^1/2 9.96 min), oxygen-15 (T _1/2 2.03 minutes) fluoro-18 (T ¹ / ₂ 109.8 minutes) iodine.

From the viewpoint of radiation exposure (the lower T _^1/2, the greater the dose of the radiopharmaceutical can be introduced into the patient's body at the same time while maintaining the same total dose), ease of transportation and preparation of radiopharmaceuticals (T _^1/2 should not be too short to for the period between the time of manufacture and the moment of introduction into the body before the study, the necessary amount of the radioisotope was stored in the preparation), and the resolution of the PET scanners (increases with decreasing radiation energy) olee optimal are radionuclides with half-lives greater than 60 minutes and low energy radiation.

In terms of the combination of nuclear-physical and chemical properties, gallium-68 is one of the most convenient radionuclides for the manufacture of radiopharmaceuticals for positron emission tomography.

This is due to the availability and ease of use of germanium-68 / gallium-68 gallium-68 generators, the long half-life of parent germanium-68 (T ^1/2 _of approximately 250 days), which ensures a long life of the generator in laboratory conditions (see Astia, M . et al. Validation of 68Ge / 68Ga generator processing by chemical purification for routine clinical application of 68Ga-DOTATOC. // Nuclear Medicine and Biology. - 2008. - Vol. 35. - P.721-724; Zhemosekov KP et al. Processing of generator-produced 68Ga for medical application. J. Nucl. Med. - 2007. - Vol.10. - P.1741-1748; McAlister DR, Horwitz EP Automated two column generator systems for medical radionuclides. Applied Radiation and Isotopes 67 (2009) 1985-1991). In this gallium-68, in contrast, has a very short half-life (T _^1/2 = 68.1 min) that allows the use of radiopharmaceuticals desired activity, without creating a significant burden on the patient dose. In addition, the gallium-68 (III) cation forms a wide range of stable complex compounds with many ligands containing oxygen, nitrogen and sulfur as donor atoms, which makes it suitable for the synthesis of a large number of chelate complexes and macromolecules for various functional purposes.

Known radiopharmaceuticals mainly contain gallium-68 in the form of complexes with bidentate ligands associated with various biospecific molecules. The most significant polydentate representatives of the bifunctional chelating agents used are the acyclic ligands N, N'-bis (2,2-dimethyl-2-mercaptoethyl) ethylenediamine-N, N'-diacetic acid (6SS), tris-aminomethylethane (TAME), diethylene triamine pentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA) and dipheroxamine (DFO), as well as macrocyclic 1,4,7-triazacyclononan-N, N ', N' '- triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane -1,4,8,11-tetraacetic acid (TETA) and 1,4,7,10-tetraazacyclodecane-N, N ', N' ', N' '' - tetraacetic acid (DOTA) and their derivatives water (articles by Maecke HR et al. 68 Ga-Labeled Peptides in Tumor Imaging, J. Nucl. Med. 46: 172S-178S (2005); Breeman WAP et al Radiolabelling DOTA-peptides with 68 Ga. Eur. J. Nucl. Med. Mol. Imaging. - 2005. - V.33. - P.478-85 and patent documents WO 2005/057589, US 20080277350, RU 2343965 C2, US 7586102 B2, WO 2004/089425 A1 and US 7728310 B2).

Despite the popularity of the general approaches to the creation of gallium-68 bioconjugates and approaches to the manufacture of biocompatible nanoparticles, so far no attempts have been made to create radiopharmaceutical preparations in the form of gallium-68 nanoparticles, or have not been successful. Meanwhile, the creation of such drugs could help improve the accuracy of the diagnosis of oncological diseases of those organs, which are characterized by the most significant accumulation of nanoparticles, namely the liver, kidneys and brain (see Semete B. et al. In vivo evaluation of the biodistribution and safety of PLGA nanoparticles as drug delivery systems. Nanomedicine: Nonotechnology, Biology, and Medicine, - 6 (2010) - P.662-671). In addition, the creation of particles of this type could be an important step towards the creation of targeted radiopharmaceuticals based on nanoparticles containing, along with the isotope tag, various biospecific molecules, such as antibodies and receptor ligands, characteristic of normal and malignant cells.

One of the problems that prevents the creation of such nanoparticles is that the eluates of commercially available germanium-68 / gallium-68 generators, along with gallium-68, contain a large number (7 ÷ 10 orders of magnitude more) of competing cations that prevent the formation of gallium complexes -68 (III). Presence of Cd (II) impurities in the working solution; Co (II); Cu (II); In (III); Fe (II); Fe (III); Lu (III); Ni (II); Zn (II) already in the amount of 1 μM can be a serious problem (Breeman WAP et al Radiolabelling DOTA peptides with 68 Ga. Eur. J. Nucl. Med. Mol. Imaging. - 2005. - V.33. - P.478- 85). In addition, the eluates obtained from the generator have a low pH (usually get 5.0 ml of a solution with a pH of 1.0), and after dilution with a buffer to pH 5.0, a significant volume of solution with a very low concentration of gallium-68 is obtained. Thus, the use of nanoparticles for the manufacture of gallium-68 radiopharmaceuticals without additional purification and concentration of the eluate is possible only if their sorption capacity is sufficient to bind not only gallium-68, but also all interfering cations, and their specificity for gallium is 68 will be sufficient for almost complete binding of gallium-68 from solutions with a concentration of less than 1 · 10 ^-6 M.

DETAILED DESCRIPTION OF THE INVENTION

The terms and expressions used in this text are given the following meaning, unless the context otherwise implies.

DIPEA - Diisopropylethylamine

EDC - N- (3-Dimethylaminopropyl) -N'-ethyl carbonate

lgβ ₁ - stability constant of the complex compounds in solution

PDI - Polydispersity Index

PLGA - a copolymer of lactic and glycolic acid

PSD - particle size distribution

Resomer 202S is a biodegradable copolymer of D- and L-lactic acid with a molecular weight of 10-18 kDa, a characteristic viscosity of 0.16-0.24 dl / g, a glass transition temperature of 38-42 ° C, free carboxyl groups are esterified.

Resomer 502H is a biodegradable copolymer of lactic and glycolic acid (50:50) with a molecular weight of 7-17 kDa, a characteristic viscosity of 0.16-0.24 dl / g, a glass transition temperature of 42-46 ° C, there are free carboxyl groups

Resomer 752H is a biodegradable copolymer of lactic and glycolic acid (75:25) with a molecular weight of 7-17 kDa, a characteristic viscosity of 0.14-0.22 dl / g, a glass transition temperature of 42-46 ° C, there are free carboxyl groups

ZAVeD - average particle size

ZP - Zeta Potential

Biodegradable polymer - a polymer that, when introduced into the human body under the action of enzymes, is completely broken down into harmless non-cumulative metabolites that can be excreted by the body's excretory systems.

Biocompatibility is a property of the material, which manifests itself in the fact that when introduced into the body, the material does not cause intolerance reactions or allergic reactions.

DMSO - dimethyl sulfoxide

Acid Index - the number of milliequivalents of KOH needed per gram of polymer to neutralize free acidity

Polylactide is a biocompatible biodegradable polymer selected from the group consisting of a copolymer of lactic and glycolic acid and a copolymer of D and L-lactic acid.

Nanoparticles (abbreviated as LF) are fine particles that form colloidal systems, in particular suspensions, emulsions, aerosols, gels and sols.

The remaining terms and expressions are used in the usual sense known to those skilled in the art.

An object of the present invention is to provide a method for producing highly dispersed biocompatible particles for the manufacture of diagnostic radiopharmaceuticals based on gallium-68 radionuclide for positron emission tomography.

The technical result consists in the creation of a non-toxic (at effective concentrations) radiopharmaceutical that provides effective binding of gallium-68 (III) cations from highly diluted eluates contaminated with metal cations and a high concentration of gallium-68 (III) in bound form.

Free and bound to gallium-68 particles is distributed differently in the body, which makes it easy to distinguish high-contrast clusters of bound gallium-68 with clear contours from blurry zones enriched with free gallium-68. Due to the increase in the content of gallium-68 (III) in the dosage form ready for administration compared with solutions of gallium-68 (III), it became possible to introduce it into the bloodstream in a bolus regimen - by short injections in a small volume of fluid.

In addition, it can be expected that due to the insignificant penetration of nanoparticles into the interstitial fluid, lymph and lymph nodes, the lymphotoxicity of gallium-68 in the particle-bound form will be lower than that of unbound analogues.

The present invention is based on the fact that, as it was unexpectedly discovered, the capacity of polylactide particles with non-covalently bound gallium (III) complexes far exceeds the capacity of particles with ligands covalently bonded to the surface of polylactide particles.

The above problem is solved due to the fact that in the method of producing biocompatible fine particles for in situ manufacturing of diagnostic tools for positron emission tomography by combining these particles with a solution containing gallium-68 (III) cations

(a) use at least bidentate aromatic ligands having donor atoms of a lone pair of electrons to form at least two coordination bonds with a gallium (III) cation, namely, oxygen atom (s) directly bonded to aromatic ring, and / or nitrogen atom (s) in the aromatic ring, wherein the stability constant lgβ1 of the complexes of said ligands with a gallium (III) cation in water under normal conditions is at least 5.0;

(b) using a solution containing lactic acid copolymers selected from the group consisting of copolymers of D- and L-lactic acid and copolymers of lactic and glycolic acid in a low-polar solvent;

(c) using an aqueous solution of a stabilizer;

(d) (i) combine the solution of ligand (a) in a low-polar solvent with solution (b) and at least three volumes of solution (c) and mix vigorously, or

(ii) combine the solution of ligand (a) in a polar solvent with solution (b), mix the mixture vigorously to obtain a highly homogeneous mixture, add at least three volumes of solution (c) and mix vigorously to obtain a highly homogeneous mixture;

(e) evaporating the highly homogeneous mixture obtained in stage (d) to obtain a suspension;

(e) filtering the suspension obtained in step (e) by means of a filter with a pore size of less than 30 μm to obtain a filtrate containing the above particles.

In a preferred embodiment of the method, a lyoprotectant is added to the filtrate obtained in step (e), frozen and lyophilized. Glucose, trehalose, lactose, sucrose and / or mannitol, preferably 1% mannitol, can be added as a lyoprotectant.

The term “lyoprotectant” refers to a compound that, when incorporated into a lyophilizable composition, will protect chemical compounds from the negative effects of freezing and evacuation, such as those typically associated with lyophilization, such as damage, adsorption and loss from the vacuum used in lyophilization.

The present invention is not limited to the use of a particular lyoprotectant; examples of suitable lyoprotectants include, but are not limited to, hydrocarbons such as saccharides, mono-, di- or polysaccharides, for example glucose, galactose, fructose, sucrose, trehalose, maltose, lactose, amylose, amylopectin, cyclodextrins, dextran, inulin, soluble starch, hydroxyethyl starch (HES), sugar alcohols, for example mannitol, sorbitol, and polyglycols, such as polyethylene glycols. A list of substances with a lyoprotective effect is given in Acta Pharm, Technol. 34 (3), pp. 129-139 (1988), the contents of which are incorporated herein by reference. These lyoprotective agents can be used individually or as mixtures of one or more compounds.

Also, the present invention is not limited to any particular amount of lyoprotector used. However, the optimal weight concentration of lyoprotective agents in the emulsion before lyophilization is from about 1 to about 25%, preferably from about 2 to about 20%, and even more preferably from about 5 to about 10%.

The freezing in step (g) may (but not necessarily) be carried out to a temperature of from -196 to -60 ° C, preferably to a temperature of -75 ° C.

At least 90% of the particles in the lyophilisate obtained in step (g) can have (but not necessarily) a size of 100 ÷ 800 nanometers, and when dispersed in a pharmaceutically acceptable carrier or solvent, the particles form a stable suspension suitable for intravenous administration in which at least 90% of nanoparticles have a size of 200 ÷ 800 nanometers.

Ligands can be (but not necessarily) selected from the group consisting of anthraquinones, flavones, hydroxyquinolines, triphenylmethanes having substituents containing an oxygen atom or containing a nitrogen atom in the aromatic ring.

Preferably, the ligands are selected from the group consisting of alizarin, quinalizarin, alizarin red, quercetin, 8-hydroxyquinoline and aluminone.

Said copolymer may have (but not necessarily) an intrinsic viscosity of 0.10 ÷ 0.30 dl / g.

Said copolymer may be (but not necessarily) a copolymer of D, L-lactic and glycolic acid with a lactic acid: glycolic acid ratio of from 5: 1 to 1: 5.

Preferably, said copolymer is hydrophilic and has an acid index above 1 meq KOH, more preferably above 1.2, even more preferably 1.5 meq KOH per gram of copolymer.

A low-polar solvent in which lactic acid copolymers are dissolved may be (but not necessarily) selected from the group consisting of ethyl acetate, chloroform, dichloromethane, or a mixture thereof.

As used herein, the term “low-polar solvent” refers to water-immiscible solvents, that is, to such solvents that when it is mixed with water, two separate phases are formed. Water-immiscible solvents are generally known in the art as apolar or non-polar solvents, in contrast to polar solvents (such as water). Non-water-miscible solvents are generally almost insoluble in water. For the purposes of the present invention, organic solvents suitable for emulsification with an aqueous solvent are those with a solubility in water of less than about 10 g / L. Preferably, the solubility of said solvent in water is about 1.0 g / L or less, more preferably about 0.2 g / L or less, and much more preferably about 0.01 g / L or less. Particularly preferred solvents are those with a solubility in water of 0.001 g / L or less. In particular, insoluble organic solvents (e.g. perfluorocarbons) can have a solubility of up to about 1.0 · 10-6 g / L (for example, perfluorooctane 1.66 · 10-6 g / L).

The organic solvent is preferably lyophilizable, i.e. said solvent has a sufficiently high vapor pressure at lyophilization temperatures, for example between -30 ° C and 0 ° C, to allow efficient and complete evaporation / sublimation within acceptable times, for example 24-48 hours. Preferably, the vapor pressure of the organic solvent is above about 0.2 kPa at 25 ° C.

The organic solvent can be selected from a wide range of solvents of any chemical nature, i.e. not miscible with water and lyophilized, as described above, and which are preferably liquid at room temperature (25 ° C). If a solvent with a boiling point lower than room temperature is used, the container containing the emulsifying mixture can advantageously be cooled below the boiling point of the specified solvent, for example, to 5 ° C or 0 ° C.

Suitable organic solvents include, but are not limited to, alkanes, such as branched or preferably straight (C5-C10) alkanes, for example pentane, hexane, heptane, octane, nonane, decane; alkenes such as (C5-C10) alkenes, for example 1-pentene, 2-pentene, 1-octene; cycloalkanes such as (C5-C8) cycloalkanes optionally substituted with one or two methyl groups, for example cyclopentane, cyclohexane, cyclooctane, 1-methylcyclohexane; aromatic hydrocarbons such as benzene and benzene derivatives substituted with one or two methyl or ethyl groups, for example benzene, toluene, ethylbenzene, 1,2-dimethylbenzene, 1,3-dimethylbenzene; alkyl ethers and ketones, such as dibutyl ether and diisopropyl ketone; halogenated hydrocarbons or ethers such as chloroform, carbon tetrachloride, 2-chloro-1- (difluoromethoxy) -1,1,2-trifluoroethane (enflurane), 2-chloro-2- (difluoromethoxy) -1,1,1- trifluoroethane (isoflurane), tetrachloro-1,1-difluoroethane and, in particular, perfluorinated hydrocarbons or ethers such as perfluoropentane, perfluorohexane, perfluoroheptane, perfluoromethylcyclohexane, perfluorooctane, perfluoronononeterfluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorodifluorofluorofluorofluorofluorofluorofluorodifluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorodperfluorofluorofluorofluorofluorofluoro ethyl perfluoroisobutyl e Fir and mixtures thereof.

Mixing in stage (d) (i) can be carried out (but not necessarily) 3 ÷ 10 minutes on a capacitive homogenizer at 19000 ÷ 30,000 rpm of the rotor, and then 3 ÷ 10 minutes on a high pressure homogenizer at a pressure of 20,000 ÷ 30,000 psi.

Mixing in stage (g) (ii) is preferably carried out 1.5 ÷ 10 minutes on a capacitive homogenizer at 19000 ÷ 30,000 rpm of the rotor.

The highly homogeneous mixture in step (e) can be evaporated (but not necessarily) on a rotary evaporator.

The filtering in step (e) can be carried out (but not necessarily) on a glass filter with a pore size of essentially 10 micrometers.

As the stabilizer in stage (c), an aqueous solution of polyvinyl alcohol can be used (but not necessarily), preferably a 1.0% w / v aqueous solution of polyvinyl alcohol with a molecular weight of 9.0 ÷ 80 kDa.

In one embodiment of the method, the particles obtained in step (e) are washed and then placed in a solution containing gallium-68 (III) cations.

In yet another embodiment of the method, the pH of the solution containing gallium-68 (III) cations is adjusted to a pH of 3.0-8.0, preferably to a pH of about 5.0.

Particles may contain (but not necessarily) a stoichiometric excess of ligand and metal cations in a solution containing gallium-68 (III) cations.

The particles may additionally conjugate (but not necessarily) with avidin and / or streptavidin to impart the ability to selectively bind to biospecific biotinylated ligands, preferably monoclonal antibodies.

The mentioned particles could additionally be conjugated (but not necessarily) with biospecific ligands.

Mentioned biospecific ligands may be (but not necessarily) selected from the group consisting of monoclonal antibodies, active fragments of peptide hormones, active fragments of integrin-binding proteins, and an RGD peptide.

Mentioned biospecific ligands may be selected (but not necessarily) from the group consisting of monoclonal antibodies specific for blood coagulation factors, preferably tissue thromboplastin.

Mentioned biospecific ligands may be selected (but not necessarily) from the group consisting of monoclonal antibodies specific for vascular endothelial growth factor (VEGF).

In another aspect, the present invention relates to a method for manufacturing a radiopharmaceutical for positron emission tomography, in which particles obtained by the above method are placed in a solution containing gallium-68 (III) with a pH of 3.0-8.0.

Said particles may be (but not necessarily) washed before being placed in said solution.

In another aspect, the invention relates to the use of particles obtained by the above method, in which the said particles are combined with a solution containing gallium-68 (III) cations and introduced into the human body before conducting positron emission tomography.

In a preferred embodiment, said particles may be (but not necessarily) washed before being placed in said solution.

In another aspect, the invention relates to biocompatible fine particles for the manufacture of a radiopharmaceutical for positron emission tomography, characterized in that they are obtained in accordance with the above method.

In another aspect, the invention relates to the use of particles obtained in accordance with the above method for the manufacture of a radiopharmaceutical for positron emission tomography.

Positron emission tomography can (but not necessarily) be combined with computed tomography.

Positron emission tomography can also (but not necessarily) be combined with magnetic resonance (nuclear magnetic) tomography.

As a contrast, the aforementioned particles with pre-sorbed gadolinium (III) cations can (but not necessarily) be used in said magnetic resonance imaging.

In another aspect, the invention relates to a diagnostic radiopharmaceutical composition for positron emission tomography, characterized in that it contains an effective amount of gallium-68 (III) associated with particles made by the above method.

An effective amount per body weight of 50 ÷ 90 kg corresponds to an activity of 2 ÷ 5 mCi.

In one preferred embodiment, after combining, the solution is incubated, centrifuged, the supernatant removed and resuspended in a pharmaceutically acceptable solution for parenteral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the NMR spectrum of the modified alizarin in example 1.

Figure 2 shows the spectrum of Resomer 752H covalently modified with alizarin, as in example 1.

Figure 3 shows the absorption spectra of xylenol orange and its complex with gallium (III) in an aqueous-organic medium (water-DMSO, pH <1.0 (0.1 n H ₂ SO ₄ )).

Figure 4 shows a calibration graph for determining free gallium (water-DMSO, pH <1.0 (0.1 n H ₂ SO ₄ )); wavelength of 550 nm.

Figure 5 shows a calibration graph for determining the total gallium content (water-DMSO, pH <1.0 (0.1 n H ₂ SO ₄ )); wavelength of 550 nm.

6 shows microphotographs of the samples of example 2.

DETAILED DESCRIPTION OF THE INVENTION

The content of gallium, which is a complex in the composition of polylactide particles, as well as the degree of inclusion of gallium (III) in polylactide particles, is determined by spectrophotometry of solutions using an indicator - xylenol orange, forming colored complexes with gallium (III) at pH 1.0 or less . This method is based on the fact that in an acidic environment, gallium (III) complexes with the analyzed ligands are destroyed and do not interfere with the determination of gallium content in the form of its complex with an indicator. The absorption is measured at a wavelength of 550 nm (see figure 3).

The concentration of free (not bound to polylactide particles) gallium (III) is determined according to a previously constructed calibration curve (absorbance is measured at a wavelength of 550 nm) (Fig. 1 and Fig. 2).

To identify a possible interfering effect of the intrinsic absorption of organic reagents, the concentration in the absence and presence of organic reagents is determined. The concentration of organic reagents in the analyzed solution corresponded to the maximum theoretically possible content of organic reagents in the preparation.

The validation results of the method for determining gallium (III) on the example of polylactide particles with quercetin, quinalizarin and alizarin are presented in table 1+.

Table 1 No organic reagents added. Introduced, mcg / ml Found, mcg / ml S _r ,% 3.46 3.49 1.3 1,66 1,61 2 With the addition of organic reagents (0.1 ml of solution ^* ) 1.66 + quercetin 1,63 3 1.66 + quinalizarin 1.81 2.2 1.66 + alizarin 1,58 3 Note: the concentration of quercetin, quinalizarin and alizarin was 1.86 · 10 ^-5 mol / ml, 1.25 · 10 ^-5 mol / ml and 0.94 · 10 ^-5 mol / ml, respectively.

As follows from table 1, the presence of quinalizarin in solution leads to an overestimation of the results of determining the concentration of gallium. Therefore, to determine gallium in solutions containing this reagent, the additive method is used.

To determine the concentration of free gallium (III) in a suspension of polylactide particles, the lyophilisate is resuspended in a volume of water equal to 0.5 from the source, quantitatively transferred to a centrifuge tube, polylactide particles are separated by centrifugation (Avanti J-301, 180C, 20,000 rpm, 30 min ) and take the supernatant. Next, to a mixture of 0.1% aqueous indicator solution (0.2 ml), 1 N. H ₂ SO ₄ (0.1 ml) and 0.5 ml of dimethyl sulfoxide add 0.2 ml of the supernatant, mix, incubate for 30 min at room temperature and determine the concentration of free gallium (III) according to a previously constructed calibration schedule (figure 4).

To determine the total content of gallium (III) in polylactide their particles are dissolved in dimethyl sulfoxide and according to a previously constructed calibration schedule for solutions of gallium (III) in a mixture of water: DMSO = 1: 1 (vol.: vol.) (figure 5). In this case, take into account that the concentration of the polylactide solution in 50% dimethyl sulfoxide should not exceed 1 mg / ml.

To determine the total gallium (III) content in the lyophilisate of polylactide particles, the lyophilisate obtained from 1 or 2 ml, suspensions of polylactide particles are dissolved in 5.0 or 10.00 ml of dimethyl sulfoxide, respectively. Into volumetric flasks with a capacity of 10.00 ml add 2 ml of a 0.1% aqueous solution of the indicator, 1 ml of H ₂ SO ₄ (1 N), an aliquot of the suspension of a sample of PLGA particles (from 1 to 5 ml, if necessary) and adjust the content of dimethyl sulfoxide in a flask up to 5.00 ml. Next, the volume of the solution in the flask was adjusted to the mark with distilled water, stirred, and after 30 minutes the optical density of the solutions was measured.

To determine the total gallium (III) content in the precipitates of polylactide particles after centrifugation, the precipitate remaining in the centrifuge tube after sampling the supernatant is resuspended and lyophilized again. After lyophilization, the contents of the vial are dissolved in dimethyl sulfoxide (based on 5 ml of dimethyl sulfoxide per precipitate in the vial obtained from 1 ml of the original lyophilisate). 2 ml of a 0.1% aqueous solution of the indicator, 1 ml of 1 n H ₂ SO ₄ and 5 ml of a solution of PLGA particles in dimethyl sulfoxide are added to a 10 ml flask (if there was only 5 ml in the bottle, it is transferred quantitatively). The solution was adjusted to the mark with water, stirred, and after 30 minutes, absorbance was measured at 550 nm.

The above method for determining gallium (III) in a lyophilisate was validated using a lyophilisate of polylactide particles containing quinalizarin. A slight interfering effect of quinalizarin's own color on the results of determining the concentration of gallium was found. Therefore, to determine gallium in lyophilisates of polylactide particles containing quinalizatrin, the additive method is used. The validation results of the method are presented in table 2.

table 2 Introduced, mcg / ml Found, mcg / ml Sample 1181 0.443 Sample 1181 + additive (0.700) 1,184

Thus, the error is 3.4%, and the method allows with sufficient accuracy to determine the content of gallium in samples of polylactide particles.

The degree of binding is calculated as the percentage ratio of the content of gallium (III) in the precipitate of polylactide particles to the content of gallium (III) in the supernatant obtained after centrifugation of a suspension of polylactide particles after their incubation in a solution of gallium (III).

EXAMPLE 1 (COMPARATIVE)

Preparation and study of polylactide particles containing a covalently bonded ligand forming stable complexes with gallium (III)

Stage 1

Obtaining modified alizarin for covalent binding to polylactide particles

Dissolve 0.6 g of alizarin 1 (2.5 mmol), 1.0 g (6.3 mmol) of carbethoxypiperazine, 0.14 g of KOH and 0.07 g of paraform in 2.5 ml of water and 2.5 ml of ethanol and stirred at 75 ° C for 24 hours. Then add another 0.1 g of paraform and stir for another 24 hours at the same temperature. After this, the solvent is distilled off, 30 ml of water are added to the residue and acidified with acetic acid until the color of the reaction mixture changes from violet to tan. The precipitate of compound 2 is filtered off, washed with water and mixed with 15 ml of water and 15 ml of HCl. The resulting solution is boiled with stirring for 4 hours. After cooling, the solution was filtered, the filtrate was evaporated to dryness. 20 ml of ethanol was added to the residue and evaporated again. After that, the residue is heated with 10 ml of ethanol to a boil. 30 ml of acetonitrile is added to the boiling solution. The precipitate formed is filtered off, washed with acetonitrile and dried. 227 mg of product is obtained.

Modified alizarin (220 mg, 0.54 mmol) was mixed with a resomer 360 mg Resomer 752H 3 (approximately 0.097 mmol of free carboxyl groups), 192 mg EDC (0.65 mmol), and 258 mg of DIPEA (2 mmol) were mixed in 8. 0 ml of dichloromethane for 3 days. The mixture was poured into 100 ml of water and 50 ml of ethyl acetate. The organic phase is separated and washed 8 times in 100 ml of distilled water. At each washing, 10 ml of ethyl acetate is added. Rinse until the wash solution is no longer stained. After that, the organic phase is washed 3-4 times with 100 ml of 1% HCl and then 1 time with 100 ml of water. The organic phase is dried with sodium sulfate and evaporated to a volume of 10 ml. To the residue, 30 ml of hexane are added with stirring. The precipitate formed is filtered and dried. 205 mg of polymer are obtained.

From a comparison of the NMR spectra (solvent - DMSO, frequency 400.46 MHz) of modified alizarin 2 and the resomer 752H with grafted alizarin it follows that the mass content of alizarin in the product is approximately 5.8%. In addition, the product contains minor impurities of hexane and ethyl acetate.

Stage 2

Obtaining polylactide particles containing covalently linked alizarin

A solution of polylactide (200 mg) and 50 mg of alizarin-modified polylactide obtained in step 1 in 5 ml of dichloromethane was mixed with 25 ml of a 0.5% solution of polyvinyl alcohol (9-10 thousand Daltons), homogenized for 2 min on a Turax homogenizer with 23600 rpm, then - 5 min on an Avestin homogenizer at 15000-20000 psi. Dichloromethane was rotor driven to a pressure of 15 mbar, filtered on a glass filter with 10 μm pores, 2.5% mannitol was added, frozen at -70 ° C and lyophilized on an ALPHA 2-4LSC apparatus (Martin Christ GmbH).

Stage 3

Sorption of gallium (III) polylactide particles obtained in stage 2

The polylactide particles obtained in stage 2 were resuspended and washed three times with distilled water from free alizarin by precipitation-resuspension in distilled water (centrifugation for 30 minutes at 13.2 thousand rpm and a temperature of 18 ° C in a 5415R centrifuge (Eppendorf, Germany) )

Next, the particle precipitate was resuspended in 1 ml of acetate buffer at pH 5.0, a solution of gallium (III) sulfate with a concentration of 12.84 mg / ml was added and stirred on a magnetic stirrer for 2 hours at 250 rpm. The supernatant and sediment were separated by centrifugation for 30 minutes in a Beckman centrifuge at 20,000 rpm and a temperature of 18 ° C.

Stage 4

Determination of gallium (III) content in the precipitate of polylactide particles obtained in stage 3 and in the supernatant

Alizarin-modified polylactide particles (samples nos. 1324 and 1325) were prepared as described above using Resomer 752H / Resomer752H-alizarin (5: 1) and Resomer 502H / Resomer502H-alizarin (5: 1) polymers, respectively. Sorption of gallium (III) is carried out in a solution of gallium (III) sulfate containing a known excess of gallium (III) with respect to the alizarin groups (about 3: 1 and 2: 1 for samples 1324 and 1325, respectively). The content of free gallium (III) in the supernatant and gallium (III) associated with covalently modified alizarin polylactide particles was determined spectrophotometrically, as described above. The results are presented in table 3.

Table 3 No. ZP, mV ZAVeD *, nm PDI About. PSD, nm (%) The content of gallium (III), μg / mg PLGA 1324 -0.456 ± 0.186 160.1 ± 0.1708 0.097 ± 0.009 169 (100%) 0.23 187.2 0.21 1325 -2.82 ± 0.349 173.6 ± 0.6557 0.097 ± 0.007 (one hundred%) * - dimensions are measured by diluting the sample 50 times.

EXAMPLE 2

Obtaining polylactide particles with non-covalently bound ligands capable of forming stable complexes with gallium (III)

Bidentate ligands are used that satisfy the following requirements:

(1) ligands belong to the class of arenes in which oxygen atoms directly bonded to the aromatic ring and / or nitrogen atoms of the aromatic ring serve as donors of a lone electron pair to form two coordination bonds with the gallium (III) cation;

(2) the stability constant of lgβ1 is not less than 5.0.

It is desirable to use ligands that form complexes with gallium (III) with an extraction coefficient of non-polar solvents immiscible with water from solutions in water, polar solvents, or mixtures thereof, greater than 10, and it is especially desirable when the additional free ligand is soluble in a polar solvent better, than its complex with gallium (III).

Preferred classes of ligands are anthraquinones, flavones, hydroxyquinolines and triphenylmethanes.

Specific examples of suitable ligands are alizarin, quinalizarin, alizarin red, quercetin, 8-hydroxyquinoline and aluminone (table 4).

Table 4 Title Formula Gallium (III) complex stoichiometry: ligand

Alizarin 1: 3

Quinalizarin 1: 3

Quercetin 1: 3

8-hydroxyquinoline 1: 3

Stage 1

Obtaining polylactide particles containing non-covalently bound gallium complexes with ligands

To obtain polylactide particles, 2 methods were used.

The first way. Combine 5 ml of an organic solvent (ethyl acetate, chloroform or dichloromethane) containing 50 mg / ml Resomer 752H or Resomer 202S, and a ligand (based on polymer: ligand 50: 1 ÷ 1000: 1 m / m) and 25 ml of an aqueous solution of polyvinyl alcohol with a molecular weight of 9-10 kDa (1%, w / v). The resulting mixture is homogenized for 5 minutes on an Ultra-Turrax homogenizer at 23,600 rpm, then for 5 minutes on an Avestin high-pressure homogenizer at a pressure of 20,000-25,000 psi. Then remove the solvent on a rotary evaporator. The resulting suspension is filtered through a glass filter with a pore size of 10 μm, a lyoprotector, 1% mannitol is added, 2 ml are poured into vials, frozen at -70 ° C and lyophilized. Samples 1163-1191 were obtained by this method.

The second way. Dissolve 250 mg of Resomer 502H in 3 ml of dichloromethane. To the resulting solution was added 2.0 ml of an aqueous solution containing 5–25 mg of alizarin red C, the mixture was homogenized for 2 min on a Turrax homogenizer at 23,600 rpm and transferred to a glass with 25 ml of an aqueous solution of polyvinyl alcohol with a molecular weight of 9-10 kDa (1%, w / v). Re-homogenize the mixture for 5 minutes on a Turrax homogenizer at 23,600 rpm, then remove the solvent on the rotor. The suspension is filtered through a glass filter with a pore size of 10 μm, the volume is measured and 1% mannitol is added. Samples are poured into vials (2 ml each), frozen at -70 ° C and lyophilized. Samples 1324-1331 were obtained by this method.

Stage 2

Obtaining a solution of the gallium (III) complex and the ligand bound to polylactide particles

The polylactide particles obtained in step 1 of example 2 were resuspended and processed in accordance with step 3 of example 1 to obtain a supernatant and sediment. Next, the characteristics of the particles are determined in accordance with step 4 of example 1.

The characteristics of some PLGA particles containing gallium (III) complexes with ligands are shown in Tables 6 and 9.

Table 7 presents the results of determining the content of gallium (III) in polylactide particles after sorption.

Table 8 presents the results of determining the degree of binding of gallium (III) polylactide particles with alizarin red S and aluminon before and after washing for 2 hours.

Photographs of polylactide particles after washing are presented in Figure 6. It can be clearly seen that the particles are round and there is a clear contrast rim around - the surface is covered with a contrast agent, apparently a gallium complex with ligands.

Table 6 No. Polymer Ligand ** ZP, MV ZAVeD *, nm PDI About. PSD,% 1324 Resomer 502H Alizarin Red S -1.51 ± 0.160 151.1 ± 1.679 0.087 ± 0.014 156 (100%) 1325 Resomer 502H aluminone -21.4 ± 2.41 157.5 ± 1.721 0.122 ± 0.014 166 (100%) * - dimensions are measured by diluting the sample 50 times; ** - ligand: polymer ratio 10: 1 w / w.

Table 7 No. The content of gallium (III), mcg / vial * The degree of inclusion,% ** In the supernatant Sediment Sediment and supernatant 1324 80.3 87.6 167.8 56.0 1325 86.5 91.8 178.3 52.6 1326 32.8 141.2 174.0 82.0 1327 48.9 95.0 143.9 73,2 1328 61.5 99,4 160.9 66.3 1329 29.92 121.7 151.6 83.6 1330 55.96 80.1 136.1 69.3 1331 80.7 68.8 149.5 55.8 1324 (washed) 88.9 61.6 150.5 51,2 1325 (washed) 83,2 63.1 146.3 54,4 * - the calculated value of 209.4 μg / vial. ** - the ratio of the amount of gallium (III) in the sediment to the total amount of gallium in the sediment and in the supernatant, taking into account the loss of sorption in the vial.

Table 8 No. Washing Ligand The inclusion of gallium (III),% 1322 before washing Alizarin Red S 84.9 1322 after washing Alizarin Red S 64,2 1323 before washing aluminone 87.9 1323 after washing aluminone 66,4

Comparison of the effectiveness of polylactide particles in example 1 and 2

It was expected that the degree of gallium binding by particles with a non-covalent bond of the ligand and the polymer core will be significantly lower than in the case of a ligand covalently bonded to the polylactide forming the particle core. However, it was unexpectedly discovered that such particles bind gallium (III) better than the particles of example 1, as evidenced by the data in tables 3 and 9. The relatively small inclusion of gallium (III) in the samples of example 1, apparently, is associated with a low sorption capacity of the surface particles.

The specific sorption capacity of the polylactide particles of the invention with respect to gallium (III) cations is about 2 times higher than that of the comparative particles of Example 1, and can reach 80 ÷ 100 μg of gallium (III) per 10 mg of polylactide. Moreover, the residual content of gallium (III) in a sample of particles according to the invention with an initial content of 100 μg gallium (III) / mg of polymer during incubation in blood plasma in the first 15 minutes and then will always be higher than that of comparative particles with an initial content of 40 mg gallium (III) / mg polymer.

Table 9 No. Process parameters The composition of polylactide particles The inclusion of gallium (III),% ZAVeD, nm (decomposed 100 times) PDI Polymer Solvent Ligand The number of ligand-gallium (III) complex, μg / mg PLGA Gallium (III) complex PLGA, μg / mg The content of gallium (III) total, μg / ml lyophilisate The content of gallium (III), μg / mg PLGA 1163 Resomer 752H Ethyl acetate Quercetin 27.1 19.5 14.3 1.4 94 154.1 ± 2.562 0.206 ± 0.009 1164 Resomer 752H Ethyl acetate Quinalizarin 18.3 11,4 9.2 0.9 88 162.4 ± 2.173 0.219 ± 0.016 1165 Resomer 752H Ethyl acetate Alizarin 14.7 6.8 5,6 0.6 96 183.9 ± 2.608 0.342 ± 0.047 1190 Resomer 202S Chloroform 8-hydroxy-quinoline 167.2 134.1 185 18.5 85 167.3 ± 2.083 0.123 ± 0.011 1191 Resomer 202S Methylene chloride 8-hydroxy-quinoline 167.2 134.1 110 11.0 84 151.3 ± 1.511 0.087 ± 0.005 Note: stabilizer - polyvinyl alcohol 1% w / v, molecular weight 9-10 thousand Daltons.

Claims (33)

1. A method of producing biocompatible highly dispersed polylactide particles for in situ manufacturing of diagnostic tools for positron emission tomography by combining these particles with a solution containing gallium-68 (III) cations, in which:
(a) use at least bidentate aromatic ligands, namely quercetin, quinalizarin, alizarin and 8-hydroxyquinoline;
(b) using a solution containing lactic acid copolymers selected from the group consisting of copolymers of D- and L-lactic acid and copolymers of lactic and glycolic acid in a low-polar solvent;
(c) use an aqueous solution of a stabilizer, namely polyvinyl alcohol with a molecular weight of 9.0 ÷ 80 kDa;
(d) combine the solution of ligand (a) in a polar solvent with solution (b), mix the mixture vigorously to obtain a highly homogeneous mixture, add at least three volumes of solution (c) and mix vigorously to obtain a highly homogeneous mixture;
(e) evaporating the highly homogeneous mixture obtained in stage (d) to obtain a suspension;
(e) filtering the suspension obtained in step (e) by means of a filter with a pore size of less than 30 μm to obtain a filtrate containing the above particles;
(g) a lyoprotectant is added to the filtrate obtained in step (e), frozen and lyophilized. 2. The method according to claim 1, in which glucose, trehalose, lactose, sucrose and / or mannitol, preferably 1% mannitol, are added as a lyoprotector. 3. The method according to claim 1, in which at stage (g) freezing is carried out to a temperature of from -196 to -60 ° C, preferably to a temperature of -75 ° C. 4. The method according to claim 1, wherein in the lyophilisate obtained in step (g), at least 90% of the particles have a size of 100 ÷ 800 nm, and when dispersed in a pharmaceutically acceptable carrier or solvent, a stable suspension suitable for intravenous administration is formed, which at least 90% of the nanoparticles have a size of 200 ÷ 800 nm. 5. The method according to claim 1, wherein said copolymer has an intrinsic viscosity of 0.10 ÷ 0.30 dl / g. 6. The method according to claim 1, wherein said copolymer is a copolymer of D, L-lactic and glycolic acid with a ratio of lactic acid: glycolic acid from 5: 1 to 1: 5. 7. The method according to claim 1, wherein said copolymer is hydrophilic and has an acid index above 1 meq KOH, more preferably above 1.2, even more preferably 1.5 meq KOH per gram of copolymer. 8. The method according to claim 1, wherein the low-polar solvent in which the copolymers of lactic acid are dissolved is selected from the group consisting of ethyl acetate, chloroform, dichloromethane, or a mixture thereof. 9. The method according to claim 1, in which the mixing in stage (d) is carried out for 3 ÷ 10 minutes on a capacitive homogenizer at 19000 ÷ 30,000 rpm of the rotor per minute, and then 3 ÷ 10 minutes on a high pressure homogenizer at a pressure of 20,000 ÷ 30,000 psi. 10. The method according to claim 1, in which the mixing in stage (g) is carried out 1.5 ÷ 10 min on a capacitive homogenizer at 19000 ÷ 30000 rpm of the rotor per minute. 11. The method according to claim 1, wherein the highly homogeneous mixture in step (e) is evaporated on a rotary evaporator. 12. The method according to claim 1, in which the filtering in stage (e) is carried out on a glass filter with a pore size of essentially 10 μm. 13. The method according to claim 1, in which as the stabilizer in stage (C) use an aqueous solution of polyvinyl alcohol, preferably a 1.0% w / v aqueous solution of polyvinyl alcohol with a molecular weight of 9.0 ÷ 80 kDa. 14. The method according to claim 1, in which the particles obtained in step (e) are washed and optionally placed in a solution containing gallium-68 (III) cations. 15. The method according to 14, in which the pH of the solution containing gallium-68 (III) cations is adjusted to a pH of 3.0 ÷ 8.0, preferably to a pH of about 5.0. 16. The method according to 14, in which the particles contain a stoichiometric excess of ligand and metal cations in a solution containing gallium-68 (III) cations. 17. The method of claim 1, wherein said particles are further conjugated to avidin and / or streptavidin to confer the ability to selectively bind biospecific biotinylated ligands, preferably monoclonal antibodies. 18. The method according to claim 1, in which the said particles are additionally conjugated with biospecific ligands. 19. The method of claim 18, wherein said biospecific ligands are selected from the group consisting of monoclonal antibodies, active fragments of peptide hormones, active fragments of integrin-binding proteins, and an RGD peptide. 20. The method of claim 18, wherein said biospecific ligands are selected from the group consisting of monoclonal antibodies specific for coagulation factors, preferably tissue thromboplastin. 21. The method of claim 18, wherein said biospecific ligands are selected from the group consisting of monoclonal antibodies specific for vascular endothelial growth factor (VEGF). 22. A method of manufacturing a radiopharmaceutical for positron emission tomography, in which particles obtained in accordance with the method according to any one of claims 1 to 16, are placed in a solution containing gallium-68 (III) with a pH of 3.0 ÷ 8.0 . 23. The method of claim 22, wherein said particles are washed before being placed in said solution. 24. The method of using the particles obtained by the method according to any one of claims 1 to 16 for conducting positron emission tomography, in which said particles are combined with a solution containing gallium-68 (III) cations, and introduced into the human body before conducting positron emission tomography. 25. The method according to paragraph 24, wherein said particles are washed before being placed in said solution. 26. Biocompatible fine particles for the manufacture of a radiopharmaceutical for positron emission tomography, characterized in that they are obtained in accordance with the method according to any one of claims 1 to 16. 27. The use of particles obtained in accordance with the method according to any one of claims 1 to 16, for the manufacture of a radiopharmaceutical for positron emission tomography. 28. The application of claim 27, wherein the positron emission tomography is combined with computed tomography. 29. The application of claim 27, wherein the positron emission tomography is combined with magnetic resonance (nuclear magnetic) tomography. 30. The application of clause 29, in which, as a contrast in said magnetic resonance imaging, said particles with pre-sorbed gadolinium (III) cations are used. 31. Diagnostic radiopharmaceutical composition for positron emission tomography, characterized in that it contains an effective amount of gallium-68 (III) associated with particles made by the method according to any one of claims 1 to 16. 32. The composition according to p, in which the effective amount per body weight of 50 ÷ 90 kg corresponds to an activity of 2 ÷ 5 mCi. 33. The composition according to p, in which, after combining, the solution is incubated, centrifuged, the supernatant is removed and resuspended in a pharmaceutically acceptable solution for parenteral administration.

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