RU2799325C2 - LYOPHILIZATE FOR OBTAINING DIAGNOSTIC RADIOPHARMACEUTICAL DRUG BASED ON RADIONUCLIDE 99mTc - Google Patents

Abstract

FIELD: radiopharmaceutics.

SUBSTANCE: invention relates to a lyophilisate for the preparation of a diagnostic radiopharmaceutical drug based on ^99mTc radionuclide. A lyophilisate for the production of a diagnostic radiopharmaceutical based on the ^99mTc radionuclide, consisting of a mixture of the following: a reducing agent — tin dichloride dihydrate (SnCl₂⋅2H₂O), soligand — tricine (C₆H₁₃NO₅), carrier of radionuclide atoms — PSMA-tropic molecule Hynic-iPSMA, solicand EDDA (C₆H₁₂N₂O₄), as well as sodium gluconate characterized by the composition including 15 mg of tricine, 0.015 mg of tin dichloride dihydrate, 20 mg of sodium gluconate, 5 mg of EDDA, and 0.025 mg of Hynic-iPSMA.

EFFECT: obtaining an injection solution of a diagnostic radiopharmaceutical drug based on ^99mTc-EDDA/Hynic-iPSMA metal complex with a radiochemical yield of at least 98% and a higher specific activity than stated in world practice.

2 cl, 9 dwg, 2 tbl, 2 ex

Description

The invention relates to the field of nuclear medicine, in particular to radiopharmaceutics for visualizing the localization of cells overexpressing prostate-specific membrane antigen receptors (PSMA-overexpressing cells) of prostate cancer using single-photon computed tomography (SPECT-CT) or planar scintigraphy (PS).

Currently, among the male population of Russia, prostate cancer in terms of incidence ranks second in prevalence and is second only to malignant tumors of the pulmonary system. According to statistics for 2019, 15.7% of all malignant diseases of the male population are prostate cancer (PCa), and the growth rate over the past ten years has been about 80% (number of diseases per hundred thousand population).

In addition to the instrumental methods for diagnosing this disease already used in clinical practice (determination of the level of PSA in the blood, ultrasound, MRI), currently in world practice, and also with some delay in Russia, for the purpose of obtaining information on the localization of foci of malignant neoplasms (MN) ) in prostate cancer, including metastasis, a diagnostic method using targeted tumorotropic radiopharmaceutical drugs (RFLP) is used, the distribution of which is visualized, depending on the nuclear-physical characteristics of the radionuclide, using the method of positron emission computed tomography (PET- CT) in the case of using a positron-emitting radionuclide, or by the method of single-photon emission computed tomography or planar scintigraphy, in the case of using a γ-emitting radionuclide in the photon energy range ≈ 100 - 250 keV.

PSMA is a type II transmembrane glycoprotein consisting of 750 amino acids, which is distributed on the surface of PCa cells and is highly overexpressed by them, in particular in androgen-independent, advanced and metastatic stages of the disease. The latter fact is the most important, since in almost all cases PCa becomes androgen-independent over time. Thus, PSMA has the potential to be used as a promising target for the diagnosis of this disease, since it is common at all stages and has selective overexpression (only in the prostate), is present on the cell surface, but does not enter the bloodstream, and has an enzymatic or signaling activity (EA Patent 037512 B1, Improved 18f-labeled prostate specific membrane antigen (PSMA) inhibitors and their use as imaging agents in prostate cancer) (FIG. 1). It has been established that up to 10 ⁶ PSMA molecules are present in a PCa cell, which contributes to the possibility of selective accumulation of the radionuclide on the surface of PCa cells through the use of radionuclide-containing transport target molecules with increased affinity for PSMA. The molecule of the general structure EDDA/Tricine ^-99m Tc-Hynic-2Nal-Lys-urea-Glu ( ^99m Tc-Hynic-iPSMA), which is the main diagnostic agent in a radiopharmaceutical drug obtained from a set of lyophilized reagents.

In world practice, for targeted delivery of various radionuclides to PCa cells, as well as to metastases, compounds containing the Glu-urea-Lys urea motif responsible for binding to PCa cells, namely, zinc-containing domains in PSMA (HyunsooHa, HongmokKwon, TaehyeongLim , JaebongJang, Song-KyuPark, YoungjooByun, "Inhibitorsofprostate-specificmembraneantigeninthediagnosisandtherapyofmetastaticprostatecancer - areviewofpatentliterature", / Expertopinionontherapeuticpatents // 2021, doi:10.1080/13543776.2021.1878145).

This motif remains unchanged, and the world variety of different molecules used to deliver radionuclides to PCa cells is achieved by modifying the linker in the composition of such a molecule, as well as a chelator (in the case of using a metal radionuclide) or a fragment for introducing a halogen ion (in the case of using a halogen radionuclide). , in particular, ¹⁸ F). These molecules differ in the linkor - a large part of the molecule between the unchanged Glu-urea-Lys motif and the associated radionuclide. The structure of the linker largely influences the behavior of the molecule in the body.

Further, the use of molecules containing a chelating agent will be considered in more detail, without affecting molecules with a fragment for introducing a halogen ion. At the same time, we will consider PSMA-active molecules containing chelating agents for binding specifically the technetium-99 radionuclide, without affecting other diagnostic radionuclides, and as a result, chelators that form complex compounds with radionuclides that are not within the scope of our scientific interests.

The most common method of radionuclide diagnosis of the localization of PSMA-overexpressing PCa cells is the method of visualizing the distribution of the radionuclide in the patient's body using single-photon emission computed tomography (SPECT-CT) or planar scintigraphy (PS) (Kumar A., Kireev V.S., "Review Russian market of nuclear medicine”, / Fundamental Research // No. 2, 134-138, 2018, doi: 10.17513/fr.42088). The main difference from the PET-CT study is the lower accuracy of the method, or rather, the resulting image of the biodistribution of the γ-emitting radionuclide in the patient's body, which is primarily due to the lower energy of the emitted / detected γ-quanta compared to the energy generated during positron annihilation and electron, as well as the absence of a duplicate γ-quantum at an angle of 180°, as in PET/CT imaging.

At the same time, in quantitative terms, the availability of equipment for SPECT-CT and PS imaging is almost an order of magnitude greater compared to PET / CT (Kumar A., Kireev V.S., "Overview of the Russian market of nuclear medicine", / Basic research // No. 2, 134-138, 2018, doi: 10.17513/fr.42088), which is an indisputable advantage of this imaging method compared to PET-CT examination. Also, one study on SPECT-CT, and even more so with PS, on average, is much cheaper than a similar study on PET-CT.

The most common diagnostic radionuclide for SPECT/CT imaging is the metastable isomer of technetium ^99m Tc (T _1/2 = 6.01 h), the deexcitation of which and the transition to ⁹⁹ Tc occurs with the emission of a γ-quantum with an energy of about 140 keV, which allows to detect the localization of this decay in the patient's body by SPECT-CT or PS, and as a result, to visualize the location of malignant neoplasms, including metastases, using tumoritropic RFLP based on this radionuclide, in particular, as part of a complex with molecules with increased affinity to PSMA receptors described above.

In world practice, several PSMA-active complex compounds with ^99m Tc are known, containing the Glu-urea-Lys fragment and providing visualization of the localization of prostate cancer cells by SPECT/CT or PS. For example, technetium- and rhenium-bis(heteroaryl) complexes and methods of their application for PSMA inhibition (RU 2532912 C2) are known, including a transport molecule containing Glu-urea-Glu (similar to Glu-urea-Lys) fragment, the complex is molecules with radionuclides of technetium ( ^99m Tc) or rhenium ( ¹⁸⁶ Re, ¹⁸⁸ Re) metals, as well as the use of such complexes for visualization of PSMA-overexpressing PCa cells, or their therapy in the case of using metal complexes with rhenium radioisotopes. A special case of this invention is the ^99m Tc-MIP-1404 molecule, with the general formula:

.

.

Clinical studies have been conducted on this complex compound (Kevin Mark Slawin et al, “A phase II study of ^99m Tc-trofolastat (MIP-1404) SPECT/CT to identify and localize prostate cancer in high-risk patients undergoing radical prostatectomy (RP) and extended pelvic lymph node dissection (EPLND) compared to histopathology: An interim analysis.”2014 GenitourinaryCancerSymposium 2014 abstracts, / JournalofClinicalOncology // Vol. To obtain this radiopharmaceutical drug, it is necessary to first convert the ^{pertechnetate} anions into the tricarboxylic form, and in general, a more complicated method for obtaining the target metal complex molecule than in the solution claimed by us. transfer of pertechnetate anions with an activity of about 1500 MBq, obtained from the generator in the form of ^99m TcO ₄ ^- into a tricarbonyl intermediate reactive form [ ^99m Tc (CO) ₃ (H ₂ O) ₃ ] ⁺ , after which, at the second stage, the interaction of tricarbonyl technetium- 99m with a MIP-1404 molecule (100 μg) when heated to 100°C for 30 minutes, after which, in the third stage, the solvent is evaporated, followed by the dissolution of the resulting dry radioactive residue in a mixture of trifluoroacetic acid and dichloromethane for 45 minutes at room temperature to remove the protective tert-butyl groups, after which, at the fourth stage, the resulting reaction mixture was concentrated using a rotary evaporator, followed by purification on an HPLC column, after which the target product was dissolved in an isotonic solution of 0.9% sodium chloride. As a result, a solution suitable for injection was obtained, in which the radiochemical purity of the formed metal complex ^99m Tc-MIP-1404 was at least 95%, and the radiochemical yield reached 70% (ShawnM.Hillier, KevinP. Maresca, GenliangLu, RossD. Merkin, John C. Marquis, Craig N. Zimmerman, William C. Eckelman, John L. Joyal, and John W. Babich, " ^99m Tc-LabeledSmall-MoleculeInhibitorsofProstate-pecificMembraneAntigenforMolecularImagingofProstateCancer", / THEJOURNALOFNUCLEARMEDICINE // Vol. 54 (8), 1369-13 76, 2013, doi: 0.2967/jnumed.112.116624).

However, the known solution is quite laborious in obtaining the target metal complex, both in time and in the number of operations, and secondly, the low radiochemical yield and radiochemical purity of the final product. Therefore, in the decision we defend, it was decided not to resort to the concept of chelation of technetium-99m in the form of a tricarbonyl cation, but to use a faster and simpler method - reduction with tin halides, in particular tin dichloride.

For example, the procedure for obtaining the compound ^99m Tc-PSMA-I&S (StephanieRobu, MargretSchottelius, MatthiasEiber, TobiasMaurer, JürgenGschwend, MarkusSchwaiger, and Hans-JürgenWester, “PreclinicalEvaluationandFirstPatientApplicationof ^99m Tc-PSMA -I&SforSPECTImagingandRadioguidedSurgeryinProstateCancer", / THEJOURNALOFNUCLEARMEDICINE // Vol. 58 (2), 235-242, 2017, doi: 10.2967/jnumed.116.178939), used to visualize the localization of PSMA-overexpressing PCa cells by SPECT-CT or PS, in which the technetium metal complex is used as an imaging agent. 99m, also containing a Glu-urea-Lys fragment - namely: ^99m Tc-MAS3-PSMA ( ^99m Tc-PSMA-I&S), with the general formula:

.

.

In this development, technetium binds to a sulfur atom and three nitrogen atoms that are part of the MAS3 chelator, which is mercaptoacetyl-tri-serine. This chelator forms a bond with technetium-99m when heated to 90-100°C for 20 minutes, while, as noted above, there is no need to convert the pertechnetate anion to the tricarbonyl form, and for the purposes of binding technetium-99m to the MAS3-PSMA molecule, the reducing agent is tin dichloride.

However, the disadvantages of the preparation technique include, firstly, the rather complex peptide structure of the transport molecule MAS3-PSMA, and, secondly, during the radiolabeling reaction, from 10-30% of the activity of technetium-99m does not bind to the target molecule, but settles in the form colloid, i.e. the radiochemical yield does not reach 95% and lies in the range of 65-85%, which entails the need to include an additional stage of product purification on the SPE cartridge, as well as a loss of activity - up to 30%, which can be a significant disadvantage given the limited shelf life of the technetium generator -99m. The authors of the publication have finalized the composition of a set of lyophilized reagents, consisting of one bottle, and providing, as stated, the formation of side colloidal technetium-99m with a yield of no more than 99% due to the addition of sodium tartrate. As a result, the set of lyophilisates consists of one vial, the transport molecule was taken in the amount of 26-39 µg, the activity of technetium-99m was 1000-1200 MBq. Also, from the advantages of this development - a more convenient way to obtain a finished injection form than when receiving ^99m Tc-MIP-1404, using tin chloride as a reducing agent.

The next option for creating a radiopharmaceutical drug based on the technetium-99m metal complex and a transport molecule containing a Glu-urea-Lys fragment is the ^99m Tc-PSMA-Hynic molecule (EP 3721907 A1). This patent protects a group of similar compounds of the general structure PSMA-L1 -L2-Hynic, as well as their metal complexes with technetium-99m. The most promising development is the ^99m Tc-EDDA/PSMA-T4 molecule, with the general formula:

where, as in the example above for the ^99m Tc-MAS3-PSMA metal complex, technetium-99m from sodium pertechnetate obtained from the generator undergoes reduction with tin chloride and enters the metal complex in the reduced input, forming coordination bonds with the nitrogen atom of the hydrazine group of the Hynic chelator -, as well as with a collegand, the role of which is performed by a combination of EDDA (ethylenediaminedioacetic acid) and tricine. The PSMA-T4 transport molecule (PSMA-LTrp-4Amc-Hynic) has a motif unchanged for this type of Glu-urea-Lys compounds. The patent also protects the composition of the reagent kit, which consists of one vial and provides, with the addition of sodium pertechnetate-99m solution, as well as when heated to 100°C for 15-30 minutes, the formation of ^{the 99m} Tc-PSMA-T4 complex with a radiochemical yield of more than 90 %. The set of lyophilizates consists of one vial, the transport molecule was taken in the amount of 20 μg, the activity of technetium-99m was 300 - 1500 MBq. This kit is registered in Russia as a component for a medical device - the Polgentek generator manufactured by Polatom (Poland) and the owner of the RU registration certificate is Medicare LLC.

Known complexes of technetium and rhenium with bis(heteroaryls) and methods for their use (RU 2539584 C2). The invention relates to a compound with the general formula:

or a pharmaceutically acceptable salt or solvate thereof. The values of the radicals are as follows: R ^t - H, C ₁ -C ₈ alkyl group, ammonium ion, alkali or alkaline earth metal ion; R ₈₄ - unsubstituted C _1-8 alkyl; R - C _1-8 hydroxyalkyl, C _1-8 alkoxyalkyl, C _1-8 aminoalkyl, (CH ₂ ) ₈ (NHC(S)NH)Ph(SO ₂ NH ₂ ), (CH ₂ ) _d Ph(SO ₂ NH ₂ ), (CH ₂ ) ₅ C(O)NH-(1-acetylpyrrolidin-2-yl)boric acid, (1-acetylpyrrolidin-2-yl)boric acid, (CH ₂ ) ₄ CH(NH ₂ )CO ₂ H, (CH ₂ ) ₃ CH(NH ₂ )CO ₂ H, (CH ₂ ) ₂ CH(NH ₂ )CO ₂ H, -(CH ₂ ) _d -R ₈₀ , -C(O)(CH ₂ ) _d -R ₈₀ or amino acid radical; R ₈₀ - carboxylate, C _6-10 aryl, 3-6 membered heterocyclyl, amino acid; d is an integer in the range 0 to 12 inclusive; and R ₈₂ , R ₈₃ , R ₈₅ and R ₈₆ are hydrogen, or substituted or unsubstituted alkyl, ether, ester, CH ₂ CH ₂ OCH ₂ CH ₃ , CH ₂ CH(OSH ₃ ) ₂ , -(CH ₂ ) _d -R ₈₀ , or (CH ₂ ) _d R ₈₇ ; where R ₈₇ is a phosphonate or phosphinate. Also proposed is a complex containing the above compound and the radionuclide technetium-99m, rhenium-186 or rhenium-188.

However, as in patent RU 2532912 C2, the formation of a metal complex requires the tricarbonyl cation of technetium-99m, the preparation of which is a rather laborious task.

The closest is a radiopharmaceutical for detecting overexpression of the prostate-specific membrane antigen Tc-EDDA/HYNIC-iPSMA (EA 201892668 A1), with the general formula:

In this case, as in the examples above for ^99m Tc-EDDA/PSMA-T4 and ^99m Tc-MAS3-PSMA, the reduction of technetium-99m occurs with tin dichloride, without the formation of a tricarbonyl intermediate, as for ^99m Tc-MIP-1404 which is an advantage. At the same time, the ^99m Tc-EDDA/Hynic-iPSMA metal complex is based on the concept of the formation of coordination bonds with the nitrogen atom of the hydrazine group of the Hynic chelator, as well as with the co-soligant, the role of which is played by the combination of EDDA (ethylenediaminedioacetic acid) and tricine, as in the example above with PSMA-T4 (this kind of chelation proceeds faster than using the MAS3 chelator), but in the structure of the linker of the Hynic-iPSMA molecule (PSMA-L2Nal-Hynic), unlike PSMA-T4, there is no tranexamic acid fragment (4Amc), and also, instead of tryptophan (LTrp), 2-naphthylalanine (L2Nal) is used. It is due to this modification in the linker structure that the resulting ^99m Tc-EDDA/Hynic-iPSMA metal complex exhibits the most preferable pharmacological properties compared to ^99m Tc-EDDA/PSMA-T4, namely, for example, the IC50 value for ^99m Tc-EDDA/Hynic -iPSMA is almost 30 times the concentration for ^99m Tc-EDDA/PSMA-T4. The patent also protects the composition of a set of reagents (lyophilizates), consisting of one vial and providing, with the addition of 1 ml of 0.2 M phosphate buffer solution with pH = 7 and the subsequent addition of a solution of sodium pertechnetate-99m, the formation of the complex ^99m Tc-EDDA/Hynic- iPSMA with a radiochemical yield of more than 98%. The set of lyophilisates consists of one vial, the transport molecule was taken in the amount of 37.5 μg, the activity of technetium-99m was not indicated.

However, when carrying out experimental work, following the recommendations of the patent application, the radiochemical yield did not exceed 72%.

That is why, taking into account all the advantages and advantages of the ^99m Tc-EDDA/Hynic-iPSMA metal complex compared to other solutions, it was decided to refine the lyophilized reagent and the conditions for the formation of the ^99m Tc-EDDA/Hynic-iPSMA metal complex to achieve the target radiochemical yield of 98% (Table 1).

Table 1 Comparison of PSMA-active metal complexes based on technetium-99m metal complex IC50 value (nM) Set of lyophilizates Comments ^99m Tc-MIP-1404 1.07 ± 0.89 [5] several vials The best binding to cells, but the most complex synthesis, many reagents, poor radiochemical yield, many stages and the ratio of the transport molecule: technetium-99m (100 µg : 1500 MBq) ^99m Tc-MAS3-PSMA 39.7 [6] one vial Medium cell binding, simple synthesis from a kit, a lot of colloidal technetium-99m is formed, due to which a low radiochemical yield, the ratio of the transport molecule: technetium-99m (26-39 µg: 1000-1200 MBq) ^99m Tc-EDDA/PSMA-T4 80 (EP 3721907 A1 page 12) one vial Worst binding to cells, simple synthesis and satisfactory radiochemical yield, the best ratio of transport molecule : technetium-99m (20 μg : 1500-MBq) ^99m Tc-EDDA/Hynic-iPSMA 2.9 ± 0.7 (EA 201892668 A1 page 9) one vial The best combination of good cell binding and ease of radiosynthesis, low radiochemical yield under the conditions according to the patent for the invention - reaction without heating, ratio of transport molecule: technetium-99m (37.5 μg: no information on activity in MBq)

There is also an article that describes in more detail the method for the formation of the ^99m Tc-EDDA/Hynic-iPSMA metal complex using a set of lyophilisates, in which the activity of technetium-99m is taken in the amount of 555-740 MBq per 1 ml, the solution is heated for 10 minutes at 95°C , resulting in a radiochemical yield of 98% (FranciscoOsvaldoGarcía-Pérez, JennyDavanzo, SergioLópez-Buenrostr1, ClaraSantos-Cuevas, GuillerminaFerro-Flores, MiguelAJímenez-Ríos, AnnaScavuzzo, ZaelSantana-Ríos, SevastiánMedina-Ornelas, "Head toheadcomparisonperformanceof ^99m Tc-EDDA/ HYNIC-iPSMASPECT/CTand ⁶⁸ Ga-PSMA-11 PET/CTaprospectivestudyinbiochemicalrecurrenceprostatecancerpatients", / AmJNuclMedMolImaging // Vol. 8 (5), 332-340, 2018). At the same time, even taking into account the achievement of an excellent radiochemical yield of 98%, using heating (in contrast to the method according to patent EA 201892668 A1), the ratio of the transport molecule: technetium-99m (37.5 μg: 555-740 MBq) does not lead to the best value of the specific activity of the finished product.

The objective of the invention is to create a lyophilizate suitable for obtaining an injection solution of a diagnostic radiopharmaceutical drug based on the ^99m Tc-EDDA/Hynic-iPSMA metal complex with a radiochemical yield of at least 98% and with a higher specific activity achieved by using a smaller number of Hynic-iPSMA molecules in the kit and the higher possible activity of the [ ^99m Tc] sodium pertechnetate solution obtained from the generator or extractor (up to 1480 MBq).

The problem is solved by the fact that, as well as in the well-known solution (EA 201892668 A1), one vial contains a lyophilizate of a mixture of a reducing agent - tin chloride (SnCl ₂ ⋅ 2H ₂ O), a soligand - tricine (C ₆ H ₁₃ NO ₅ ), a carrier of radionuclide atoms - PSMA-tropic Hynic-iPSMA molecule, EDDA cosoligant (C ₆ H ₁₂ N ₂ O ₄ ), as well as sodium gluconate, instead of mannitol, and this kit is used to obtain a diagnostic radiopharmaceutical drug based on the ^99m Tc-EDDA/Hynic- metal complex iPSMA.

The peculiarity of the proposed method is that it contains 15 mg of tricine, 0.015 mg of tin dichloride dihydrate, 20 mg of sodium gluconate, 5 mg of EDDA, 0.025 mg of Hynic-iPSMA, with a total activity of the resulting solution of 1480 MBq.

The invention is illustrated by a detailed description, tables, examples and illustrations which show:

Fig. 1- An example of some molecules containing the Glu-urea-Lys motif in their composition, as well as a radionuclide incorporated into the molecule due to either a covalent bond by the exchange mechanism, or due to the formation of a complex. (These molecules differ in the linkor - most of the molecule between the unchanged Glu-urea-Lys motif and associated radionuclide The structure of the linker largely influences the behavior of the molecule in the body.

Fig. 2 - Diagram: dynamics of changes in the activity of ^99m Tc-HYNIC-PSMA and hydrolyzed ^99m Tc.

Fig. 3 - Diagram: dynamics of changes in the concentration of ^99m Tc-Hynic-iPSMA in the 22Rv1 tumor of BALB/c nu/nu ( nude ) mice with prostate cancer after intravenous administration (in % of the administered dose per 1 g of tissue).

Fig. 4 - Diagram: dynamics of changes in the concentration of ^99m Tc-Hynic-iPSMA in the blood of BALB/c nu/nu ( nude ) mice with prostate cancer after intravenous administration (in % of the administered dose per 1 g of tissue).

Fig. 5 - Diagram: dynamics of ^99m Tc-Hynic-iPSMA concentration in the muscle of BALB/c nu/nu ( nude ) mice with prostate cancer after intravenous administration (in % of the administered dose per 1 g of tissue).

Fig. 6 - Diagram: dynamics of changes in the concentration of ^99m Tc-Hynic-iPSMA in the prostate of BALB/c nu/nu ( nude ) mice with prostate cancer after intravenous administration (in % of the administered dose per 1 g of tissue).

Fig. 7 - Chart: Differential accumulation ratios (DACs) of ^99m Tc-Hynic-iPSMA in prostate tumor Nude mice with respect to blood after intravenous administration of the drug.

Fig. 8 - Diagram: Differential accumulation ratios (DACs) of ^99m Tc-Hynic-iPSMA in prostate tumor Nude mice relative to muscle after intravenous administration of the drug.

Fig. 9-Diagram: Differential accumulation ratios (DACs) of ^99m Tc-Hynic-iPSMA in prostate tumor Nude mice relative to normal prostate after intravenous administration of the drug.

The invention is carried out as follows.

In a 250 ml double-necked round-bottom flask equipped with a magnetic stirrer, place 100 ml of water for injection, add 1500 mg of tricine, stir until completely dissolved, then add 1.5 mg of stannous chloride dihydrate, mix thoroughly for 5 minutes, then add 2000 mg of sodium gluconate, stirred until completely dissolved, then add 500 mg of EDDA, mix thoroughly, then add 2.5 mg of Hynic-iPSMA, then stir for 5 minutes. The resulting solution is filtered under vacuum through a cellulose acetate sterilizing filter with a pore size of 0.22 μm. The filtered solution is packaged with an automatic pipette with a nominal value of 100-1000 μl into 10 ml vials of 1 ml each (total 100 vials). The whole process is carried out in a stream of argon. Then the vials are placed in the freezer chamber on a shelf cooled to minus 20°C. A pressure of 0.1-0.2 mm Hg is created in the chamber. using a vacuum pump. Under these conditions, freeze-drying is carried out for 43 hours, after which the chamber is filled with dry argon to atmospheric pressure. Then the vials are removed from the chamber, sealed with rubber stoppers and rolled with aluminum caps (GOST R 51314-99). The bottle is labeled. The lyophilization mode is detailed below.

Method of freeze drying of reagents.

Before packaging the solutions of the components to obtain a set of lyophilizates, a sublimation unit is turned on and the shelves are cooled to minus 30°C. Vials with packaged solutions are placed on cooled shelves, the doors of the sublimation chamber are hermetically closed and the solutions in the vials are kept frozen for 30 minutes. Then turn on the condenser, bring the temperature of the condenser to 50-55°C, turn on the vacuum pump. After reaching a vacuum of 0.3 mm Hg. turn off the cooling of the shelves and freeze-dry in this mode (floating mode). In floating mode, after 20 hours the vacuum in the sublimation chamber reaches 4⋅10 ^-3 mm Hg. The temperature of the shelves gradually rises to minus 12°C 1 hour after the start of drying, after 3 hours the temperature of the shelves rises to minus 8°C, after 10 hours - up to 0°C, after 20 hours to plus 10°C, after 40 hours the temperature in the chamber rises to plus 24-27°C. At a temperature of plus 24-27°C, drying is carried out for 4 hours. The total duration of drying is 44 hours. After that, the vacuum pump is turned off, the fitting of the sublimation chamber is opened, and the sublimation chamber is filled with argon through a silicone hose to atmospheric pressure, filtering argon through a filter with a size pore 0.22 µm. After that, the door of the sublimation chamber, which is located in a sterile room, is opened, the pallets with vials are removed from the chamber, the vials are quickly closed with rubber stoppers and crimped with aluminum caps. Labels are attached to the vials, placed in a refrigerator and stored at a temperature of plus 2-8°C.

In the vial, the reagents are in lyophilized form (Table 2). The contents of the vial are sterile and kept in an inert environment, preferably argon or nitrogen with a minimum oxygen content.

table 2 The composition of the lyophilisate vial Lyophilizate for the manufacture of RFLP based on the metal complex ^99m Tc-EDDA/Hynic-iPSMA Bottle number 1 Tricin, mg SnCl ₂ * 2H ₂ O,
mg sodium gluconate,
mg EDDA,
mg Hynic-iPSMA,
mg 15 0.015 20 5 0.025

The vial contains a sterile lyophilisate of a mixture of a reducing agent - tin chloride (SnCl ₂ ⋅ 2H ₂ O), a soligand - tricine (C ₆ H ₁₃ NO ₅ ), a carrier of radionuclide atoms - PSMA-tropic molecule Hynic-iPSMA, EDDA soligant (C ₆ H ₁₂ N ₂ O ₄ ), as well as sodium gluconate. Tin dichloride 2-aqueous is a reducing agent of technetium-99m to a lower valence state, since technetium-99m in the highest oxidized state (oxidation state +7), obtained in the form of [ ^99m Tc] pertechnetate ion, does not form a complex with the carrier (Hynic-iPSMA ), tricine is necessary for stabilization of reduced technetium and further cross-liganding function together with EDDA, as well as the formation of a stable metal complex, sodium gluconate acts as a buffer and cross-linker agent.

All reagents are freely available commercially. The Hynic-iPSMA molecule is synthesized by peptide synthesis.

The vial has a one year shelf life.

Experimental part.

Example 1

Obtaining a potential diagnostic RFLP based on the ^99m Tc-EDDA/Hynic-iPSMA metal complex from the product claimed for protection - a set of lyophilisates described above.

The work is carried out under aseptic conditions in compliance with radiation safety standards.

To a 10 cm ³ lyophilisate vial containing a lyophilized mixture of 15 mg tricine, 0.015 mg tin dichloride dihydrate, 20 mg sodium gluconate, 5 mg EDDA, 0.025 mg Hynic-iPSMA, add up to 1480 MBq of [ ^99m Tc] sodium pertechnetate solution obtained from the generator or extractor, in 0.5-2 ml of saline, stirred until the precipitate is completely dissolved. The mixture is left in a protective container for 5 minutes.

After keeping the resulting solution for 5 minutes, the vial with the resulting mixture is heated for 25-30 minutes at a temperature of 95°C.

After heating, the mixture is cooled to room temperature. The volume of the solution was adjusted to 4.0 ml by adding saline. If necessary, the resulting solution is filtered through a syringe nozzle with a pore size of 0.22 μm into a sterile vial with a capacity of 10 cm ³ .

Radiochemical impurities in the form of unbound and hydrolyzed ^99m Tc do not exceed 2.0%. The stability of RFLP ^99m Tc-Hynic-iPSMA remains at the level of 97-98% within 48 hours (Fig. 2).

Quality control is carried out by TLC followed by radiometric measurement of sections of the TLC plate.

Example 2

Study of the biodistribution of the ^99m Tc-Hynic-iPSMA metal complex in laboratory animals.

BALB/c nu/nu ( nude ) mice with transplanted 22Rv1 prostate cancer cells were used for experimental work.

The dynamics of changes in the concentration of ^99m Tc-Hynic-iPSMA in tumors of mice is presented in (Fig. 3). The measurements were carried out after intravenous administration of RFLP, the control was carried out in % of the administered dose per 1 g of tissue over time. As early as 5 min after injection, the accumulation of ^99m Tc-Hynic-iPSMA in the tumor was 2.88 ± 0.24%/g and remained almost unchanged for the next hour. The maximum concentration ^{of 99m} Tc-Hynic-iPSMA (3.91 ± 0.35%/g) was registered within 3 hours after administration, decreasing by the end of the study to 1.81 ± 0.10%/g 48 hours after RFLP injection .

In the blood, the highest recorded concentration ^{of 99m} Tc-Hynic-iPSMA was 3.81 ± 0.44%/g within 5 minutes after administration. However, already after 1 hour, the concentration of the drug in the blood decreased by more than 4 times to 0.86 ± 0.03%/g and continued to decrease to 0.11 ± 0.01%/g 48 hours after the RFLP injection (Fig. 4) .

A low content of ^99m Tc-Hynic-iPSMA was registered in muscle tissue: only 1.21 ± 0.13%/g within 5 minutes after drug administration and 0.01-0.32%/g at subsequent times (Fig. 5 ).

The maximum concentration of ^99m Tc-Hynic-iPSMA in the prostate was 2.31 ± 0.26%/g within 5 minutes after intravenous injection of the drug. In subsequent periods (1-48 hours), the specific content of ^99m Tc-Hynic-iPSMA did not exceed 1%/g (0.07-0.93%/g) (Fig. 6).

For diagnostic RFLP, it is not so much the value of its specific content in the tumor that is important, but the relative accumulation in it in relation to the surrounding healthy tissues, primarily blood and muscle tissue. When analyzing the numerical values of the coefficients of differential accumulation (DAC) of the tumor/internal organs, it was found that the lowest values of the DAC were noted 5 minutes after the introduction of ^99m Tc-Hynic-iPSMA. Thus, within 5 minutes after injection, the content of ^99m Tc-Hynic-iPSMA in the tumor was lower than in the blood (Fig. 7).

In subsequent periods, due to the accumulation of ^99m Tc-Hynic-iPSMA in the tumor and excretion from the internal organs and tissues, an increase in the CDN values was observed, and for most organs and tissues these values were significantly higher than 1. The maximum tumor/blood CDN values reached 19.11 ± 2.50 and tumor/prostate - 36.48 ± 8.87 after 24 hours (Fig. 7 and Fig. 9), tumor/muscle - 182.50 ± 22.07 after 48 hours (Fig. 8).

The claimed invention allows to obtain a diagnostic radiopharmaceutical drug with a higher specific activity of ^99m Tc-EDDA/Hynic-iPSMA than stated in world practice, because the possibility of obtaining 1480 MBq of the product with a radiochemical yield of at least 98% is achieved by reducing the amount of the Hynic-iPSMA molecule to 25 µg, which is achieved by changing the concentration of some excipients, as well as using sodium gluconate instead of mannitol.

Claims (2)

1. Lyophilizate for obtaining a diagnostic radiopharmaceutical drug based on the radionuclide ^99m Tc, consisting of a mixture of a reducing agent - tin dichloride dihydrate (SnCl ₂ ⋅ 2H ₂ O), a soligand - tricine (C ₆ H ₁₃ NO ₅ ), a carrier of radionuclide atoms - PSMA- tropic molecule Hynic-iPSMA, EDDA soligand (C ₆ H ₁₂ N ₂ O ₄ ), as well as sodium gluconate, characterized in that it contains 15 mg of tricine, 0.015 mg of tin dichloride dihydrate, 20 mg of sodium gluconate, 5 mg of EDDA, 0.025 mg Hynic-iPSMA. 2. The lyophilisate according to claim 1, characterized in that the total activity of the obtained solution of the diagnostic radiopharmaceutical drug reaches 1480 MBq.

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