RU2588144C1 - Method of producing synthetic metal-polymer complexes of radioisotope gallium-68 - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to chemistry of high-molecular compounds and nuclear medicine, namely to method of producing synthetic metal-polymer complexes of gallium-68 radioisotopes. Complex includes water-soluble copolymer of N-vinylpyrrolidone with allyl-or N-vinylamine with molecular weight of 5,000-50,000 Da, containing N-vinylpyrrolidone m = 95-60 mol. % and allyl- or N-vinylamine n = 5-40 mol. %. Method involves adding copolymer chelate node, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA) (z = 1-20 mol%) to obtain polymer carrier, carrier polymer interaction with added chelate unit with radionuclide, represented by positron emitter gallium-68, in water solution in oxygen or in inert gas atmosphere at temperature of 60-95 °C at pH 1-5.5 and carrier polymer concentration of 5-20 wt% for 15-30 minutes to produce metal-polymer conjugate of following structure:

, where n, m, z are numbers of repeating units allyl-(X = 1) or N-vinylamine (X = 0), N-vinylpyrrolidone and DOTA respectively.

EFFECT: method enables to create new synthetic macromolecular radiopharmaceuticals (RP) for more accurate and reliable early diagnosis of new growths by positron emission tomography (PET).

1 cl, 2 dwg, 4 ex

Description

The invention relates to the field of chemistry of macromolecular compounds and nuclear medicine and sets the task of creating synthetic biologically active polymer carriers labeled with gallium-68 radionuclide, for the production of radiopharmaceuticals (RFPs) for the rapid diagnosis of neoplasms of the body by positron emission tomography (PET). PET is a modern non-invasive medical diagnostic technology based on the introduction of positron-emitting radionuclides in biologically active molecules. This approach allows one to obtain highly specific images of tissues, organs, and molecular processes occurring in vivo. Over the past decade, interest in the clinical use of drugs labeled with ^68u Ga has sharply increased in the world. This interest is caused by the development of commercial generators ^68Ge / ⁶⁸ Ga, allowing to obtain a radionuclide without the use of a cyclotron. ⁶⁸ Ga (T _1/2 = 67.71 min) decays by electron capture (ES) (10%) and emission of positrons (90%, Eβ + = 1900 keV). The decay is accompanied by the emission of annihilation gamma quanta (Eγ = 511 keV, 180% yield) and gamma quanta with an energy Eγ = 1077.4 keV (yield 2.93%). Such energy characteristics of ⁶⁸ Ga make it possible to obtain the radiopharmaceutical of the required activity, without creating a significant dose load on the body. In addition, cation ⁶⁸ Ga ³⁺ can form stable complex compounds with various ligands, which makes it suitable for the synthesis of a large number of biologically active complexes, labeled small molecules and macromolecules of various functional purposes.

To date, a large number of various compounds labeled with ⁶⁸ Ga have been studied, from ⁶⁸ Ga-citrate and ⁶⁸ Ga-apotransferrin to visualize infectious foci and inflammations to complex high molecular weight bio-conjugates labeled with ⁶⁸ Ga and stabilized with non-radioactive elements [Melpomeni Fania, Joao P. Andre and Helmut R. Maecke Contrast Media Mol. Imaging, 2008, V. 3, P. 67-77 41. Abstracts of 1st World Congress on Ga-68 and Peptide Receptor Radionuclide Theapy (PRRNT). // World Journal of Nuclear Medicine, 2011, V. 10 (1), P. 25-107].

, Pauliina Virsu, Petteri Lankinen, Tatu J.

, Anu Autio, Gennady G. Yegutkin, Juhani Knuuti, Sirpa Jalkanen, Anne Roivainen. Nuclear Medicine and Biology, 2009, V. 36, P. 631-641].Peptides, proteins, antibodies, etc. are used as carriers for ⁶⁸ Ga. [WO 1999056792 A1, 1999, US Pat Appl 20130310537, 2013, US 7011816 B2, 2006 Tiina Ujula, Satu

, Pauliina Virsu, Petteri Lankinen, Tatu J.

, Anu Autio, Gennady G. Yegutkin, Juhani Knuuti, Sirpa Jalkanen, Anne Roivainen. Nuclear Medicine and Biology, 2009, V. 36, P. 631-641].

Currently, cyclic peptides - analogues of somatostatin labeled with ⁶⁸ Ga (DOTA-TATE, DOTA-TOC), and an analog of the cyclic peptide - octreotide - DOTA-NOC [WO 2013029616 A1, 2013, US 7122622 B2, 2006, WO 2012138251 A1, 2012], which exhibit high accumulation in tumor tissues of various morphologies.

However, the above analogues are rather the exception, since the introduction of metals into the composition of low molecular weight biomolecules, as a rule, leads to a violation of their transport properties. In this case, non-selective accumulation of radioactivity in the organs and tissues of the body, muscle tissue and blood flow occurs, which reduces the contrast of the imaging of tumors. The disadvantage is the complicated procedure for obtaining the initial peptides and the vaccination of a polyfunctional chelate node.

A reasonable alternative to peptides are macromolecules with a high molecular weight (proteins, antibodies). In this case, the introduction of a heavy metal does not significantly affect the properties of the resulting conjugate.

, Pauliina Virsu, Petteri Lankinen, Tatu J.

, Anu Autio, Gennady G. Yegutkin, Juhani Knuuti, Sirpa Jalkanen, Anne Roivainen. Nuclear Medicine and Biology, 2009, V. 36, P. 631-641], сложные конъюгированные системы, содержащие лейкоциты, связанные с водорастворимыми полимерами и полидентатным хелатным узлом [US 20120107232 А1, 2012]. ⁶⁸ Ga-labeled macromolecules are known, such as antibodies containing a chelate ligand (for example, DOTA - 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) [WO 2003048207 A2, 2002], proteins [ ⁶⁸ Ga-DOTA-VAP-P1 (VAP-P1 is a selective analogue of the protein) Tiina Ujula, Satu

, Pauliina Virsu, Petteri Lankinen, Tatu J.

, Anu Autio, Gennady G. Yegutkin, Juhani Knuuti, Sirpa Jalkanen, Anne Roivainen. Nuclear Medicine and Biology, 2009, V. 36, P. 631-641], complex conjugated systems containing leukocytes associated with water-soluble polymers and a polydentate chelate site [US 20120107232 A1, 2012].

Common disadvantages of natural macromolecules are: a complex procedure for the isolation, purification and identification of precursors; uncontrolled administration of chelating nodes to bind radioisotopes due to the presence of a large number of free active functional amino and carboxyl groups; extremely high cost of end radiopharmaceuticals.

The closest prototype in terms of technical nature and the achieved result is a conjugated copolymer of liposome fraction with hydrophilic polymers (polyglycine, polyethylene glycol, polypropylene glycol, polymethacrylamide, etc.) with a molecular weight of 1-3 kDa and a grafted polydentate chelate unit [US 20120107232 A1, 2012]. The method for producing the copolymer is as follows: a) isolation of the liposome fraction, b) grafting of the chelate site (DOTA) to the hydrophilic polymer spacer, c) introduction of the obtained copolymer into liposomes, d) carrying out radiochemical synthesis, e) purification of the target conjugate by size exclusion chromatography.

The disadvantages of this method is the complex procedure for producing liposomes and their conjugation with a polymer, the complexity of radiochemical synthesis and the need for preparative purification and, as a result, low radiochemical yield of the target product of the drug. In addition, the synthesis of such radiopharmaceuticals requires the involvement of highly qualified personnel and the organization of well-equipped laboratories, which affects the cost.

The technical problem and the positive result of the proposed method is to obtain metal-polymer complexes of gallium-68 for the diagnosis of neoplasms of the body using synthetic metal-polymer complexes of the radioisotope gallium-68, including a water-soluble copolymer of N-vinylpyrrolidone with allyl- or N-vinylamine with a molecular weight of 5- 50 kDa, N-vinylpyrrolidone content m = 95-60 mol. % and allyl or N-vinylamine n = 5-40 mol. % This is achieved by the fact that a chelating unit is introduced into the copolymer, such as DOTA - 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate z = 1-20 mol. % with butoxycarbonyl protection, using the method of activated esters followed by deprotection and obtaining the desired carrier polymer, while the positron emitter gallium-68 is used as the introduced radionuclide. The interaction of the carrier polymer with the introduced chelate unit with a radioactive isotope is carried out in an aqueous solution in an oxygen atmosphere or in an inert gas at a temperature of 60-95 ° C, at a pH of 1-5.5 and a concentration of the carrier polymer of 5-20 mass. % for 15-30 minutes to obtain a metal-polymer conjugate of the following structure:

Metal-polymer complexes, characterized in that copolymers with a molecular weight (5-70 kDa) and a DOTA chelate content of 1-20 mol. Are used as water-soluble high molecular weight carriers. %

The method for producing water-soluble copolymers with chelating units of DOTA is characterized in that the conjugation of DOTA (BOC) ₃ with a copolymer of vinylpyrrolidone with vinyl / allylamine was carried out by the method of activated esters with subsequent removal of BOC protection.

The method is characterized in that the interaction of the carrier polymer with a ⁶⁸ Ga radioactive generator eluate with a polymer concentration of 8-15 mass. % was carried out at various pH values in an inert gas atmosphere at a temperature of 90 ° C for 15-30 minutes, followed by rapid purification of the drug on a gel filtration cartridge.

The result is a polymer water-soluble system, labeled with diagnostic PET radionuclide ⁶⁸ Ga, for the diagnosis of neoplasms of the body with multifunctional biological activity.

In FIG. 1. presents the profile of gel filtration separation of VP-AA-DOTA- ⁶⁸ Ga on a Sephadex G-25 superfine sorbent (radioactivity detection). Sample volume 0.5 ml, eluent 0.9% NaCl, time min. * 0.1. The yield of the target product is 65%. Peak 1 - VP-AA-DOTA- ⁶⁸ Ga

In FIG. 2. presents the chromatogram [VP-AA-DOTA- ⁶⁸ Ga] (MM 39 kDa) fraction isolated on a gel filtration cartridge with the Sephadex G-25 superfine sorbent. Column Ultrahydrogel Linear 0.78 × 30 cm 0.6 × 4 cm, eluent 0.2 M NaCl, 0.8 ml / min. Upper chromatogram - UV detection (210 nm) 11.9 min, lower - radioactivity detection - 12.18 min. The delay between the detectors is 0.25 minutes.

The essence of this method of obtaining radiopharmaceuticals is disclosed below by examples of its experimental implementation.

.Synthesis of copolymer VP-AA. The VP-AA copolymer was synthesized by the method of radical copolymerization of monomers in a 50% ethanol solution in the presence of 1 mass. % initiator - 2,2′-azo-bis-isobutyronitrile at 60 ° C for 48 hours. The molar ratio of VP: AA in the initial mixture was 70:30 mol. % The resulting copolymer was purified from low molecular weight impurities by dialysis against water. Used Spectra / Por 7 dialysis membranes from SpectrumLab. Inc. (USA), allowing to remove compounds with M≤1000. The polymer was isolated by freeze drying. Received a copolymer containing 7.0 mol. % AA links $${[\eta ]}_{0.1MC{H}_{3}COOH}=0.16dl/g$$

.

, d=1.014; Т_кип=65°С (4 мм рт. ст.), $${\eta }_D^{20}=1.4920$$

, d=1.015 соответственно.Synthesis of VP-VA copolymers. The monomers N-VP (N-vinylpyrrolidone) and N-VFA (N-vinylformamide) were purified by vacuum distillation T _bale = 69 ° C (3 mm RT. Art.), $${\eta }_D^{\mathrm{twenty}}=1.5120$$

, d = 1.014; T _bale = 65 ° C (4 mm RT. Art.), $${\eta }_D^{\mathrm{twenty}}=1.4920$$

, d = 1.015, respectively.

The polymerization was carried out in ethyl and propyl alcohol at 60 ° C, filled in ampoules in an argon atmosphere. As the initiator used dinitrilazobisisobutyric acid (DAK) 1-2 mass. % by weight of loaded monomers. The resulting copolymers were isolated by precipitation in diethyl ether. The precipitate was dried in vacuo at room temperature to constant weight.

The hydrolysis of VP-VFA copolymers was carried out with 1 N HCl at a temperature of 90 ° C for 7 hours. The hydrolyzed copolymer was purified by dialysis relative to water, then isolated by freeze drying. The completeness of hydrolysis was determined by the argentometric method from the content of Cl ^- ion in the copolymer.

.Received a copolymer containing 10.0 mol. % VA units $${[\eta ]}_{0.1MC{H}_{3}COOH}=0.23dl/g$$

.

The content of amino groups in the obtained copolymers was determined by spectroscopy, spectrophotometrically from an absorption band of 420 nm, characteristic of complexes of primary amines with 2,4,6-trinitrobenzenesulfonic acid, and by potentiometric titration with a 0.1 N HCl solution.

The copolymers of VP-AA-DOTA, VP-VA-DOTA were synthesized by the method of activated esters using N-hydroxysuccinimide (GOSI). The copolymer was dissolved in DMF, 0.01 equiv. (relative to the total number of VP fragments and AA / VA units in the polymer chain) DOTA (BOC) ₃ , GOSI and diisocyclohexylcarbodiimide (DCC) in DMF. The reaction mixture was kept for 2 hours at room temperature to obtain a polymer with 1 mol. % of groups conjugated to DOTA. The polymer was precipitated in cold ether and dried under reduced pressure. To remove BOC protection and obtain free carboxyl groups of the DOTA ligand, the preparation was treated with a mixture of trifluoroacetic acid: water (95: 5 vol.%, 2 hours). The solution was evaporated under reduced pressure, treated with a 5% solution of triethylamine in DMF and precipitated into chilled ether, dried under reduced pressure, re-dissolved in water, dialyzed against distilled water twice and subjected to freeze drying.

The radiochemical synthesis procedure was as follows.

Example 1. To an aliquot of 20 μl of an aqueous solution containing 80 μg of VP-AA-DOTA copolymer placed in a microtest tube (Eppendorf) filled with nitrogen was added 250 μl of a ⁶⁸ Ga generator eluate with an activity of 17-25 MBq. The resulting solution was diluted with 750 μl of a 0.2 M sodium acetate solution (pH = 5.5). The microtube with the reaction mixture was sealed and heated for 15 min at 95 ° C. The volume of the cartridge was selected so that the target metal-polymer conjugate was practically not retained (eluted with V _{o the} retained volume). After that, the reaction mixture was transferred onto a cartridge with a Sephadex-25 superfine sorbent (10 × 80 mm) and was eluted with a 0.9% NaCl solution using a peristaltic pump at a rate of 0.3 ml / min. During elution, fractions of V _o (containing a metal-polymer complex) from 1.5 ml of eluate to 2.8 ml of eluate and V _e (containing part of free ⁶⁸ Ga ³⁺ ) were collected (most were irreversibly sorbed on a column) from 2.8 to 7.8 ml of eluate. In this case, the preparative separation of the peak of the target metal-polymer conjugate and the assessment of the radiochemical yield of the reaction (PXB) were performed. RHV was 60-75%, and the radiochemical purity (RHC) of the target product, which was monitored by HPLC on an UltrahydrogelLinear 0.78 × 30 cm exclusion column (eluent 0.2 M NaCl), reached 97-99%.

Example 2. To an aliquot of 20 μl of an aqueous solution containing 30 μg of a VP-AA-DOTA copolymer placed in a microtest tube (Eppendorf) filled with nitrogen was added 250 μl of a ⁶⁸ Ga generator eluate with an activity of 17-25 MBq. The resulting solution was diluted with 750 μl of 0.2 M HCl (pH 2). The microtube with the reaction mixture was sealed and heated for 15 min at 90 ° C. After purification by gel filtration, the RXB of the drug was 30%, and the RHC was 97%.

Example 3. To an aliquot of 20 μl of an aqueous solution containing 60 μg of VP-VA-DOTA copolymer placed in a microtest tube (Eppendorf) filled with nitrogen was added 250 μl of a ⁶⁸ Ga generator eluate with an activity of 17-25 MBq. The resulting solution was diluted with 750 μl of phosphate buffer (pH 7.5). The microtube with the reaction mixture was sealed and heated for 15 min at 60 ° C. After purification by gel filtration, the RXB of the drug was 10%, and the RXC - 8%.

Example 4. To an aliquot of 20 μl of an aqueous solution containing 100 μg of VP-VA-DOTA copolymer placed in a microtest tube (Eppendorf) filled with nitrogen was added 250 μl of a ⁶⁸ Ga generator eluate with an activity of 17-25 MBq. The resulting solution was diluted with 750 μl of a 0.2 M sodium acetate solution (pH 5.5). The microtube with the reaction mixture was sealed and heated for 30 min at 95 ° C. After purification by gel filtration, the RHB of the drug was 80%, and the RHC was 97%.

Thus, the developed method for producing water-soluble copolymer complexes of ⁶⁸ Ga based on copolymers of N-vinylamides (N-vinylpyrrolidone (VP)), N-vinylamine (VA) and N-allylamine (AA) with a grafted DOTA chelating unit allows one to create new synthetic macromolecular ones RFP for a more accurate and reliable diagnosis of neoplasms of the body by the PET method, which indicates the compliance of this technical solution with all the required criteria of the invention protected by a patent.

Claims (1)

A method of producing synthetic metal-polymer complexes of gallium-68 radioisotopes, including a water-soluble copolymer of N-vinylpyrrolidone with allyl or N-vinylamine with a molecular weight of 5000-50000 Yes, the content of N-vinylpyrrolidone m = 95-60 mol. % and allyl or N-vinylamine n = 5-40 mol. %, characterized in that a chelate unit, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA) is introduced into the copolymer z = 1-20 mol. %, to obtain a carrier polymer, wherein a gallium-68 positron emitter is used as the radionuclide, the interaction of the carrier polymer with the introduced chelate unit with the radionuclide is carried out in an aqueous solution in an oxygen atmosphere or in an inert gas at a temperature of 60-95 ° C, at a pH of 1-5.5 and a concentration of carrier polymer of 5-20 mass. % for 15-30 minutes to obtain a metal-polymer conjugate of the following structure:

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