Method for purifying radionuclide-labeled drugs

Abstract

To provide methods for removing peptides not labeled with a radionuclide from radionuclide-labeled agents.SOLUTION: Disclosed is a method for purifying an agent labeled with a radionuclide from a solution where the agent comprises a chelator that forms a complex with the radionuclide. The method comprises passing the solution through a column packed with a weakly acidic cation exchange resin, then passing an acidic solution through the column to extract the radionuclide-labeled agent. The radionuclide may be a tetravalent one, e.g., zirconium. The labeled agent comprises a trivalent chelator, DOTA or NOTA as the chelator. The weakly acidic cation exchange resin comprises carboxyl groups as the functional group. The labeled agent is a prostate specific antigen binding agent, a somatostatin receptor binding agent, or a fibroblast activation protein binding agent.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for purifying radionuclide-labeled agents. [Background technology]

[0002] In recent years, peptides labeled with radionuclides (radionuclide-labeled peptides) have been practically used as radiodiagnostic agents and radiotherapeutic agents. Radionuclide-labeled peptides are attracting particular attention as promising nuclear medicine diagnostic and therapeutic agents for cancer. Examples of peptides being studied include DOTA-TATE, which binds to the somatostatin receptor; PSMA-617, which binds to prostate-specific antigen; and FAPI-04 and FAPI-2286, which are fibroblast activation protein (FAP) binding agents. In addition, many derivative substances with modified chemical structures are being investigated for each of these peptides. Peptides targeting the somatostatin receptor have already been put into practical use as nuclear medicine diagnostic and therapeutic agents for neuroendocrine tumors. Peptides targeting prostate-specific antigen are expected to be used as nuclear medicine diagnostic and therapeutic agents for prostate cancer, and some have already begun to be put into practical use. FAP is attracting attention as a versatile target antigen for cancer because it is overexpressed in cancer-associated fibroblasts (CAFs) but hardly expressed in normal cells (see Non-Patent Literature 1).

[0003] Radioactive nuclide-labeled peptides are labeled by complexing a metallic radionuclide with a peptide containing an appropriate chelate. In the labeling reaction, while a high proportion of metallic radionuclides are labeled to the peptide, some remain unreacted as free radionuclides in the labeling reaction solution. Conventionally, purification is performed using reversed-phase solid-phase extraction columns such as C18 columns to remove unreacted free radionuclides. That is, peptides adsorb to the column due to hydrophobic interactions, while metallic radionuclides do not, allowing for selective adsorption of peptides to the column. Peptides adsorbed on the reversed-phase column can be eluted by passing ethanol through the reversed-phase column. Purification is performed by selectively adsorbing the peptide onto the reversed-phase column and then eluting it, thereby removing free radionuclides. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] “Development of Quinoline-Based Theranostic Ligands for the Targeting of Fibroblast Activation Protein”, JOURNAL OF NUCLEAR MEDICINE, 2018, 59 (9) 1415-1422. [Overview of the project] [Problems that the invention aims to solve]

[0005] However, in the above-described prior art, there was a problem that peptides not labeled with radionuclides could not be removed. That is, since peptides are selectively adsorbed onto the reversed-phase column by hydrophobic interaction of the peptides with the reversed-phase column, both labeled peptides (hereinafter referred to as labeled peptides) and unlabeled peptides (hereinafter referred to as unlabeled peptides) are adsorbed onto the hydrophobic column without being distinguished. Since unlabeled peptides cannot be used as radiodiagnostic agents or radiation internal therapeutic agents, it is desirable to remove them. Therefore, the present inventor has found the necessity for developing a technique capable of removing peptides not labeled with radionuclides in radionuclide-labeled agents.

[0006] The present invention has been made in view of the above, and an object thereof is to provide a method for purifying a radionuclide-labeled agent capable of removing peptides not labeled with radionuclides in the radionuclide-labeled agent.

Means for Solving the Problems

[0007] In order to solve the above-described problems and achieve the object, a method for purifying a radionuclide-labeled agent according to one aspect of the present invention is a method for purifying a radionuclide-labeled agent for separating and purifying a radionuclide-labeled agent labeled with a radionuclide from a solution containing a labeling agent having a chelating agent that forms a complex by labeling with a radionuclide, wherein the solution is passed through a column containing a weakly acidic cation exchange resin, and then an acidic solution is passed through the column to extract the radionuclide-labeled agent.

[0008] A method for purifying a radionuclide-labeled agent according to one aspect of the present invention is, in the above invention, the radionuclide is a tetravalent radionuclide. A method for purifying a radionuclide-labeled agent according to one aspect of the present invention is, in this configuration, the radionuclide is zirconium.

[0009] The method for purifying a radionuclide-labeled drug according to one aspect of the present invention is, in the above invention, the chelating agent is a trivalent chelating agent. The method for purifying a radionuclide-labeled drug according to one aspect of the present invention is, in this configuration, the chelating agent includes DOTA or NOTA.

[0010] The method for purifying a radionuclide-labeled drug according to one aspect of the present invention is, in the above invention, the weakly acidic cation exchange resin has a carboxylic acid group as a functional group.

[0011] The method for purifying a radionuclide-labeled drug according to one aspect of the present invention is, in the above invention, the labeled drug is a prostate-specific antigen-binding drug, a somatostatin receptor-binding drug, or a fibroblast activation protein-binding drug.

Advantages of the Invention

[0012] According to the method for purifying a radionuclide-labeled drug according to the present invention, it becomes possible to remove a peptide not labeled with a radionuclide in the radionuclide-labeled drug.

Brief Description of the Drawings

[0013] [Figure 1] FIG. 1 is a schematic diagram for explaining a method for purifying a radionuclide-labeled drug according to an embodiment of the present invention. [Figure 2] FIG. 2 is a schematic diagram for explaining a method for purifying a radionuclide-labeled drug according to the prior art.

Modes for Carrying Out the Invention

[0014] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings of the following embodiments, the same or corresponding parts are denoted by the same reference numerals. Further, the present invention is not limited by the embodiments described below.

[0015] First, in order to facilitate understanding of the embodiments of the present invention, the diligent research conducted by the inventors to solve the above-mentioned problems will be described. In this specification, linear compounds containing amide bonds are broadly referred to as "peptides." That is, in this specification, "peptides" are not limited to peptides composed solely of natural amino acids.

[0016] Figure 2 illustrates a conventional method for purifying radionuclide-labeled drugs. As shown in Figure 2, in the conventional method for purifying radionuclide-labeled drugs, activation (hereinafter referred to as conditioning) is first performed by passing pure water through the column. The quenchable liquid consisting of water that has passed through the column is discarded. This process can be omitted if the column does not require conditioning. It is also possible to use a mixture of ethanol (C2H5OH) and pure water (ethanol aqueous solution) instead of pure water.

[0017] Next, the radioactive nuclide-labeled ligand solution is passed through the column. This causes the radioactive nuclide-labeled ligand to adsorb onto the column. The quenched liquid containing the free radioactive nuclides that have passed through the column is discarded. Subsequently, the column is washed by passing pure water through it. The quenched liquid after washing the column is discarded. Alternatively, an ethanol aqueous solution with a concentration of 10-30% and a mixture of pure water can be used instead of pure water.

[0018] After washing the column with pure water, a highly concentrated ethanol aqueous solution, for example, one with a concentration of 70% or more and mixed with pure water, is passed through the column to elute the radionuclide-labeled ligand adsorbed to the column. The ethanol aqueous solution from which the radionuclide-labeled ligand has been eluted is recovered after passing through the column. Here, considering the administration of a solution containing radionuclide-labeled ligand (radionuclide-labeled ligand solution) to the human body, it is undesirable to administer ethanol to the human body. Therefore, the recovered ethanol solution is evaporated to dryness to remove the ethanol. That is, the ethanol solution that has passed through the column is evaporated to dryness while still containing the radionuclide-labeled ligand. After that, the dried radionuclide-labeled ligand is redissolved in physiological saline to form a formulation. The column is then discarded.

[0019] However, the conventional purification method for radionuclide-labeled drugs shown in Figure 2 has the problem that it is not possible to remove peptides that are not labeled with radionuclides. That is, because peptides selectively adsorb to the reversed-phase column due to the hydrophobic interaction of the peptides with the reversed-phase column, both labeled and unlabeled peptides are adsorbed to the hydrophobic column without distinction. If unlabeled peptides are present, phenomena such as self-blocking can occur, where the unlabeled peptides inhibit the binding of the labeled peptide to cancer cells, for example, when binding to cancer cells in PET (positron emission tomography) scans. From this point of view, the inventors of the present invention have found a need to remove peptides that are not labeled with radionuclides in radionuclide-labeled drugs.

[0020] Here, the inventors investigated the case in which a radionuclide is labeled to a fibroblast activation protein (FAP) inhibitor (FAPI), such as the one shown in the following chemical formula (1), which is capable of labeling radionuclides, in the conventional method for purifying radionuclide-labeled agents described above. As the radionuclide-labeled agent, a prostate-specific antigen-binding agent, a somatostatin receptor-binding agent, or a fibroblast activation protein-binding agent can be used.

[0021] [ka]

[0022] In this case, the binding yield (radiochemical yield) may be less than 100%, and it is necessary to remove radionuclides that are not bound to the ligand (free radionuclides). Therefore, conventionally, in the purification of radionuclide-labeled drugs used as radiopharmaceuticals, purification is generally carried out by solid-phase extraction using a hydrophobic solid-phase extraction column such as a C18 column. A C18 column (also called an ODS column) is a type of column used for high-performance liquid chromatography (HPLC) analysis, and has an octadecylsilyl (ODS) group (C) as shown in the following chemical formula (2). 18 H 37 The stationary phase is packed with chemically bonded porous spherical silica gel (Si) whose surface is modified with Si. As a result, free radionuclides do not adsorb to the column, while radionuclide-labeled agents are adsorbed to the column, and the radionuclide-labeled agents adsorbed to the column can be recovered.

[0023] [ka]

[0024] Here, in the purification of a radionuclide-labeled agent, there may be a labeled peptide labeled with a radionuclide while there may also be an unlabeled peptide not labeled with a radionuclide. When removing free radionuclides as described above, the labeled peptide is selectively adsorbed onto a column by utilizing the hydrophobicity of the peptide. Therefore, both the labeled peptide and the unlabeled peptide are adsorbed onto the column without distinction. In this regard, according to the findings of the present invention, it is desirable to remove unlabeled peptides because they cannot be used as radiodiagnostic agents or radiation internal therapeutic agents.

[0025] Therefore, the present inventor further conducted intensive studies. That is, as chelating agents contained in the peptide, for example, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic Acid: DOTA) shown in the following general formula (3), or trivalent chelating agents such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic Acid: NOTA) shown in general formula (4) were adopted. Further, as the radionuclide, a tetravalent radionuclide such as zirconium (Zr) was adopted. In these cases, it was conceived that the labeled peptide could be separated by ionic interaction in a state of forming a complex with the radionuclide.

[0026]

Chemical formula

[0027] Specifically, the inventors passed a labeling reaction solution containing a labeled peptide labeled with a radionuclide such as radiozirconium, unreacted free radionuclides such as radiozirconium, and an unlabeled peptide through a column containing a weakly acidic cation exchange resin. As a result, the inventors found that the labeled peptide could be selectively extracted by the cation exchange column. That is, by adsorbing the radionuclide-labeled peptide onto the cation exchange column, selectively retaining it, and then eluting it, the target substance, the radionuclide-labeled peptide (hereinafter referred to as the radionuclide-labeled peptide), could be recovered from the free radionuclide and the unlabeled peptide.

[0028] Based on these experiments, the inventors conducted research and focused on the fact that when a trivalent chelating agent, such as DOTA, in which one of the carboxylic acids is amide-bonded, binds to a tetravalent radionuclide such as zirconium, it forms a +1-valent complex. In other words, they focused on the fact that a +1-valent complex is formed when a trivalent chelating agent binds to a tetravalent radionuclide, whereas unlabeled peptides do not have a positive charge. From this point, the inventors conceived a method for separating radionuclide-labeled drugs by utilizing the difference in valency between labeled peptides that form a +1-valent complex and unlabeled peptides that do not have a positive charge, and devised a method for separation by cation exchange.

[0029] Furthermore, the inventors have investigated and found that a carboxyl group (COO) can be used as a functional group for cation exchange resins. - It was found that it is preferable to use a so-called weakly acidic cation exchange resin having a carboxyl group (COO). When the inventors conducted experiments, it was confirmed that when a radionuclide-labeled peptide was passed through a weakly acidic cation exchange resin column, the radionuclide-labeled peptide was retained in the weakly acidic cation exchange resin column due to intermionic interactions. Subsequently, when an acidic solution was passed through the column to wash it, the carboxyl group (COO) in the weakly acidic cation exchange resin was retained. - The carboxylic acid (COOH) loses its charge and thus loses its ion exchange properties, allowing the radionuclide-labeled peptide to be eluted from the column. One embodiment described below was devised by the inventors through the above-mentioned diligent research.

[0030] Next, before describing a method for producing a radionuclide-labeled agent according to one embodiment of the present invention, a method for producing a radionuclide complex will be described. In this embodiment, an example of a method for producing a radionuclide complex will be described when zirconium is used as the radionuclide and DOTA is used as the chelating agent contained in the peptide.

[0031] First, a DOTA solution containing a compound, such as DOTA, is introduced into a microcentrifuge tube as a radionuclide labeling agent at a predetermined concentration. Next, a nearly neutral buffer solution is introduced into the microcentrifuge tube. For example, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) with a pH of 7.0 can be used as the buffer solution, and its concentration is typically between 0.25 mol / L and 1.2 mol / L, preferably between 0.25 mol / L and 1 mol / L. Next, an organic solvent, such as DMSO, is introduced into the microcentrifuge tube. The order in which the DOTA solution, buffer solution, and organic solvent are introduced into the microcentrifuge tube is not limited to the order described above, and various orders are possible.

[0032] After introducing the DOTA solution, buffer solution, and organic solvent into the microtube, add the following to the reaction solution inside the microtube: 89 Acidic solution containing Zr ( 89 A mixed solution is generated in a microtube by introducing a Zr-containing acidic solution. In this embodiment, the acidic solution is preferably a strong acid, specifically hydrochloric acid (HCl), but is not necessarily limited. In the microtube, DOTA solution, buffer solution, organic solvent, and 89 After mixing with a Zr-containing acidic solution, the mixture is heated to a predetermined temperature and maintained for a predetermined time. This process involves DOTA and 89 Zr reacted, 89 A zirconium complex is obtained in which DOTA is bound to Zr. In this way, a radionuclide-labeled ligand solution containing the radionuclide complex is obtained.

[0033] Next, a method for purifying the radionuclide-labeled agent will be described. Figure 1 is a diagram illustrating the method for purifying the radionuclide-labeled agent according to this embodiment. In the method for purifying the radionuclide-labeled agent according to one embodiment, a column containing, for example, 100 mg of a weakly acidic cation exchange resin shown in the following chemical formula (5) as the solid phase (e.g., CBA resin: manufactured by GL Sciences Co., Ltd.) is used. CBA resin is a solid phase in which carboxyl groups are bonded to silica gel. In addition, as the labeling agent, for example, FAPI-04 shown in the above chemical formula (1) is used.

[0034] [ka]

[0035] As shown in Figure 1, in one embodiment of the method for purifying a radionuclide-labeled agent, the column is first conditioned by passing physiological saline through it. The quenchable solution consisting of physiological saline that has passed through the column is discarded. This step can be omitted if the column does not require conditioning.

[0036] Next, the radionuclide-labeled ligand solution is passed through a column containing a weakly acidic cation exchange resin. This causes the radionuclide-labeled ligand to adsorb onto the column. In this case, it is thought that the weakly acidic cation exchange resin and the radionuclide-labeled peptide interact ions, as shown in the following chemical formula (6-1). The permeate containing free radionuclides that have passed through the column is discarded. Subsequently, the column is washed by passing approximately 10 mL of, for example, 20% phosphate-buffered saline (PBS) through the column as a washing solution. Physiological saline may be used instead of phosphate buffer. The permeate after washing the column is discarded. After the column has been washed with phosphate-buffered saline, approximately 3 mL of, for example, 20 mmol / L aqueous phosphoric acid solution is passed through the column as an eluent. This causes the carboxyl groups (COO) in the weakly acidic cation exchange resin to adsorb, as shown in the following chemical formula (6-2). -The ion exchange activity is lost when the carboxylic acid (COOH) is converted to a carboxylic acid (COOH) and loses its charge. As a result, the radionuclide-labeled peptide is eluted from the column, and the radionuclide-labeled ligand adsorbed to the column is eluted into the phosphoric acid aqueous solution.

[0037] [ka]

[0038] The phosphoric acid aqueous solution from which the radionuclide-labeled ligand has been eluted is recovered after passing through the column. Subsequently, an appropriate amount of sodium hydroxide (NaOH) aqueous solution is added to the recovered phosphoric acid aqueous solution to neutralize it. This adjusts the pH of the recovered phosphoric acid aqueous solution to between 5 and 8, making it a neutral solution that can be administered to humans. In this way, it is formulated as a radionuclide-labeled agent. This completes the purification process of the radionuclide-labeled agent according to this embodiment.

[0039] When the inventors measured the presence of unlabeled peptides in the radionuclide-labeled ligands obtained as described above, it was confirmed that the unlabeled peptides were below the detection limit and had been sufficiently removed. Furthermore, when the recovery rate of the labeled peptides was measured, it was confirmed that a recovery rate of approximately 85% could be secured.

[0040] (modified version) Furthermore, it is possible to apply this method to various radionuclide-labeled agents in addition to those mentioned above. For example, ligands containing chelating agents, such as those shown in chemical formula (7) below, can also be used.

[0041] [ka]

[0042] In the case of the radionuclide labeling agent shown in chemical formula (7), it is conceivable that ionic interactions occur with the weakly acidic cation exchange resin of the weakly acidic cation exchange resin column, as shown in chemical formula (8) below.

[0043] [ka]

[0044] Furthermore, a column containing 500 mg of CBA resin as a solid phase (manufactured by GL Sciences Co., Ltd.) was used as the column containing the weakly acidic cation exchange resin. In addition, approximately 10 mL of 20% PBS was used as the washing solution, and approximately 30 mL of 20 mmol / L phosphoric acid aqueous solution was used as the eluent. When the inventor measured the presence of unlabeled peptides in the radionuclide-labeled ligand obtained in the manner described above, it was confirmed that the unlabeled peptides were below the detection limit and were sufficiently removed. Furthermore, when the recovery rate of the labeled peptides was measured, it was confirmed that a recovery rate of approximately 65% ​​could be secured. With this, the purification process of the radionuclide-labeled agent according to this embodiment is completed.

[0045] According to the embodiment described above, unlabeled peptides that are not labeled with radionuclides can be removed from radionuclide-labeled drugs including DOTA and NOTA. Furthermore, since disposable columns can be used, there is the advantage of easy quality control.

[0046] Although one embodiment of the present invention has been described in detail above, the present invention is not limited to the above-described embodiment, and various modifications are possible based on the technical idea of ​​the present invention. For example, the column products listed in the above-described embodiment are merely examples, and different products may be used as needed, and the present invention is not limited by the description and drawings that constitute part of the disclosure of the present invention in this embodiment.

[0047] In the embodiment described above, zirconium is used as the radioactive nuclide. 89While Zr was used, other tetravalent radionuclides can also be used, and while DOTA or NOTA are used as the trivalent chelating agent, other trivalent chelating agents can also be used. In other words, based on the technical concept of this invention, any combination of radionuclides and chelating agents that forms a charged complex can be used.

Claims

1. A method for purifying a radionuclide-labeled drug, comprising separating and purifying a radionuclide-labeled drug from a solution containing a labeled peptide having a chelating agent that labels a radionuclide and constitutes a complex, The aforementioned solution is passed through a column containing a weakly acidic cation exchange resin, and then an acidic solution is passed through the column to extract the radionuclide-labeled agent. The radioactive nuclide and the chelating agent produce the +1 valent complex. A method for purifying radionuclide-labeled drugs.

2. The aforementioned radioactive nuclide is a tetravalent radioactive nuclide. A method for purifying a radionuclide-labeled agent according to claim 1.

3. The radioactive nuclide is zirconium. A method for purifying a radionuclide-labeled agent according to claim 2.

4. The chelating agent is a trivalent chelating agent. A method for purifying a radionuclide-labeled agent according to claim 1.

5. The chelating agent includes DOTA or NOTA. A method for purifying a radionuclide-labeled agent according to claim 4.

6. The aforementioned weakly acidic cation exchange resin has a carboxylic acid group as a functional group. A method for purifying a radionuclide-labeled agent according to claim 1.

7. The labeled peptide is a prostate-specific antigen-binding peptide containing PSMA-617, a somatostatin receptor-binding peptide containing DOTA-TATE, or a fibroblast-activating protein-binding peptide containing FAPI-04 or FAPI-2286. A method for purifying a radionuclide-labeled agent according to claim 1.

8. The radioactive nuclide is a tetravalent radioactive nuclide, and the chelating agent is a trivalent chelating agent. A method for purifying a radionuclide-labeled agent according to claim 1.

9. The radioactive nuclide is zirconium, and the chelating agent comprises DOTA or NOTA. A method for purifying a radionuclide-labeled agent according to claim 1.

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