Radionuclide-labeled polymer, preparation method therefor, and usethereof

Abstract

Disclosed herein are a radionuclide-labeled polymer, a preparation method therefor, and a use thereof. The radionuclide-labeled polymer has the following structure: G-L1-L2-P; where G is a group containing a radioactive nuclide, comprising a chelating group and a radioactive nuclide, the chelating group chelates the radioactive nuclide, L1 and L2 are independently a chemical bond or a linking group; and P is a temperature-sensitive structural unit.

Description

A radionuclide-labeled polymer, its preparation method and application

[0001] This application claims priority to Chinese patent application No. 2024115715157, filed on November 6, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to a radionuclide-labeled polymer, its preparation method, and its application. Background Technology

[0003] With the rapid development of biomedical technology, biocompatible hydrogels have been widely used in various biomedical fields such as filling, repair, drug delivery, and interventional embolization. For example, hydrogels have been used as an advanced material for vascular embolization in the treatment of tumors with rich blood supply, such as hepatocellular carcinoma.

[0004] There is currently no method to sustainably and in real-time track the status and location of implanted hydrogels, posing a significant challenge to the in vivo performance evaluation of implantable hydrogels. While existing angiography techniques can visualize vascular embolism to some extent, the imaging time is limited, making it impossible to continuously observe the status and location of implanted hydrogels and thus failing to meet the imaging requirements of hydrogel materials with longer implantation periods. Summary of the Invention

[0005] To address the issue that existing hydrogel materials cannot meet the imaging requirements of long implantation periods, this disclosure provides a radionuclide-labeled polymer, its preparation method, and its applications. The radionuclide-labeled polymer provided in this disclosure can be used as a tracer for hydrogels without altering the properties of the hydrogel itself. It exhibits good compatibility with hydrogels, excellent binding stability, and maintains high radioactivity purity both in vivo and in vitro for more than 11 days. It can be used as a tracer for in vivo implantation of hydrogels to continuously and in real-time track the position and status of the implanted hydrogel, and can be used for in vivo performance evaluation of implantable hydrogels.

[0006] This disclosure provides a radionuclide-labeled polymer having the following structure: G-L1-L2-P;

[0007] Wherein, G is a group that chelates a radionuclide, which includes a chelating group and a radionuclide, wherein the chelating group chelates with the radionuclide;

[0008] L1 and L2 are independently chemical bonds or linking groups;

[0009] P represents a temperature-sensitive structural unit.

[0010] In one scheme, G consists of a chelating group and a radioactive nuclide.

[0011] In one particular scheme, the radionuclide is 89 Zr;

[0012] The chelating group is a chelating group formed by a bifunctional chelating agent.

[0013] In one embodiment, L1 is a chemical bond or a linking group as shown in formula (I-1):

[0014] In equation (I-1), x and y are independently selected from integers from 0 to 3;

[0015] R1 is C 1-5 alkylene Or chemical bonds;

[0016] R e for Substituted or unsubstituted C 6-10 aryl, or, substituted or unsubstituted C 5-10 heteroaryl;

[0017] R d C 1-5 alkylene Or chemical bonds;

[0018] R b It is hydrogen, hydroxyl or C 1-5 alkyl;

[0019] X is O or S; e represents the connection site between G and L1, and i represents the connection site between R1 and R2. e The connection site, f represents the connection site between L1 and L2;

[0020] The L2 is a chemical bond, or a group selected from those shown in formula (II-1) or (II-2):

[0021] In equation (II-1), z is an integer in the range of 0-10;

[0022] In equation (II-2), R2 and R3 are independently hydrogen and C. 1-5 Alkyl, substituted or unsubstituted C 6-10 aryl, or, substituted or unsubstituted C 5-10 heteroaryl, amide, or thiol; f represents the connection site between L1 and L2, and h represents the connection site between L2 and P;

[0023] u is an integer in the range of 0-10;

[0024] Each R 1-1 R e-1 and Rd-1 Independent is C 1-5 alkylene,

[0025] L1 and L2 are not chemical bonds at the same time.

[0026] In one embodiment, P is a chitosan structural unit, an acrylamide structural unit, or a group as shown in formula (III-1):

[0027] In equation (III-1), a and c are independently selected from the range 2-130; b is in the range 10-80; and R is selected from hydrogen and C. 1-5 alkyl.

[0028] In this disclosure, in the compound shown in formula (I), each C 1-5 The alkylene groups are independently selected from methylene,

[0029] In this disclosure, each C 6-10 The arylene group is either phenylene or naphthylene, for example, phenylene.

[0030] In this disclosure, each C 5-10 The heteroatom of the heteroaryl group is independently selected from one or two of N, O and S, and the number of heteroatoms is independently one, two or three, preferably N or O, and the number of heteroatoms is one or two, such as pyridine, pyrimidine or pyran.

[0031] In this disclosure, each C 1-5 The alkyl group is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl, such as methyl, ethyl, or isopropyl.

[0032] In this disclosure, each substituted or unsubstituted C 6-10 aryl, or, substituted or unsubstituted C 5-10 heteroaryl, or, substituted or unsubstituted C 6-10 aryl, or, substituted or unsubstituted C 5-10 The substituents in heteroaryl groups are independently C 1-3 Alkyl, halogen, hydroxyl, or cyano groups; the C 1-3 The alkyl group is methyl, ethyl, or propyl;

[0033] The halogen is F, Cl, Br or I.

[0034] The chelating group may be a chelating group formed by a bifunctional chelating agent, such as DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, DFO, or TACN.

[0035] In one embodiment, the chelating group is

[0036] In one embodiment, L1 is the linking group shown in formula (I-2) or (I-3):

[0037] In equation (I-2), R1 is... Or C 1-5 alkylene groups, i indicates connection to Re; R e For substituted or unsubstituted C 6-10 aryl, or, substituted or unsubstituted C 5-10 Hybrid aryl; where e indicates connection to G, and f indicates connection to L2;

[0038] In equation (I-3), each n is independently selected from an integer in the range of 0-10, and R b It can be hydrogen or hydroxyl.

[0039] In one particular scheme, in equation (I-2), R1 or methylene i indicates that it is related to R e Connection site.

[0040] In one particular scheme, in equation (I-2), R e For substituted or unsubstituted C 6-10 arylene compounds, such as phenylene compounds;

[0041] In one embodiment, in formula (I-2), R1 is a methylene group or... And R e For phenyl, i indicates that it is related to R e connect.

[0042] In a certain scheme, in equation (I-3), each n is independently 1, 2, 3, 4, or 5, R b It is a hydroxyl group.

[0043] In one scheme, L1 is e indicates a connection to G, and f indicates a connection to L2.

[0044] In one scheme, in equation (II-1), z is 0, 1, 2 or 3.

[0045] In one scheme, in formula (II-2), both R2 and R3 are methyl groups.

[0046] In one scheme, u in equation (II-2) is 0, 1, 2 or 3.

[0047] In one scheme, in formula (II-2), R2 and R3 are both methyl groups, and u is 0.

[0048] In one scheme, L2 is (For example )or

[0049] In a certain scheme, in equation (III-1), a and c are independent integers in the range of 80-130, and b is an integer in the range of 30-60; for example, a or c is 80 or 101, and b is 27 or 56.

[0050] In one embodiment, the acrylamide structural unit comprises groups represented by formula (III-2):

[0051] In equation (III-2), R c It can be hydrogen, -CH-(CH3)2, -C-(CH3)3, or -(CH2). m -NH2, where m is an integer between 0 and 5, R a Independently, it can be -CH-(CH3)2, -C-(CH3)3, or -(CH2). m -NH2 can form a divalent group by losing a hydrogen atom;

[0052] p or q are independent integers in the range of 20-200.

[0053] In one scheme, in equation (III-2), R c It can be -CH-(CH3)2 or -(CH2). m -NH2, where m is 1, 2, 3, 4 or 5.

[0054] In one scheme, in equation (III-2), R c It is -CH-(CH3)2 or -(CH2)2-NH2.

[0055] In one scheme, in equation (III-2), R a -(CH2) m -NH2 loses hydrogen to form a divalent group, where m is an integer between 0 and 5, for example -(CH2)2-NH-.

[0056] In a certain scheme, in equation (III-2), p is an integer in the range of 100-150, and q is an integer in the range of 20-40; for example, when R c When it is -CH-(CH3)2, p is 110-125, q is an integer in the range of 20-25 or R. c When it is -(CH2)2-NH2, p is an integer in the range of 120-135, and q is an integer in the range of 25-35.

[0057] In one scheme, P is

[0058] In one embodiment, P is derived from the polymer represented by formula (IV):

[0059] In equation (IV), the definitions of a, b, c, and R are as described above.

[0060] In one embodiment, the polymer in formula (IV) has an average relative molecular weight of 8000-13000 Da, for example 8600 Da-11500 Da, preferably 8600 Da or 11500 Da.

[0061] In some embodiments, the P is derived from the polymer represented by formula (V):

[0062] In equation (V), R c It can be hydrogen, -CH-(CH3)2, -C-(CH3)3, or -(CH2). m -NH2, where m is an integer between 0 and 5, p is an integer between 100 and 150, and q is an integer between 20 and 40.

[0063] In one embodiment, the polymer satisfies the following schemes (1)-(5):

[0064] Option (1):

[0065] The chelating group is

[0066] The P is Where R is a methyl group, a is an integer in the range of 75-110, and b is an integer in the range of 25-70;

[0067] L1 is Where e represents a connection to G, and f represents a connection to L2;

[0068] The L2 is f represents the connection site between L1 and L2, and h represents the connection site between L2 and P;

[0069] Option (2):

[0070] The chelating group is

[0071] The P is Where R is a methyl group, a is an integer in the range of 75-110, and b is an integer in the range of 25-70;

[0072] L1 is Where e represents a connection to G, and f represents a connection to L2;

[0073] The L2 is f represents the connection site between L1 and L2, and h represents the connection site between L2 and P;

[0074] Option (3):

[0075] The chelating group is

[0076] The P is Where R is a methyl group, a is an integer in the range of 75-110, and b is an integer in the range of 25-70;

[0077] L1 is Where e represents a connection to G, and f represents a connection to L2;

[0078] The L2 is f represents the connection site between L1 and L2, and h represents the connection site between L2 and P;

[0079] Option (4):

[0080] The chelating group is

[0081] The P is

[0082] L1 is Where e represents a connection to G, and f represents a connection to L2;

[0083] The L2 is a chemical bond;

[0084] Option (5):

[0085] The chelating group is

[0086] The P is

[0087] L1 is a chemical bond;

[0088] The L2 is a chemical bond.

[0089] In some of the schemes disclosed herein, the chemical bonds are covalent bonds.

[0090] In one embodiment, the radionuclide-labeled polymer has any of the following structures:

[0091] Where a, b, c, p, q, R, R a and R c As previously stated. For example:

[0092] In one embodiment, the radionuclide-labeled polymer has any of the following structures:

[0093] Where a, b, c, p, q, R, R a and R c As previously stated. For example:

[0094] In some embodiments, the radionuclide-labeled polymer has any of the following structures:

[0095] This disclosure also provides a polymer as shown in Formula A:

[0096] Q-L1-L2-P

[0097] A;

[0098] Wherein, L1, L2, and P are defined as described above, and Q is a chelating group, which is described above.

[0099] In one embodiment, the polymer as shown in Formula A has any of the following structures:

[0100] a and c are independently selected from the range 2–130; b is in the range 10–80, and R is selected from hydrogen and C. 1-6 alkyl;

[0101] R c It can be hydrogen, -CH-(CH3)2, -C-(CH3)3, or -(CH2). m -NH2, where m is an integer between 0 and 5, R a It can be -CH-(CH3)2, -C-(CH3)3, or -(CH2). m-NH2 can be formed by losing a hydrogen atom to form a divalent group.

[0102] p or q are independent integers in the range of 20-200.

[0103] In one embodiment, the polymer as shown in Formula A has any of the following structures:

[0104] a, b, c, p, q, R, Rc, and Ra are as described above.

[0105] In some embodiments, the polymer shown in Formula A has any of the following structures:

[0106] For example, polymers of formula (A) are selected from the following polymers:

[0107] This disclosure also provides a method for preparing a radionuclide-labeled polymer, comprising the following steps: mixing and incubating the polymer as shown in formula (A) above with a solution containing a radionuclide to obtain a radionuclide-labeled polymer.

[0108] In one embodiment, the radionuclide-labeled polymer in the preparation method is as described above.

[0109] In one embodiment, the preparation method includes the following steps:

[0110] S1. Provide an oxalic acid solution containing radioactive metal ions to obtain mixed solution 1;

[0111] S2. Incubate mixed solution 1 with Na2CO3 solution at room temperature to obtain mixed solution 2;

[0112] S3. Mix the mixed solution 2 with the polymer and buffer solution shown in formula (A), incubate, and obtain the product after the reaction is complete;

[0113] In step S1, the solvent for the oxalic acid solution is water;

[0114] In step S2, the solvent for the Na2CO3 solution is water.

[0115] Preferably, the preparation method further includes:

[0116] S0. Prepare the polymer to be labeled;

[0117] Step S0 includes the following steps:

[0118] S01. Synthesizing amino derivatives of temperature-sensitive polymers;

[0119] S02. The amino derivative is subjected to a chelating reaction with a chelating agent to generate the polymer shown in formula (A), wherein the polymer shown in formula (A) is a chelate of a polymer and a chelating group.

[0120] In one particular scheme, S01 includes the following steps:

[0121] (1) In anhydrous dichloromethane, amino-protected amino acids react with the above temperature-sensitive polymer in the presence of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to obtain crude product;

[0122] (2) In anhydrous dichloromethane, the crude product from step (1) undergoes a deprotection reaction to obtain an amino derivative of the temperature-sensitive polymer.

[0123] In one particular scheme, S02 includes the following steps:

[0124] (3) In the bicarbonate solution, the amino derivative in step (2) undergoes an addition reaction with the chelating agent to obtain the polymer A to be labeled;

[0125] The amino-protected amino acid is valine protected by a Boc group;

[0126] The deprotection reaction is a deprotection of Boc;

[0127] The bifunctional chelating agent is one or more of DFO or DOTA.

[0128] This disclosure also provides a gel composition comprising a radionuclide-labeled polymer and a hydrogel formulation as described above.

[0129] The hydrogel formulations of this application may be conventional hydrogel formulations used in the art for in vivo vascular embolization, in vivo hemostasis, drug delivery, in vivo tissue filling or in vivo tissue repair, and preferably include temperature-sensitive gelling agents and gelling agents.

[0130] In one embodiment, the temperature-sensitive gelling agent is one or more of poloxamer, N-isopropylacrylamide polymer, chitosan, or polyethylene glycol block copolymer.

[0131] In one embodiment, the gelling agent is one or more of sodium alginate, hydroxymethyl cellulose, and CaCl2.

[0132] In one embodiment, the hydrogel formulation further includes a developer, such as iohexol.

[0133] In one embodiment, based on the total volume of the hydrogel formulation, the mass-to-volume ratio of the temperature-sensitive gelling agent is 0.12-0.2 g / mL, the mass-to-volume ratio of the gelling agent is 0.005-0.5 g / mL, preferably 0.14-0.18 g / mL for the temperature-sensitive gelling agent and 0.005-0.3 g / mL for the gelling agent. In another embodiment, the hydrogel formulation comprises: 0.2-1 g / mL iohexol, 0.02-0.1 g / mL sodium alginate, 0.12-0.2 g / mL poloxamer, 0.005-0.1 g / mL hydroxymethyl cellulose or CaCl2, and water.

[0134] In one embodiment, the gelling agent is prepared by the following method: dissolving sodium alginate and hydroxymethyl cellulose in an aqueous solution of iohexol, heating to dissolve, and cooling to obtain the gelling agent; wherein the mass ratio of iohexol to water in the aqueous solution of iohexol is (1-5):3, for example 2:3.

[0135] In one embodiment, the hydrogel formulation comprises one or more of sodium alginate, poloxamer, and hydroxymethyl cellulose, preferably the hydrogel formulation is composed of iohexol, sodium alginate, poloxamer, hydroxymethyl cellulose, and water.

[0136] In one embodiment, the mass-to-volume ratio of the radionuclide-labeled polymer to the hydrogel formulation is 0.01 mg / mL to 2 mg / mL, preferably 0.01 mg / mL to 1.5 mg / mL, more preferably 0.06 mg / mL to 1 mg / mL, for example 0.06 mg / mL.

[0137] In one embodiment, the gel composition, based on its volume, comprises 0.06 mg / mL of the radionuclide-labeled polymer, 0.4 g / mL of the iohexol, 0.02 g / mL of the sodium alginate, 0.14 g / mL of the poloxamer, 0.005 g / mL of the hydroxymethyl cellulose, and the balance being water.

[0138] In one embodiment, the sol-gel transition temperature of the gel composition is 25-37°C, preferably 25.0-28.3°C, for example 25.5-27.5°C.

[0139] This disclosure also provides a radionuclide-labeled polymer, which is prepared by the preparation method described above.

[0140] This disclosure also provides a tracer comprising a polymer labeled with a radionuclide as described above.

[0141] In one embodiment, the tracer is made by adding a polymer labeled with a radionuclide as described above to the hydrogel formulation as described above. The tracer is a PET / CT imaging agent or a SPECT imaging agent, so that the in vivo distribution of the gel composition can be observed by PET / CT imaging.

[0142] In one embodiment, the tracer is used to evaluate the embolic properties of the gel composition as described above.

[0143] This disclosure also provides an application of the gel composition as described above, wherein the application is the use of the gel composition in the preparation of materials for in vivo vascular embolization, in vivo hemostasis, or in vivo tissue repair.

[0144] In one embodiment, the application is in the preparation of an embolic agent, wherein the embolic agent may be an embolic agent for transcatheter arterial chemoembolization in the treatment of tumors.

[0145] This disclosure also provides a method for tracing an implantable hydrogel, comprising the following steps:

[0146] (1) Provide a hydrogel, and add the polymer labeled with radionuclides as described above;

[0147] (2) Implanted into the subject's body;

[0148] (3) Perform PET / CT imaging at multiple preset time points, check the imaging effect, and / or perform radioactivity counting.

[0149] This application also provides a method for radiolabeling a hydrogel formulation, comprising:

[0150] Provides a polymer labeled with radionuclides as described above;

[0151] The radionuclide-labeled polymer is added to the hydrogel formulation as described above.

[0152] In one embodiment, the temperature-sensitive gelling agent and the radionuclide-labeled polymer have the same temperature-sensitive structural unit.

[0153] Preferably, when the temperature-sensitive gelling agent is poloxamer, P in the radionuclide-labeled polymer includes the structure shown in formula (III-1);

[0154] When the temperature-sensitive gelling agent is an N-isopropylacrylamide polymer, P in the radionuclide-labeled polymer includes the structure shown in formula (III-2).

[0155] In one protocol, the subjects are humans, primates, or non-primate mammals.

[0156] In one protocol, the subject is an experimental animal model.

[0157] Except as for the foregoing, when used in the specification and claims of this application, the following terms shall have the following meanings unless otherwise specifically indicated:

[0158] In this document, "temperature sensitivity" refers to the property of gelling from a liquid state or solidifying from a liquid state to a gel or solid state with increasing temperature. "Hydrogel formulation" refers to a water-soluble or hydrophilic polymer or a composition containing such polymers, including gel-like substances formed by chemical or physical cross-linking of the composition. The hydrogel formulation may include temperature-sensitive polymers, such as poloxamer or acrylamide polymers, or other known temperature-sensitive polymers in the art. Therefore, the hydrogel formulation exhibits temperature-responsive properties, such as transforming from a sol state to a gel state at a certain temperature. The hydrogel formulation can be used as a material for in vivo vascular embolization, in vivo hemostasis, drug delivery, in vivo tissue filling, or in vivo tissue repair. The hydrogel formulation can be any hydrogel material for medical, pharmaceutical, or cosmetic use.

[0159] In this article, "phase transition temperature" refers to the temperature or temperature range at which a polymer undergoes a phase transition.

[0160] The term "polymer" refers to a molecule formed by the combination of two or more oligomer units. These chemical units are typically linked by covalent bonds. The two or more chemical units in a polymer can be entirely identical; in this case, the polymer is called a homopolymer. They can also be combinations of different units; these polymers are called copolymers.

[0161] The term "chemical bond" refers to the collective term for the strong interaction forces between two or more adjacent atoms (or ions) within a pure molecule or crystal, generally ionic bonds, covalent bonds, and metallic bonds.

[0162] The term "linking group" refers to the portion of an organic compound that remains after it loses one or more atoms, acting as a linker. It is a general term for groups and bases that act as links. Linking groups typically contain covalent bonds, but they are not chemical bonds themselves.

[0163] The term "heteroaryl" refers to an aromatic system containing one, two, three, or four heteroatoms selected from N, O, or S. Heteroaryl groups include, but are not limited to, thiazolyl, thiophene, pyridinyl, and pyrimidinyl.

[0164] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this disclosure.

[0165] All reagents and raw materials used in this disclosure are commercially available.

[0166] The beneficial effects of this disclosure are as follows: This disclosure provides a radionuclide-labeled polymer that can be mixed with thermosensitive hydrogels without changing the physicochemical properties of the hydrogel itself, and can still maintain the original viscosity and thermosensitive curing properties of the hydrogel. The radionuclide labeling signal can exist stably in vivo for a long time, maintain a long half-life, and the continuous imaging cycle can reach more than 11 days. It can be used for in vivo tracing of hydrogel embolization agents, and can conveniently and continuously monitor the distribution of hydrogel embolization in vivo without the need for multiple angiography.

[0167] The radionuclide-labeled polymer disclosed herein covalently links the polymer unit with a chelating group, and then chelates the radionuclide through the chelating group, resulting in better binding force with the hydrogel formulation. When used for radiolabeling of implantable hydrogels, it exhibits better stability and in vivo imaging time compared to non-covalent linkage methods such as direct addition of zirconium salt chelation or hydrogen bonding. Attached Figure Description

[0168] Figure 1 shows dog B01. 89 Dynamic transverse sectional view of Zr-labeled poloxamer in vivo over 5 hours;

[0169] Figure 2 shows dog B01. 89 Dynamic coronal plane plot of Zr-labeled poloxamer in vivo over 5 hours;

[0170] Figure 3 shows dog B01. 89 In vivo dynamic transverse sectional view of Zr-labeled poloxamer 1d;

[0171] Figure 4 shows dog B01. 89 Dynamic coronal plane plot of Zr-labeled poloxamer 1d in vivo;

[0172] Figure 5 shows dog B01. 89 Dynamic transverse sectional view of Zr-labeled poloxamer 5d in vivo;

[0173] Figure 6 shows dog B01. 89 Zr-labeled poloxamer 5d in vivo dynamic coronal plane map;

[0174] Figure 7 shows dog B01. 89 In vivo dynamic transverse sectional view of Zr-labeled poloxamer 7d;

[0175] Figure 8 shows dog B01. 89 Dynamic coronal plane plot of Zr-labeled poloxamer 7d in vivo;

[0176] Figure 9 shows dog B01. 89 In vivo dynamic transverse sectional view of Zr-labeled poloxamer 11d;

[0177] Figure 10 shows dog B01.89 Dynamic coronal plane plot of Zr-labeled poloxamer 11d in vivo;

[0178] Figure 11 shows dog B04. 89 Dynamic transverse sectional view of Zr-labeled poloxamer in vivo over 5 hours;

[0179] Figure 12 shows dog B04. 89 Dynamic coronal plane plot of Zr-labeled poloxamer in vivo over 5 hours;

[0180] Figure 13 shows dog B04. 89 In vivo dynamic transverse sectional view of Zr-labeled poloxamer 1d;

[0181] Figure 14 shows dog B04. 89 Dynamic coronal plane plot of Zr-labeled poloxamer 1d in vivo;

[0182] Figure 15 shows dog B04. 89 Dynamic transverse sectional view of Zr-labeled poloxamer 5d in vivo;

[0183] Figure 16 shows dog B04. 89 Zr-labeled poloxamer 5d in vivo dynamic coronal plane map;

[0184] Figure 17 shows dog B04. 89 In vivo dynamic transverse sectional view of Zr-labeled poloxamer 7d;

[0185] Figure 18 shows dog B04. 89 Dynamic coronal plane plot of Zr-labeled poloxamer 7d in vivo;

[0186] Figure 19 shows dog B04. 89 In vivo dynamic transverse sectional view of Zr-labeled poloxamer 11d;

[0187] Figure 20 shows dog B04. 89 Dynamic coronal plane plot of Zr-labeled poloxamer 11d in vivo;

[0188] Figure 21 is 89 Calibration curve of Zr activity meter test values ​​and γ counter test values. Detailed Implementation

[0189] The present disclosure is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present disclosure. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0190] Example:

[0191] Material:

[0192] Polymer 1:

[0193] Manufacturer: BASF CORPORATION, Model No. P 407 Geismar.

[0194] Polymer 2:

[0195] Manufacturer: BASF CORPORATION, Model No. P 188 Geismar.

[0196] Chitosan: average relative molecular weight 5000 Da, product number C434552, non-animal origin, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0197] Iohexol: Manufacturer: Shanghai Sitaili Pharmaceutical Co., Ltd.

[0198] Sodium alginate: Manufacturer: Qingdao Mingyue Alginate Tissue Engineering Materials Co., Ltd.

[0199] 89 Zr:

[0200] Manufacturer: PerkinElmer, Inc.

[0201] Purity and content: The total activity concentration was 19.54 mCi / mL after opening the box.

[0202] Formula composition: 89-Zr dissolved in 1 mol / L oxalic acid solution.

[0203] Storage conditions: Store at room temperature in a 2mL V-bottom screw-cap glass bottle in a lead container, taking care to prevent ionizing radiation.

[0204] N-isopropylacrylamide monomer: sourced from Shanghai Aladdin, catalog number I106818.

[0205] N-(3-aminopropyl)methacrylamide monomer (APAM monomer): Shanghai Aladdin, item number N129096.

[0206] Beagle dog. Animal source: Nanjing Chaimen Biotechnology Co., Ltd.

[0207] Unless otherwise specified, all other reagents are commercially available.

[0208] Preparation Example 1: Radiolabeled Polymer 89 Preparation of Zr-DFO-P1-1

[0209] 1.1 Synthesis of thermosensitive polymer and DFO chelate

[0210] (1) Polymer amino activation

[0211] 1.086 g of Boc-Val-OH (Boc-L-valine), 1.91 g of DCC (N,N'-dicyclohexylcarbodiimide), and 1.22 g of DMAP (4-dimethylaminopyridine) were dissolved in 40 mL of ultra-dry anhydrous DCM (dichloromethane) at a molar ratio of 1:2:2. The mixture was reacted at room temperature under argon protection for half an hour. 5.75 g of polymer 1 (P1-1) was added, and the reaction continued for 2 days. The DCM was concentrated by rotary evaporation, precipitated with ice-cold ether, and the white precipitate was collected by filtration. The precipitate was redissolved with 3 mL of DCM and precipitated again with ice-cold ether. This precipitation and filtration process was repeated three times. The precipitate was redissolved in ultrapure water at a ratio of 1 mg:1 mL (heated). The mixture was dialyzed for three days, and lyophilized to obtain 4.13 g of Boc-protected polymer P1-1-Val-Boc, with a yield of 71.9%.

[0212] (2) De-Boc protection

[0213] 4g of polymer P1-1-Val-Boc was weighed and dissolved in 20mL of DCM. The mixture was placed in an ice-water bath and 45mL of trifluoroacetic acid was added under argon protection. The mixture was stirred and reacted for 2 hours. The solvent was removed by rotary evaporation. The product was re-dissolved in chloroform. The product was then evaporated to dryness and dialyzed in hot water for three days. The product was lyophilized to obtain 3.2g of deprotected polymer P1-1-Val, with a yield of 80%.

[0214] (3) Preparation of DFO chelates

[0215] 15.3 mg of polymer P1-1-Val was dissolved in 4 mL of 0.1 M NaHCO3 (sodium bicarbonate) solution. 2 mg of p-SCN-Bn-DFO (CAS No.: 1222468-90-7) (200 μL DMSO) was added slowly dropwise at a molar ratio of 1:2. The reaction was carried out at room temperature for 1 h. After rotary evaporation, the product was dissolved in hot water and dialyzed for three days. After lyophilization, 10.2 mg of polymer DFO-P1-1 was obtained, with a purity of 99.3% and a yield of 66.7%.

[0216] 1.2 89 Synthesis of Zr-labeled polymers

[0217] 89 Zr-DFO-P1-1

[0218] (1) Prepare the labeling solution

[0219] 89 Zr oxalic acid solution: zirconium [ 89 Zr] dispersed in 1M oxalic acid solution (pH < 4) (PerkinElmer, Inc.);

[0220] Preparation of 0.5M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer: First, add 18 mL of ultrapure water to 20 mL of 1M HEPES buffer and check its pH value. If the pH value is <7.1, adjust the pH value using 1M NaOH; if the pH value is >7.3, adjust the pH value using 1M H2SO4. Once the pH value of the HEPES buffer is within the range of 7.1 to 7.3, continue to add a certain volume of ultrapure water until the total volume is 40 mL.

[0221] Preparation of polymer DFO-P1-1 solution (1 mg / mL): Accurately weigh 0.6 mg of polymer DFO-P1-1 and dissolve it in 600 μL of 10 mM pH 8.4 borate buffer solution.

[0222] (2) 89 Zr-labeled reaction

[0223] A. Accurately measure 84 μL 89 Pour the Zr oxalic acid solution into a glass reaction flask;

[0224] B. Add 1M oxalic acid solution to the glass reaction flask to make the total volume of the reaction flask 200μL;

[0225] C. Accurately pipette 90 μL of 2M Na2CO3 solution into the reaction flask and incubate at room temperature for 3 min;

[0226] D. While gently shaking the reaction flask, dissolve 0.3 mL of 0.5 M HEPES (pH 7.1–7.3) and 600 μL of the prepared polymer DFO-P1-1 solution (1 mg / mL), then add 0.7 mL of 0.5 M HEPES to the reaction flask. (Note: The pH of the reaction solution should be between 6.8 and 7.2 to obtain the best labeling efficiency. Also, do not use a syringe with a metal needle when preparing the solution.)

[0227] E. Incubate at room temperature for 1 hour, while gently shaking the reaction flask;

[0228] F. After the reaction is complete, purify the reaction product using an ultrafiltration centrifuge tube to remove free... 89 Zr (washing solution: 10mM pH8.4 borate buffer solution; 500μL*2 times).

[0229] Preparation Example 2: Radiolabeled Polymer 89 Preparation of Zr-DFO-P1-2

[0230] The polymer 1 (P1-1) in Preparation Example 1 was replaced with polymer 2 (P1-2), and the amount of polymer 2 was calculated based on its average relative molecular mass. The reaction was carried out according to the steps of Preparation Example 1.

[0231] Preparation of radiolabeled polymers (Example 3) 89 Preparation of Zr-DOTA-P1-1

[0232] The reaction was carried out according to the steps of Preparation Example 1, except that p-SCN-Bn-DFO (CAS No.: 1222468-90-7) in Preparation Example 1 was replaced with p-SCN-Bn-DOTA (CAS No.: 127985-74-4).

[0233] Preparation Example 4: Radiolabeled Polymer 89 Preparation of Zr-DFO-P2

[0234] Chitosan was dissolved in 0.1M acetic acid solution, and p-SCN-Bn-DFO (2 mg; 200 μL DMSO) was added. The reaction ratio of chitosan to p-SCN-Bn-DFO was 1:2, and the mixture was slowly added dropwise. The reaction was carried out at 37°C for 4 hours. After rotary evaporation, the product was dissolved in hot water and dialyzed for three days. After lyophilization, the product DFO-P2 was obtained. Then, the process was carried out according to step 1.2 in Preparation Example 1. 89 Radiolabeling of Zr 89 Zr-DFO-P2.

[0235] Preparation Example 5: Radiolabeled Polymer 89 Preparation of Zr-DOTA-P3-1

[0236] (1) Synthesis of Boc-protected N-isopropylacrylamide (NIPAM) polymer:

[0237] 3.69 g of N-isopropylacrylamide monomer (NIPAM), 1.76 g of N-hydroxysuccinimide (NHS), and 290 mg of azobisisobutyronitrile (AIBN) were dissolved in 10 mL of THF. The mixture was reacted at 60 °C for 12 h to prepare pNIPAM-NHS. Then, 80.6 g of N-Boc-ethylenediamine was added and reacted for 12 h. The mixture was then precipitated with 150 mL of diethyl ether, filtered, washed twice with diethyl ether, and dried to obtain pNIPAM-Boc with a yield of 83%.

[0238] (2) Deprotection from Boc groups:

[0239] 2.4 g pNIPAM-Boc was dissolved in 50 mL of trifluoroacetic acid aqueous solution (trifluoroacetic acid:water volume ratio of 8:2), stirred at room temperature for 2 h, dried under vacuum by rotary drying, reconstituted in water, purified by Sephadex G25 column chromatography, and freeze-dried to obtain pNIPAM with a yield of 90%.

[0240] (3) DOTA activation: Dissolve 48 mg DOTA in 2 mL of water, and dissolve 9.0 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in 0.5 mL of water. Mix the two aqueous solutions to form a reaction mixture. Adjust the pH of the reaction mixture to 5.0 with 0.1 M NaOH. Then add 6.8 mg NHS, stir in an ice bath, and react for 40 minutes to obtain DOTA-NHS.

[0241] (4) Synthesis of pNIPAM-DOTA compound: 298 mg of pNIPAM (containing 0.03 mmol of amino groups) was dissolved in a mixed solvent, which included 3 mmol of N,N-diisopropylethylamine (DIEA) and 6 mL of DMAC. The mixture was heated to 45 °C, and 0.03 mmol of DOTA-NHS was added. The reaction was carried out for 3 hours. The mixture was purified by centrifugation and filtration. The molecular weight cutoff of the centrifugation filtration membrane was 3.5 kDa. After purification, the mixture was freeze-dried to obtain the chelated ligand product DOTA-P3-1.

[0242] (5) Follow the steps in Preparation Example 1, Step 1.2. 89 Zr radiolabeling, to obtain 89 Zr-DOTA-P3-1.

[0243] In this preparation example, the N-isopropylacrylamide polymer (the product of step (2) of preparation example 5) had an average molecular weight of 17400 g / mol as determined by size exclusion chromatography (SEC).

[0244] After connecting DOTA, the D2O solution was recorded using a Varian AC400 NMR spectrometer (Varian NMR Systems, Palo Alto, CA). 1 According to the 1H-NMR, the molar ratio of the two repeating units, p:q, is approximately 0.85:0.15.

[0245] Preparation of radiolabeled polymers (Example 6) 89 Preparation of Zr-DFO-P3-2

[0246] (1) 3.37 g of N-(3-aminopropyl)methacrylamide monomer (APAM monomer) and 164 mg of azobisisobutyronitrile (AIBN) as initiator were added. The mixture was synthesized via free radical reaction in an acetone-DMSO mixed solvent (V(acetone):V(DMSO)=15:1). The polymerization reaction was carried out under vacuum at 55℃ for 24 hours. The precipitate was recovered by filtration, yielding the crude product. The crude product was dissolved in a small amount of methanol, and 15 times its volume of excess acetone was added to precipitate the product. The precipitate was dried under vacuum to obtain polyurethane-propylmethacrylamide. The average molecular weight Mn was determined to be 19200-21500 g / mol by size exclusion chromatography (SEC) using a Synchropak CATSEC-300 column.

[0247] (2) p-SCN-Bn-DFO (CAS No.: 1222468-90-7) (2 mg; 200 μL DMSO) was slowly added dropwise to 12 mg of polyurethane methacrylamide. Sodium hydroxide was added to adjust the pH to 6.0. The reaction was carried out at room temperature for 1 h. After rotary evaporation, the product was dissolved in water and dialyzed for three days. The molecular weight cutoff of the centrifuged filter membrane was 10 kDa. After lyophilization, a total of 9.7 mg of polymer was obtained, with a yield of 69.3%. The D2O solution was recorded using a Varian AC400 NMR spectrometer (Varian NMR Systems, Palo Alto, CA). 1 According to the 1H-NMR, the molar ratio of the two repeating units, p:q, is approximately 0.82:0.18.

[0248] (3) Follow the steps in Preparation Example 1, Step 1.2. 89 Zr radiolabeling, to obtain 89 Zr-DFO-P3-2.

[0249] Preparation Example 7: Radiolabeled Polymer 89 Preparation of Zr-DFO-LP1-1

[0250] In Preparation Example 1, p-SCN-Bn-DFO was replaced with compound D-1 (CAS: 1623757-39-0), and D-1 was reacted with P1-Val to prepare the DFO-coupled polymer. Then, proceeding according to step 1.2 of Preparation Example 1... 89 The Zr label.

[0251] Preparation Example 8: Preparation 89 Zr-labeled hydrogel formulations

[0252] (1) Formulation 1

[0253] Preparation of iohexol aqueous solution: Accurately measure iohexol and water for injection at a mass ratio of 2g:3g, mix well, and prepare a 40% iohexol solution.

[0254] To prepare a solution of sodium alginate and hydroxymethyl cellulose: Weigh 2g of sodium alginate and 0.5g of hydroxymethyl cellulose (HPMC-100M), and slowly add them to 100mL of 40% iohexol solution. Slowly heat the solution to 35℃ at room temperature to promote the dissolution of sodium alginate and hydroxymethyl cellulose. After complete dissolution, allow it to cool to room temperature.

[0255] Preparation of the gel composition: Accurately weigh 14g of poloxamer 407 and slowly add it to 100mL of a solution of sodium alginate and hydroxymethyl cellulose. Let it stand at 4℃ for 3-4 hours until completely dissolved to obtain formulation 1. The sol-gel phase transition temperature is 26.5℃. At 37℃, it gels within 32 seconds and transforms into a solid within 50 seconds; the viscosity is 0.9 Pa·s (25℃) and 1.2 × 10⁻⁶ Pa·s. 3 Pa.S (30℃).

[0256] (2) Formulation 2: Embrace HES polyethylene glycol liquid embolizer.

[0257] (3) Formulation 3: Peptide ProShen TM Temperature-sensitive liquid embolizing agent (copolymer of N-isopropylacrylamide and N-n-propylacrylamide).

[0258] (4) Preparation of zirconium-labeled gel composition:

[0259] Preparation 89 Zr polymer-labeled formulations: 1 mL of the formulations prepared in Examples 1-7 89 A Zr-labeled polymer solution (containing 0.6 mg of polymer) is mixed with 10 mL of formulation 1, formulation 2, or formulation 3, and then refrigerated at 4-8°C after gentle stirring.

[0260] (5) Preparation 89 Zr-free, non-chelating agent-labeled formulations: radioisotopes 89 1 mL of Zr oxalic acid solution was added to 10 mL of formulation 1, formulation 2, or formulation 3 and mixed.

[0261] Experiment 1: Performance Testing

[0262] 1.1 89 Radiochemical purity of Zr-labeled hydrogel formulations

[0263] A Mini-Scan thin-layer scanner (Eckert & Ziegler Radiopharma Inc., USA) was used with rapid thin-layer chromatography paper (Agilent ITLC strips, 1cm × 10cm) as the carrier. After spotting the sample, the system was developed with 20mM citric acid solution (pH 4.9–5.1). After the chromatography paper was dried, the thin-layer scanner was used to acquire data. After data acquisition, images were obtained for data analysis.

[0264] The performance test results of the gel compositions prepared according to the proportions of Preparation Example 8 (1) for each polymer and hydrogel formulation prepared in the preparation example are shown in Table 1 below:

[0265] Table 1

[0266] This experiment evaluated the results using thin-layer chromatography. 89 The radiochemical purity of Zr-labeled hydrogel formulations and the labeling stability at different time points are shown in the table above. 89 The Zr-labeled hydrogel formulation maintained a radiochemical purity of over 93% at different time stages, indicating that... 89 Zr-labeled hydrogel formulations exhibit good in vitro stability. Especially through… 89 Zr-labeled polymers used in hydrogel formulations all exhibited radiochemical purity greater than 93% after labeling, meeting experimental requirements, and the radiochemical purity remained greater than 93% even after 19 days. Experimental group h was directly added... 89 Zr in oxalic acid solution 89 Zr ions chelate with the hydroxyl groups of sodium alginate, promoting cross-linking of sodium alginate to form a network structure. 89 Zr wrapping, to achieve 89 Zr without chelating agent coating showed high labeling efficiency at the initial 1 day of labeling, but the radiochemical purity decreased with prolonged storage time, indicating that... 89 The content of Zr-labeled sodium alginate decreased, while the content of Zr-labeled sodium alginate increased. 89 Zr detached from the label, and the experimental group numbered h showed poor stability. The polymer provided in this example exhibits better stability and is less prone to degradation due to the chemical chelation of radioactive elements by the chelating groups and covalent bonds within the polymer. This reduces the release of free radioactive elements and prolongs the radioactive labeling and imaging time.

[0267] 1.2 89 Sol-gel rheological properties of Zr-labeled gel compositions

[0268] The tests were conducted on an AR-1000 constant stress rheometer (manufacturer: TA Instruments) with built-in temperature and gap calibration.

[0269] The rheometer is equipped with a cone / plate geometry (40 mm diameter, 4° cone angle). The degassed sample is dispensed onto a temperature-controlled Peltier plate and pre-equilibrated to 25°C. During rheological testing, the sample is covered with a water-containing solvent trap to prevent moisture loss. The temperature of the Peltier plate is controlled within ±0.1°C of the preset temperature during testing. The geometric gap is calibrated before testing. After loading excess sample, the geometry is lowered to the predetermined gap, and excess sample is removed. The sample is equilibrated for 30 seconds before starting the test. Data is processed using TA data analysis software.

[0270] Viscosity tests were performed on various hydrogel formulations using flow rheology. Steady-state flow experiments were conducted at 25°C to determine the behavior of the hydrogels under increasing shear stress. Viscosities of the samples were measured within a shear stress range of 1 Pa to 100 Pa. The thermal responsiveness of various hydrogel formulations was assessed rheologically by oscillation measurements. Temperature scans from 10°C to 40°C were performed on all hydrogel formulations. The sol-gel transition temperature was defined as the temperature at which gelation occurs. The gel point was defined as the temperature at which the storage modulus (G') equals the loss modulus (G”).

[0271] Therefore, gelation is considered to have occurred when G'>G". The temperature was increased at a rate of 1°C / min, while the oscillation pressure and angular frequency remained constant.

[0272] Performance test results of gel compositions formulated with polymers and hydrogel formulations prepared according to the preparation examples

[0273] As shown in Table 2 below.

[0274] Table 2

[0275] Therefore, the addition of radiolabeled polymers has no significant effect on the viscosity and phase transition temperature of the liquid embolizing agent, nor does it affect the rheological properties or temperature-sensitive properties of the hydrogel formulation itself. Especially when the radiolabeled polymer and the temperature-sensitive material of the hydrogel formulation have the same or similar structural units, the radiolabeled polymer and the hydrogel formulation exhibit excellent compatibility, allowing for more uniform and stable distribution within the hydrogel formulation, with even less impact on the viscosity and phase transition temperature of the hydrogel formulation itself.

[0276] Experimental Example 2: In vivo animal experiments

[0277] This experiment observed the content of radiolabeled polymers in animal blood and used PET / CT to acquire signals within a window of 110 keV ± 15% to dynamically observe the results of preparations in Examples 1-7 of this application. 89The dynamic distribution of Zr-labeled polymers after implantation into Beagle dogs according to the scheme shown in Table 3 over a certain period of time.

[0278] Beagle dogs were given general anesthesia (1.5% sodium pentobarbital, 20 mg / kg). The femoral artery was bluntly dissected and punctured under direct vision. A 5F arterial sheath was inserted, and the femoral artery and sheath were fixed with absorbable sutures. Under digital subtraction angiography (DSA) guidance, a 4F angiographic catheter was inserted from the femoral artery to the abdominal aorta. A Y-valve was connected to the end of the 4F angiographic catheter, and a 2.4F microcatheter was selectively inserted into the common hepatic artery through the positive port of the Y-valve. Gel was injected through the microcatheter, minimizing gel reflux. After embolization, the catheter and arterial sheath were removed, and the area above the femoral artery puncture site was ligated. The muscle and skin were then sutured layer by layer.

[0279] The hydrogel formulation prepared in the preparation example was used as a vascular embolization agent for implantation into Beagle dogs. The implantation protocol is shown in Table 3:

[0280] Table 3 Implantation Protocol

[0281] 2.1 Blood 89 Zr detection

[0282] The embolization agent was administered as follows: 1 mL of labeled gel per animal, with a gel specific activity of 280 μCi / mL. The dosage per dog was approximately 280 μCi. Blood was collected from the cephalic vein of the forelimb using heparin sodium anticoagulant negative pressure blood collection tubes at 5 h, 1 d, 3 d, 5 d, 7 d, and 11 d after hepatic artery embolization, with a blood volume of approximately 2 mL. The blood samples were analyzed using a WIZARD gamma counter. [Establishment / Initialization] 89 The calibration curve for Zr activity meter detection value (μCi) and γ counter detection value (CPM) is used for the conversion of activity and CPM values. The calibration curve is shown in Figure 21.

[0283] Estimate the blood volume based on the dog's total circulating blood volume and blood leakage count, animal dosage, and body weight data. 89 Zr dosage and calculation of blood concentration 89 The percentage of Zr radioactivity relative to the total dose. The circulating blood volume of a Beagle dog is 85 mL / kg. The total blood volume is calculated based on the animal's weight, and then the total blood exposure is calculated based on the total blood volume. The percentage of blood exposure is calculated by comparing this to the attenuated total dose. The percentage of blood exposure relative to the total dose is calculated as follows: body weight (kg) * 85 mL / kg * blood detection value (μCi / g) / administered dose. The blood detection value is corrected to the value at the time of administration.

[0284] Tables 4 and 5 show that on the first day of embolism, the blood of dogs B01-B14 contained... 89The percentage of Zr radioactivity relative to the total dose was less than 7% in all cases, and then gradually decreased until, 7 days after administration, the radioactivity in the blood of dogs B01-B13 was [missing value]. 89 The radioactivity of Zr accounts for approximately 0.69%-2% of the total dose administered.

[0285] Overall, it shows that in the blood 89 The low radioactivity percentage of Zr indicates that the gel formed by the test substance mainly remains in the embolization site in vivo, and also demonstrates the good stability of the test substance after labeling and long-term placement. Specifically, the implantation of Zr-containing... 89 No free Zr-labeled polymer hydrogel formulations were observed in the blood. 89 Abnormal increases or decreases in Zr indicate better stability in animals, with no obvious free Zr observed.

[0286] The polymer provided in this test example is due to... 89 Zr chemically chelates with polymers through chelating groups, compared to... 89 Zr oxalate-labeled formulations exhibit better stability, are less prone to degradation, and reduce the release of free radioactive elements.

[0287] The gel implanted in dog B14 89 Zr is a direct-mixed coating, without chemical chelation of the thermosensitive polymer. Post-implantation, the radioactivity in the blood was significantly higher than other implants, including [other types of implants]. 89 The Zr chemically chelated polymer gel showed high levels of radioactivity in the blood for 1-5 days, indicating that radioactive elements were continuously entering the bloodstream. After 5 days, most embolism sites showed... 89 Zr has entered the bloodstream, and there is no more. 89 Zr escaped, and the radioactivity in the blood decreased after 7 days. Within 11 days, 60% of the total dose of radioactivity entered the bloodstream, indicating that direct addition of free Zr... 89 Zr radiolabeled materials have poor stability and are prone to dissociation from the embolization site. Radiolabeled materials cannot stably trace the position of hydrogels for a long period of time.

[0288] In implantation 89 In animals, Zr-labeled polymer hydrogel formulations, polymers with different structures, acting as radiolabelers for the hydrogels, all exhibited low levels of blood radioactivity and good stability. Especially when the hydrogel formulation and the radiolabeled polymer contain the same thermosensitive polymer units, they have more similar viscosities and phase transition temperatures, resulting in better compatibility and tighter binding, and the radiolabeling exhibits excellent stability. For example, when a radiolabeled polymer containing alkyl ether structural units is added to formulation 1 or 2... 89 Zr-DFO-P1-1 or89 When Zr-DFO-P1-2 is used, the total percentage of radioactive counts in the blood during the detection period is significantly lower than when it is added to formulation 3; radiolabeled polymers containing acrylamide structural units. 89 When Zr-DFO-P3-2 is added to formulation 3, which uses acrylamide polymers as the thermosensitive component, it exhibits significantly better stability than when added to formulation 1.

[0289] Compared to animal B11, animal B01 only differs in the linking group of the radioactive polymer in its formulation, but animal B01 showed better tracer performance, indicating that... 89 Zr-DFO-P1-1 ratio 89 Zr-DFO-LP1-1 has better performance, possibly due to 89 The chain length of the linking group in Zr-DFO-LP1-1 is affected.

[0290] Table 4 Animal blood 89 Zr radioactivity

[0291] Table 5 Animal blood 89 Zr radioactivity percentage

[0292] 2.2 In vivo dynamic imaging study

[0293] Collection times: 5h, 1d, 5d, 7d, 11d, and 18d after embolization.

[0294] Acquisition method: Nuclear medicine computed tomography (CT) + localization CT. After general anesthesia and restraint, the animal was placed under the Discovery VCT instrument. First, a whole-body CT scan was performed to assist in the identification of tissues and organs. Then, a whole-body nuclear medicine signal scan was performed from the head to the bottom of the lower limbs.

[0295] PET-CT imaging data processing: The coronal and transverse images of the PET-CT imaging results of dogs B01-B14 were saved. Based on the imaging images and nuclear medicine images from the two locations, the region of interest (ROI) was delineated (the main organs with nuclear medicine signals were delineated), and the corresponding areas were circled. The percentage of the count of the delineated area to the total count of the whole body was recorded.

[0296] The results are shown in Figures 1-20 and Table 6.

[0297] Figures 1, 3, 5, 7, and 9 are transverse images of dog B01 at 0.5h, 1d, 5d, 7d, and 11d after implantation of the radiolabeled hydrogel preparation; Figures 11, 13, 15, 17, and 19 are transverse images of dog B04 at 0.5h, 1d, 5d, 7d, and 11d after implantation of the radiolabeled hydrogel preparation. In the transverse images, the upper left corner is the CT localization image, the upper right corner is the nuclear medicine imaging image of the localization location, the lower left corner is the image of the nuclear medicine image-rich area in the transverse section, and the lower right corner is the whole-body nuclear medicine signal image (this image does not change with the CT localization image). The localization of each area is the same location—the liver.

[0298] Figures 2, 4, 6, 8, and 10 are coronal views of dog B01 at 0.5h, 1d, 5d, 7d, and 11d after implantation of the radiolabeled hydrogel. Figures 12, 14, 16, 18, and 20 are transverse views of dog B04 at 0.5h, 1d, 5d, 7d, and 11d after implantation of the radiolabeled hydrogel. In the coronal views, the leftmost corner is the CT localization image, the second from the left is the nuclear medicine imaging of the localization location, the third from the left is the nuclear medicine image enriched area in the coronal region, and the fourth from the left is the whole-body nuclear medicine image. All regions are located in the same location—the liver.

[0299] As shown in the images, in the animal implanted with supplementary regimen A (B01), the radioactive signal was mainly distributed in the liver within the first 5 hours after implantation, with a small amount distributed in the intestine and bladder. From 1 to 11 days post-implantation, the radioactive signal remained primarily distributed in the liver, with only very small amounts distributed in other organs. Compared to B01, in the animal implanted with supplementary regimen D (B04), the radioactive signal was mainly distributed in the liver, but the distribution in the lungs, intestines, kidneys, and brain was significantly greater than in B01.

[0300] Table 6 below records the average percentage of the total count in five enriched areas (liver, lungs, intestines, kidneys, and brain) of five dogs.

[0301] Table 6. Percentage of animal organ counts relative to total body count (%)

[0302] Table 6 shows that on the day of administration, the proportion of each drug in the liver of the animals reached over 80%, indicating that the test substance embolized in the liver on the day of administration, and most of the radioactive signal was at the embolization site, indicating high labeling efficiency and almost no degradation. From day 1 to 5 of administration, the proportion of the test substance in the liver decreased slightly, possibly because some of the test substance embolized near the porta hepatis. Radiolabeled polymers that did not fully bind to the hydrogel during the administration phase were washed away by the bloodstream and migrated to other tissues such as the intestine, lung, and kidney, where they accumulated. Therefore, from day 5 to 7, slight distribution was also observed in the intestine, lung, and kidney, and the proportion of radioactive counts showed a slight upward trend; however, the proportion in the liver remained above 80%, still significantly marking the embolization site and not affecting the assessment of the embolization status. From day 11 to day 18 post-intervention, and from day 5 to day 18 post-administration, the proportion of the test substance in the liver gradually increased to 100%. Signals in the intestinal wall and other organs significantly decreased. By day 18, radioactive elements in other organs had been metabolized and excreted, decaying to no signal, with only the liver showing a signal. This indicates that the test substance remained at the embolization site in the body. 89 Zr-labeled polymers exhibit good long-term stability in vivo.

[0303] Throughout the entire experimental period, 89 Zr-labeled polymers were mainly enriched in the liver, with an average percentage between 80% and 100%, indicating that most of the radionuclides were tightly bound to the implant and did not detach. This makes them significantly distinguishable from weak free signals, making them excellent tracers for implantable hydrogels.

[0304] Specifically, in animal B01, after implantation with supplementation regimen A, the radioactive signal in the liver was significantly higher starting at 5 hours. Throughout the monitoring period (1-18 days), the liver maintained a high radioactivity intensity, consistently above 80% or 84%, while the radioactive signals in other organs such as the lungs, intestines, kidneys, and brain were very low. This indicates that in animal B01, the radiolabeled polymer and preparation 1 were tightly bound, embolizing the liver. The embolization preparation showed good stability, and the radiolabeled polymer also maintained good stability, with minimal detachment from the embolization site. Animals B02 implanted with supplementation regimen B, and animals B05 implanted with supplementation regimen E, also maintained a liver radioactivity count above 80% throughout the monitoring period. In contrast, animal B04, implanted with supplementation regimen D, showed a relatively low radioactive signal concentration in the liver, starting at 80.9% initially. This indicates that after implantation with supplementation regimen D, a small amount of radioactive signal entered the bloodstream and circulated to other organs. However, by day 18, the radioactivity in other organs outside the liver of animal B04 had been metabolized and excreted. It can be seen that polymers based on polymer P1 and linking groups 89 Zr-DFO-P1-1 reacts directly with the amino groups of chitosan to form polymers. 89The Zr-DFO-P2 structure exhibits greater stability and provides a more stable radiotracing effect in hydrogel formulations.

[0305] Although embodiments of this application have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the claims and their equivalents.

Claims

1. A radionuclide-labeled polymer having the following structure: G-L1-L2-P; in, G is a group that chelates a radioactive nuclide, comprising a chelating group and a radioactive nuclide, wherein the chelating group is chelated with the radioactive nuclide; L1 and L2 are independently chemical bonds or linking groups; P represents a temperature-sensitive structural unit; Wherein, L1 is a chemical bond or a linking group as shown in formula (I-1): In equation (I-1), x and y are independently selected from integers from 0 to 3; R1 is C 1-5 alkylene Or chemical bonds; R e for Substituted or unsubstituted C 6-10 aryl, or, substituted or unsubstituted C 5-10 heteroaryl; R d C 1-5 alkylene Or chemical bonds; R b It is hydrogen, hydroxyl or C 1-5 alkyl; X is O or S; e represents the connection site between G and L1, and i represents the connection site between R1 and R2. e The connection site, f represents the connection site between L1 and L2; and / or, The L2 is a chemical bond, or a group selected from those shown in formula (II-1) or (II-2): In equation (II-1), z is an integer in the range of 0-10; In equation (II-2), R2 and R3 are independently hydrogen and C. 1-5 Alkyl, substituted or unsubstituted C 6-10 aryl, or, substituted or unsubstituted C 5-10 heteroaryl, amide, or thiol; f represents the connection site between L1 and L2, and h represents the connection site between L2 and P; u is an integer in the range of 0-10; Each R 1-1 R e-1 and R d-1 Independent is C 1-5 alkylene, L1 and L2 are not chemical bonds at the same time.

2. The radionuclide-labeled polymer according to claim 1, wherein, The polymer satisfies one or more of the following conditions (1)-(5): (1) Each C 1-5 The alkylene groups are independently selected from methylene, (2) Each C 6-10 The arylene group is either phenylene or naphthylene; (3) Each C 5-10 The heteroatom of the heteroaryl group is independently selected from one or two of N, O and S, and the number of heteroatoms is independently 1, 2 or 3, preferably N or O, and the number of heteroatoms is 1 or 2; (4) Each C 1-5 The alkyl group is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl; (5) Each substituted or unsubstituted C 6-10 aryl, substituted or unsubstituted C 5-10 Heteroaryl, substituted or unsubstituted C 6-10 aryl, or, substituted or unsubstituted C 5-10 The substituents in heteroaryl groups are independently C 1-3 Alkyl, halogen, hydroxyl or cyano groups; The C 1-3 The alkyl group is methyl, ethyl, or propyl; The halogen is F, Cl, Br or I.

3. The radionuclide-labeled polymer according to claim 1, wherein it satisfies one or more of the following conditions (1)-(4): (1) The L1 is a linking group as shown in formula (I-2) or (I-3): in, In equation (I-2), R1 is Or C 1-5 alkylene, i indicates that it is related to R e Connection; R e Selected from substituted or unsubstituted C 6-10 aryl, substituted or unsubstituted C 5-10 Hybrid aryl; where e indicates connection to G, and f indicates connection to L2; In equation (I-3), each n is independently selected from an integer in the range of 0-10, and each R b Independently hydrogen or hydroxyl; (2) In equation (II-1), z is 0, 1, 2 or 3; (3) In formula (II-2), both R2 and R3 are methyl groups; (4) In equation (II-2), u is 0, 1, 2 or 3.

4. The radionuclide-labeled polymer according to any one of claims 1-3, wherein, The P is one or more of a chitosan structural unit, an acrylamide structural unit, or a group as shown in formula (III-1): In equation (III-1), a and c are independently selected from the range of 2-130; b is in the range of 10-80; and R is selected from hydrogen and C. 1-5 alkyl.

5. The radionuclide-labeled polymer according to claim 4, wherein, At least one of the following conditions (1)-(2) must be satisfied: (1) In equation (III-1), a and c are independent integers in the range of 80-130, and b is an integer in the range of 30-60; (2) The acrylamide structural unit includes the group shown in formula (III-2): In equation (III-2), R c It can be hydrogen, -CH-(CH3)2, -C-(CH3)3, or -(CH2). m -NH2, where m is an integer between 0 and 5, R a It can be -CH-(CH3)2, -C-(CH3)3, or -(CH2). m -NH2 can be a divalent group formed by losing a hydrogen atom; p or q are independent integers in the range of 20-200.

6. The radionuclide-labeled polymer according to any one of claims 1-4, wherein, The radioactive nuclide is 89 Zr、 68 Ga、 64 Cu、 99m Tc or Al 18 F; The chelating group is a chelating group formed by a bifunctional chelating agent. Preferably, the chelating agent is DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, DFO, or TACN.

7. The radionuclide-labeled polymer according to claim 1, wherein, It satisfies one or more of the following conditions: (1) The chelating group is (2) L1 is e indicates a connection to G, and f indicates a connection to L2; (3) L2 is f represents the connection site between L1 and L2, and h represents the connection site between L2 and P; (4) P is 8. The radionuclide-labeled polymer according to claim 1, wherein, The polymer satisfies any one of the following schemes (1)-(5): Option (1): The chelating group is The P is Where R is a methyl group, a is an integer in the range of 75-110, and b is an integer in the range of 25-70; L1 is Where e represents a connection to G, and f represents a connection to L2; The L2 is f represents the connection site between L1 and L2, and h represents the connection site between L2 and P; Option (2): The chelating group is The P is Where R is a methyl group, a is an integer in the range of 75-110, and b is an integer in the range of 25-70; L1 is Where e represents a connection to G, and f represents a connection to L2; The L2 is f represents the connection site between L1 and L2, and h represents the connection site between L2 and P; Option (3): The chelating group is The P is Where R is a methyl group, a is an integer in the range of 75-110, and b is an integer in the range of 25-70; L1 is Where e represents a connection to G, and f represents a connection to L2; The L2 is f represents the connection site between L1 and L2, and h represents the connection site between L2 and P; Option (4): The chelating group is The P is L1 is Where e represents a connection to G, and f represents a connection to L2; The L2 is a chemical bond.

9. The radionuclide-labeled polymer according to claim 1, wherein, It can be any of the following structures: in, a and c are independently selected from the range 2–130; b is in the range 10–80, and R is selected from hydrogen and C. 1-6 alkyl; R c It can be hydrogen, -CH-(CH3)2, -C-(CH3)3, or -(CH2). m -NH2, where m is an integer between 0 and 5, R a It can be -CH-(CH3)2, -C-(CH3)3, or -(CH2). m -NH2 can be a divalent group formed by losing a hydrogen atom; p or q are independent integers in the range of 20-200.

10. The radionuclide-labeled polymer according to claim 9, wherein, It can be any of the following structures:

11. A polymer as shown in formula (A): Q-L1-L2-P (A); in, The definitions of L1, L2, and P are the same as those of L1, L2, and P in any one of claims 1-10, and Q is a chelating group, which is the same as the definition of a chelating group in any one of claims 1-10.

12. The polymer according to claim 11, wherein, The polymer shown in formula (A) has any of the following structures: a and c are independently selected from the range 2–130; b is in the range 10–80, and R is selected from hydrogen and C. 1-6 alkyl; R c It can be hydrogen, -CH-(CH3)2, -C-(CH3)3, or -(CH2). m -NH2, where m is an integer between 0 and 5, R a It can be -CH-(CH3)2, -C-(CH3)3, or -(CH2). m -NH2 can be formed by losing a hydrogen atom to form a divalent group. p or q are independent integers in the range of 20-200.

13. The polymer according to claim 12, wherein, The polymer shown in formula (A) has any of the following structures:

14. A method for preparing a radionuclide-labeled polymer, comprising the following steps: mixing and incubating the polymer of formula (A) as described in any one of claims 11-13 with a solution containing a radionuclide to obtain the radionuclide-labeled polymer.

15. A gel composition comprising a polymer and hydrogel formulation labeled with a radionuclide as described in any one of claims 1-10.

16. The gel composition according to claim 15, wherein, The gel composition satisfies one or more of the following conditions (1)-(4): (1) The hydrogel formulation includes a temperature-sensitive gelling agent and a gelling agent; (2) The hydrogel formulation further includes a developing agent; (3) The mass-to-volume ratio of the radionuclide-labeled polymer to the hydrogel formulation is 0.01 mg / mL to 2 mg / mL; (4) The sol-gel transition temperature of the gel composition is 25℃-37℃.

17. The gel composition of claim 16, wherein, Based on the volume of the hydrogel formulation, the hydrogel formulation comprises: 0.2-1 g / mL iohexol, 0.02-0.1 g / mL sodium alginate 0.12-0.2 g / mL poloxamer 0.005-0.1 g / mL hydroxymethyl cellulose or CaCl2, and water; and / or In the gel composition, the mass-to-volume ratio of the radionuclide-labeled polymer to the hydrogel formulation is 0.01 mg / mL to 0.5 mg / mL.

18. The use of a radionuclide-labeled polymer as described in any one of claims 1-10 in the preparation of a tracer, wherein the tracer is used to trace the gel composition as described in any one of claims 15-17.

19. An application of the gel composition according to any one of claims 15-17, wherein the application is the use of the gel composition in the preparation of materials for in vivo vascular embolization, in vivo hemostasis, drug delivery or in vivo tissue repair.

20. A method for radiolabeling a hydrogel formulation, comprising: Provide a polymer labeled with a radionuclide as described in any one of claims 1-10; The radionuclide-labeled polymer is added to the hydrogel formulation of any one of the gel compositions described in claims 15-17.

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