13C-MRI contrast agent

Abstract

The present invention 13 An efficient measurement method for C-MRI, and a method used in said measurement method. 13 The objective is to provide a C-MRI contrast agent and a method for producing the contrast agent. [Solution] The present invention provides to the end 13 The dendrimer structure D has a functional group X containing C, and the group X is capable of forming a conjugate with a polypeptide. 1 A compound having this property is reacted with a polypeptide. 13 By preparing a polypeptide into which a functional group X containing C has been introduced. 13 A method for producing a C-MRI contrast agent, and a method that can be prepared by said method. 13 C-MRI contrast agent will be provided.

Description

[Technical Field]

[0001] The present invention 13 C-MRI contrast agent, method for manufacturing the same, and 13 C-MRI contrast agent used 13 This document concerns the measurement method of CMRI. [Background technology]

[0002] Current molecular imaging primarily utilizes nuclear medicine techniques such as positron emission tomography (PET) due to its high sensitivity. However, PET requires a cyclotron or radioactive material experimental facility, making it less convenient. On the other hand, magnetic resonance imaging (MRI) is widely used in clinical practice.

[0003] Recently, ultra-high magnetic field MRI devices such as 3 Tesla (T) and 7T have been introduced into clinical settings. With the increase in static magnetic field and the advancement of gradient magnetic field control, the signal-to-noise ratio has improved, making it possible to image nuclides other than protons using MRI. The use of contrast agents for purposes such as angiography using MRI is being investigated, and the use of contrast agents containing dendrimer structures is being considered (Non-Patent Literature 1 and 2). For example, the use of dendrimer-antibody conjugates is being investigated (Non-Patent Literature 3).

[0004] 13 Regarding C-MRI, 13 Regarding C-methionine-enriched gliomas 13 There are reports on C-MRI (Non-Patent Document 4). 13 Using a C-enriched compound 13 The measurement method of C-MRI has been studied (Patent Documents 1-3). [Prior art documents] [Patent Documents]

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Document

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0007] In recent years, high-performance MRI devices have become available, and 13 studies have also been conducted on C-MRI. However, the abundance of natural 13 C is low, and 13 low sensitivity has been a problem in molecular imaging by C-MRI. Also, 13 sufficient sensitivity cannot be obtained only by using labeled compounds such as C-labeled amino acids, and efficient 13There was a strong need for a C-labeling method. [Means for solving the problem]

[0008] 13 Regarding C-MRI, the inventors of this application have made numerous improvements by utilizing dendrimer structures. 13 By preparing a labeled compound containing C, 13 We discovered that strong peaks could be detected in C-MRI, which led to the completion of the invention. This specification encompasses the following disclosures of the invention.

[0009] [A-1] 13 A method for producing a C-MRI contrast agent, 13 C-MRI contrast agents are 13 This polypeptide is labeled by the introduction of C. The above method is at the end 13 The dendrimer structure D has a functional group X containing C, and the group X is capable of forming a conjugate with a polypeptide. 1 A compound having this property is reacted with a polypeptide. 13 The process includes producing a polypeptide into which a functional group X containing C has been introduced, The method wherein the compound contains 5 to 200 functional groups X.

[0010] [A-2]X 1 The method according to [A-1], wherein the group is a sulfanyl, azide, or group containing a CC triple bond. [A-3] Functional group X is 13 CH3S- or 13 The method described in [A-1], comprising CH3CO-.

[0011] [A-4] The polypeptide to be subjected to the reaction is X 1 Reactive group X 2 A polypeptide that has been introduced, as described in any of [A-1] to [A-3]. [A-5] Compounds of formula 1, formula 2, or formula 3:

[0012] [ka]

[0013] [In the formula, X 1 The group is selected from sulfanyl, azide, and CC triple bond-containing groups. L 1 This indicates absence or linker, D represents a dendrimer structure. R 1 is a hydrogen atom, or C 1-6 Represents alkyl, L 2 Each of these independently represents a linker. L 3 Each of these independently represents a linker. n 1 [This represents numbers between 5 and 200] The compound represented by reacts with a polypeptide. 13 The process involves producing a polypeptide into which a functional group X containing C has been introduced. 13 Method for manufacturing C-MRI contrast agents.

[0014] [A-6]L 1 C 2-10 The method described in [A-5], comprising alkylene. [A-7] The compound is represented by formula 1, L 2 However, each is independent of -COY-(C 2-10 Alkylene) is where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3 It may be substituted with one or more substituents selected from the following: Y can be directly bonded, -O-, or -NR, independently of each other. 4 -and, R 1 , R 2 , R 3 , and R 4 These are, independently, hydrogen atoms and C 1-6 It is an alkyl or nitrogen atom protecting group, -COY- is the carbonyl group -NR 1 - Bonded to the nitrogen atom, The method described in [A-5] or [A-6].

[0015] [A-8] The compound is represented by formula 2, L 3 However, each independently, C 2-10 It is alkylene, where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3 It may be substituted with one or more substituents selected from the following: R 1 , R 2 , and R 3 These are, independently, hydrogen atoms and C 1-6 Alkyl or nitrogen atom protecting group, The method described in [A-5] or [A-6].

[0016] [A-9]D contains multiple nitrogen atoms as branching points, and the branching points are -(C 2-6 Alkylene-CONR 11 -(C 2-6 Alkilen)-, or -(C 2-10 Alkilen) This represents a dendrimer structure linked by R 11 Each of these is independently a hydrogen atom or C 1-6 The method described in any of [A-1] to [A-8], wherein the material is alkyl.

[0017] The method according to any one of [A-1] to [A-9], wherein [A-10]D has a polyaminoamine (PAMAM) dendrimer structure in which branching points are linked by -C2H4-CONH-C2H4-.

[0018] [A-11] The method according to any of [A-1] to [A-10], wherein [A-11]D has a generation 1 to 4 polyaminoamine (PAMAM) dendrimer structure. [A-12]X 1That is sulfanyl, D is a dendrimer structure corresponding to a thiol obtained by reducing the disulfide bond of a dendrimer having a disulfide bond in its core. n 1 The method described in any of [A-1] to [A-11], wherein the value is between 5 and 95.

[0019] [A-13] Functional group X is formula 4, formula 5, or formula 6:

[0020] [ka]

[0021] [In the formula, R 1 and R 6 These are, independently, a hydrogen atom and C 1-6 Selected from alkyl groups, R 2 , and R 3 These are, independently, hydrogen atoms and C 1-6 Selected from alkyl and nitrogen atom protecting groups, R 5 These are, independently, hydrogen atoms and C 1-6 [Selected from alkyl and carboxyl protecting groups] A method described in any of [A-1] to [A-12], represented by [A-1].

[0022] [A-14] The method according to any of [A-1] to [A-13], wherein the functional group X is represented by formula 3. [A-15] All functional groups X in the compound are methyl 13 A method according to any of [A-1] to [A-14], including the structure of C-methionine.

[0023] [A-16] All functional groups X 13 A method according to any of [A-1] to [A-15], wherein C is detected as substantially the same signal in nuclear magnetic resonance measurements. [A-17] Compound, formula 7:

[0024] [Chemical formula]

[0025] [wherein, L 1 is an alkylene, 2-6 and X 1 is sulfanyl, D represents a dendrimer structure obtained by reducing the disulfide bond of a generation 1-3 polyaminoamine (PAMAM) dendrimer having a disulfide bond in the core part, R 2 , and R 3 are each independently selected from a hydrogen atom, a C 1-6 alkyl, and a protecting group for a nitrogen atom, n 1 represents a number from 5 to 200) by the method according to any one of [A-1] to [A-16].

[0026] [A-18] X 1 is sulfanyl, X 2 is a group containing a C-C double bond, X 1 is azido, X 2 is a group containing a C-C triple bond, or X 2 is a group containing a C-C triple bond, X 2 is azido, the method according to any one of [A-1] to [A-17].

[0027] [A-19] A polypeptide into which a functional group X is introduced is of formula 10, formula 11 or formula 12:

[0028] [Chemical formula]

[0029] [wherein, R 1 , L 1 , L 2 , L 3 , D, and n1 is as defined previously The method according to any one of [A-5] to [A-18], which forms a conjugate with the group represented by

[0030] [A-20]L 1 The method according to [A-19], wherein is linked to the polypeptide via 1,2,3-triazolyl, -S-S-, or -S-.

[0031] The method according to any one of [A-1] to [A-20], wherein the polypeptide is an antibody or an antigen-binding fragment thereof, a receptor, a ligand, or a fusion protein.

[0032] [B-1] 13 A method for producing a C-MRI contrast agent, comprising: ) 13 The C-MRI contrast agent is 13 a polypeptide labeled by the introduction of C, The method comprises Equation 8: A-S-S-B (8) [wherein A and B each independently represent a dendrimer structure D having a functional group X containing C at the terminal, and A and / or B may be bonded to S via a linker, 13 the aforesaid functional group X is contained in a total of 5 to 200 in A and B] reducing the compound represented by to prepare a thiol, reacting the thiol with a polypeptide 13 to produce a polypeptide into which a functional group X containing C is introduced, 13 A method for producing a C-MRI contrast agent.

[0033] [B-2] The method according to [B-1], wherein the functional group X 13 contains CH3S-. ​[B-3] Formula 9: >NL 1 -SSL 1 -N< (9) [In the formula, L 1 Each of these independently represents a linker, and the two nitrogen atoms are each part of dendrimer structure D of A or B. A method according to [B-1] or [B-2], comprising a structure represented by [B-1].

[0034] [B-4] A and B are, independently, given by equation 10, equation 11, or equation 12:

[0035] [ka]

[0036] [In the formula, L 1 This indicates absence or linker, D represents a dendrimer structure. R 1 is a hydrogen atom, or C 1-6 Represents alkyl L 2 Each of these independently represents a linker. L 3 Each of these independently represents a linker. n 1 [This represents numbers from 5 to 95] A method described in any of [B-1] to [B-3], represented by [B-1].

[0037] [B-5]L 1 C 2-10 The method described in [B-4], which is alkylene. [B-6] A and B are expressed in equation 10, L 2 However, each is independent of -COY-(C 2-10 Alkylene) is where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3 It may be substituted with one or more substituents selected from the following: Y can be directly bonded, -O-, or -NR, independently of each other. 4 -and, R 1 , R 2 , R 3 , and R 4 These are, independently, hydrogen atoms and C 1-6 It is an alkyl or nitrogen atom protecting group, -COY- is the carbonyl group -NR 1 - Bonded to the nitrogen atom, The method described in [B-1] or [B-5].

[0038] [B-7] A and B are expressed in equation 9, L 3 However, each independently, C 2-10 It is alkylene, where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3 It may be substituted with one or more substituents selected from the following: R 1 , R 2 , and R 3 These are, independently, hydrogen atoms and C 1-6 Alkyl or nitrogen atom protecting group, The method described in any of [B-1] to [B-6].

[0039] [B-8]D contains multiple nitrogen atoms as branching points, and the branching points are -(C 2-6 Alkylene-CONR 11 -(C 2-6 Alkilen)-, or -(C 2-10 Alkilen) This represents a dendrimer structure linked by R 11 Each of these is independently a hydrogen atom or C 1-6 The method described in any of [B-1] to [B-7], wherein the material is alkyl.

[0040] The method according to any one of [B-1] to [B-8], wherein [B-9]D has a polyaminoamine (PAMAM) dendrimer structure in which branching points are linked by -C2H4-CONH-C2H4-.

[0041] [B-10] The method according to any of [B-1] to [B-9], wherein [B-10]D has a generation 1 to 4 polyaminoamine (PAMAM) dendrimer structure. [B-11]D represents a dendrimer structure obtained by reducing the disulfide bond of a generation 1-3 polyaminoamine (PAMAM) dendrimer having a disulfide bond in the core portion, according to any of the methods described in [B-1] to [B-10].

[0042] [B-12] Functional group X is formula 4, formula 5, or formula 6:

[0043] [ka]

[0044] [In the formula, R 1 and R 6 These are, independently, a hydrogen atom and C 1-6 Selected from alkyl groups, R 2 , and R 3 These are, independently, hydrogen atoms and C 1-6 Selected from alkyl and nitrogen atom protecting groups, R 5 These are, independently, hydrogen atoms and C 1-6 [Selected from alkyl and carboxyl protecting groups] A method described in any of [B-1] to [B-11], represented by [B-1].

[0045] [B-13] The method according to any of [B-1] to [B-12], wherein the functional group X is represented by formula 3. [B-14] All functional groups X in the compound are methyl 13 A method according to any of [B-1] to [B-13], comprising the structure of C-methionine.

[0046] [B-15] All functional groups X 13 A method according to any of [B-1] to [B-14], wherein C is detected as substantially the same signal in each magnetic resonance measurement.

[0047] [B-16] A and B are each independently expressed in Equation 13:

[0048] [ka]

[0049] [In the formula, L 1 C 2-6 It is alkylene, D represents the polyaminoamine (PAMAM) dendrimer structure of generations 1-3. R 2 , and R 3 These are, independently, hydrogen atoms and C 1-6 Selected from alkyl and nitrogen atom protecting groups, n 1 [This represents numbers from 5 to 95] A method described in any of [B-1] to [B-15], represented by [B-1].

[0050] [B-17] The method according to any of [B-1] to [B-16], wherein the polypeptide is a polypeptide to which a group capable of reacting with a sulfur atom has been introduced. [B-18] The method according to any of [B-1] to [B-17], wherein the group capable of reacting with a sulfur atom is a group having a double bond or a disulfide group.

[0051] [B-19] The method according to any of [B-1] to [B-18], wherein the group capable of reacting with a sulfur atom is a maleimide group or a pyridyl disulfide group. The method according to any of [B-1] to [B-19], wherein the polypeptide is an antibody or its antigen-binding fragment, receptor, ligand, or fusion protein.

[0052] The method according to any of [B-1] to [B-20], wherein the polypeptide is an antibody selected from IgG or its antigen-binding fragment, a single-chain antibody, and a multispecific antibody or its antigen-binding fragment. [B-22] polypeptide 13 A method used as a C-MRI contrast agent, according to any of the methods described in [B-1] to [B-21].

[0053] [C-1] Formula 1, Formula 2, or Formula 3:

[0054] [ka]

[0055] [In the formula, X 1 The group is selected from sulfanyl, azide, and CC triple bond-containing groups. L 1 This indicates absence or linker, D represents a dendrimer structure. R 1 is a hydrogen atom, or C 1-6 Represents alkyl, L 2 Each of these independently represents a linker. L 3 Each of these independently represents a linker. n 1 [This represents numbers between 5 and 200] A compound represented by, or a salt thereof.

[0056] [C-2]L 1 C 2-10 Compounds containing alkylene, as described in [C-1], or salts thereof. [C-3] Expressed by Equation 1, L 2 However, each is independent of -COY-(C 2-10 Alkylene) is where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3It may be substituted with one or more substituents selected from the following: Y can be directly bonded, -O-, or -NR, independently of each other. 4 -and, R 1 , R 2 , R 3 , and R 4 These are, independently, hydrogen atoms and C 1-6 It is an alkyl or nitrogen atom protecting group, -COY- is the carbonyl group -NR 1 - Bonded to the nitrogen atom, The compound described in [C-1] or [C-2], or a salt thereof.

[0057] [C-4] Expressed by Equation 2, L 3 However, each independently, C 2-10 It is alkylene, where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3 It may be substituted with one or more substituents selected from the following: R 1 , R 2 , and R 3 These are, independently, hydrogen atoms and C 1-6 Alkyl or nitrogen atom protecting group, A compound described in any of [C-1] to [C-3], or a salt thereof.

[0058] [C-5]D contains multiple nitrogen atoms as branching points, and the branching points are -(C 2-6 Alkylene-CONR 11 -(C 2-6 Alkilen)-, or -(C 2-10 Alkilen) This represents a dendrimer structure linked by R 11 Each of these is independently a hydrogen atom or C 1-6 A method using alkyl groups [C-1] to [C-4].

[0059] [C-6]D is a compound according to any of [C-1] to [C-5], or a salt thereof, having a polyaminoamine (PAMAM) dendrimer structure in which branching points are linked by -C2H4-CONH-C2H4-.

[0060] [C-7]D is a compound according to any of [C-1] to [C-6], or a salt thereof, having a polyaminoamine (PAMAM) dendrimer structure of generation 1 to 4. [C-8]X 1 That is sulfanyl, D is a dendrimer structure corresponding to a thiol obtained by reducing the disulfide bond of a dendrimer having a disulfide bond in its core. n 1 A compound or salt thereof, as described in any of [C-1] to [C-7], wherein the ratio is 5 to 95.

[0061] [C-9] Formula 7 or Formula 8:

[0062] [ka]

[0063] [In the formula, R 6 These are, independently, a hydrogen atom and C 1-6 Selected from alkyl groups, X 1 , L 1 , D,n 1 , R 2 , and R 3 As already defined, R 5 These are, independently, hydrogen atoms and C 1-6 [Selected from alkyl and carboxyl protecting groups] A compound represented by any of the [C-1] to [C-8] compounds, or a salt thereof.

[0064] [C-10] A compound represented by formula 5, one of the compounds listed in [C-1] to [C-9], or a salt thereof. [C-11] Represented by Equation 5, In the formula, L 1 C 2-6 It is alkylene, X 1 It is sulfanyl, D represents the dendrimer structure obtained by reducing the disulfide bond of generation 1-3 polyaminoamine (PAMAM) dendrimers that have a disulfide bond in the core. R 2 , and R 3 These are, independently, hydrogen atoms and C 1-6 Selected from alkyl and nitrogen atom protecting groups, n 1 represents a number between 5 and 200, and is a compound or salt thereof listed under either [C-9] or [C-10].

[0065] [C-12]X 1 is sulfanyl, X 2 This is a group containing a CC double bond. X 1 is Azid, X 2 is a group containing a CC triple bond, or X 2 is a group containing a CC triple bond, and X 2 The compound is an azide, one of the compounds listed in [C-1] to [C-11], or a salt thereof.

[0066] [D-1] Formula 21: ASSB (21) [In the formulas, A and B are, independently, in formulas 10, 11, or 12:

[0067] [ka]

[0068] It is represented by, L 1 This indicates absence or linker, D represents a dendrimer structure. R 1is a hydrogen atom, or C 1-6 Represents alkyl L 2 Each of these independently represents a linker. L 3 Each of these independently represents a linker. n 1 [This represents numbers from 5 to 95] A compound represented by or a salt thereof.

[0069] [D-2]L 1 C 2-10 The alkylene compounds or salts thereof described in [D-1]. [D-3]L 2 However, each is independent of -COY-(C 2-10 Alkylene) is where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3 It may be substituted with one or more substituents selected from the following: Y can be directly bonded, -O-, or -NR, independently of each other. 4 -and, R 1 , R 2 , R 3 , and R 4 These are, independently, hydrogen atoms and C 1-6 It is an alkyl or nitrogen atom protecting group, -COY- is the carbonyl group -NR 1 - Bonded to the nitrogen atom, The compound or salt thereof described in [D-1] or [D-2].

[0070] [D-4]L 3 However, each independently, C 2-10 It is alkylene, where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3 It may be substituted with one or more substituents selected from the following: R 1 , R 2 , and R 3 These are, independently, hydrogen atoms and C1-6 Alkyl or nitrogen atom protecting group, A compound or salt thereof described in any of [D-1] to [D-3].

[0071] [D-5]D contains multiple nitrogen atoms as branching points, and the branching points are -(C 2-6 Alkylene-CONR 11 -(C 2-6 Alkilen)-, or -(C 2-10 Alkilen) This represents a dendrimer structure linked by R 11 Each of these is independently a hydrogen atom or C 1-6 A compound or salt thereof, which is alkyl, as described in any of [D-1] to [D-4].

[0072] [D-6] A compound or salt thereof according to any one of [D-1] to [D-5], wherein D has a polyaminoamine (PAMAM) dendrimer structure in which branching points are linked by -C2H4-CONH-C2H4-.

[0073] [D-7] A compound or salt thereof according to any of [D-1] to [D-6], wherein D has a polyaminoamine (PAMAM) dendrimer structure of generation 1 to 4. [D-8] All included 13 A compound or salt thereof described in any of [D-1] to [D-7], wherein C is detected as substantially the same signal in nuclear magnetic resonance measurements.

[0074] [D-9] A and B are each independently given by Equation 13:

[0075] [ka]

[0076] [In the formula, L 1 C 2-6 It is alkylene, D represents the polyaminoamine (PAMAM) dendrimer structure of generations 1 to 3, R2 and R3 are each independently selected from a hydrogen atom, C 1-6 alkyl, and a protecting group for a nitrogen atom, n 1 represents a number from 5 to 95] A compound according to any one of [D-1] to [D-8] or a salt thereof, represented by.

[0077] A compound according to any one of [D-1] to [D-9] or a salt thereof for use in the preparation of a conjugate with a [D-10] polypeptide. A compound according to [D-10] or a salt thereof, wherein the [D-11] polypeptide is a polypeptide into which a group capable of reacting with a sulfur atom has been introduced.

[0078] A compound according to either [D-10] or [D-11] or a salt thereof, wherein the group capable of reacting with a sulfur atom is a group having a double bond or a disulfide group.

[0079] A compound according to any one of [D-10] to [D-12] or a salt thereof, wherein the group capable of reacting with a sulfur atom is a maleimide group or a pyridyldisulfide group.

[0080] A compound according to any one of [D-10] to [D-13] or a salt thereof, wherein the polypeptide is an antibody or an antigen-binding fragment thereof, a receptor, a ligand, or a fusion protein.

[0081] A compound according to any one of [D-10] to [D-14] or a salt thereof, wherein the polypeptide is an antibody or an antigen-binding fragment thereof selected from IgG or an antigen-binding fragment thereof, a single-chain antibody, and a multispecific antibody.

[0082] [E-1] Formula 10, Formula 11 or Formula 12:

[0083]

Chemical formula

[0084] It is represented by, L 1 This indicates absence or linker, D represents a dendrimer structure. R 1 is a hydrogen atom, or C 1-6 Represents alkyl L 2 Each of these independently represents a linker. L 3 Each of these independently represents a linker. n 1 [This represents numbers between 5 and 200] A polypeptide containing a group represented by .

[0085] [E-2]L 1 C 2-10 The polypeptide described in [E-1] is alkylene. [E-3] Contains a group represented by formula 8, L 2 However, each is independent of -COY-(C 2-10 Alkylene) is where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3 It may be substituted with one or more substituents selected from the following: Y can be directly bonded, -O-, or -NR, independently of each other. 4 -and, R 1 , R 2 , R 3 , and R 4 These are, independently, hydrogen atoms and C 1-6 It is an alkyl or nitrogen atom protecting group, -COY- is the carbonyl group -NR 1 - Bonded to the nitrogen atom, The polypeptide described in [E-1] or [E-2].

[0086] [E-4] Contains a group represented by formula 9, L 3 However, each independently, C 2-10 It is alkylene, where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3 It may be substituted with one or more substituents selected from the following: R 1 , R 2 , and R 3 These are, independently, hydrogen atoms and C 1-6 Alkyl or nitrogen atom protecting group, A polypeptide listed in any of [E-1] to [E-3].

[0087] [E-5]D contains multiple nitrogen atoms as branching points, and the branching points are -(C 2-6 Alkylene-CONR 11 -(C 2-6 Alkilen)-, or -(C 2-10 Alkilen) This represents a dendrimer structure linked by R 11 Each of these is independently a hydrogen atom or C 1-6 A polypeptide that is alkyl, as described in any of [E-1] to [E-4].

[0088] [E-6] The polypeptide according to any one of [E-1] to [E-5], wherein D has a polyaminoamine (PAMAM) dendrimer structure in which branching points are linked by -C2H4-CONH-C2H4-.

[0089] [E-7]D is a polypeptide according to any of [E-1] to [E-6], having a generation 1 to 4 polyaminoamine (PAMAM) dendrimer structure. [E-8] A group represented by formula 8 or formula 9 is linked to the polypeptide via -S- or -SS-, D is a dendrimer structure corresponding to a thiol obtained by reducing the disulfide bond of a dendrimer having a disulfide bond in its core. n1 The method according to any one of [E-1] to [E-7], wherein it is 5 to 95.

[0090] All of those containing [E-9] 13 The polypeptide according to any one of [E-1] to [E-8], wherein C is detected as substantially the same signal in nuclear magnetic resonance measurement. [E-10] containing the group represented by Formula 8, and the group is Formula 13:

[0091] [Chemical formula]

[0092] [In the formula, L 1 is alkylene C 2-6 and D represents a polyaminoamine (PAMAM) dendrimer structure of generations 1 to 3, R 2 , and R 3 are each independently selected from a hydrogen atom, C 1-6 alkyl, and a protecting group for a nitrogen atom, n 1 represents a number from 5 to 200] The polypeptide according to any one of [E-1] to [E-9], represented by

[0093] [E-11] 13 The polypeptide according to any one of [E-1] to [E-IO] for use as a C-MRI contrast agent. [E-12] L 1 The polypeptide according to any one of [E-1] to [E-11], wherein L is linked to the polypeptide via 1,2,3-triazolyl, -S-S-, or -S-.

[0094] [E-13] The polypeptide according to [E-12], wherein the sulfanyl is linked to the polypeptide via -S- formed by addition to a C-C double bond and 1,2,3-triazolyl formed by reaction of an azide with a C-C triple bond.

[0095] [E-14] A polypeptide according to any of [E-1] to [E-13], which is an antibody or its antigen-binding fragment, receptor, ligand, or fusion protein.

[0096] [E-15] The polypeptide according to any of [E-1] to [E-14], wherein the polypeptide is an antibody selected from IgG or its antigen-binding fragment, a single-chain antibody, and a multispecific antibody or its antigen-binding fragment.

[0097] [F-1] Formula 1: -SA (1) [In the formulas, A is independently of each other, independently of each other, formula 10, formula 11 or formula 12:

[0098] [ka]

[0099] It is represented by, L 1 This indicates absence or linker, D represents a dendrimer structure. R 1 is a hydrogen atom, or C 1-6 Represents alkyl L 2 Each of these independently represents a linker. L 3 Each of these independently represents a linker. n 1 [This represents numbers from 5 to 95] A polypeptide containing a group represented by .

[0100] [F-2]L 1 C 2-10 A polypeptide, which is alkylene, as described in [F-1]. [F-3]L 2 However, each is independent of -COY-(C 2-10 Alkylene) is where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3It may be substituted with one or more substituents selected from the following: Y can be directly bonded, -O-, or -NR, independently of each other. 4 -and, R 1 , R 2 , R 3 , and R 4 These are, independently, hydrogen atoms and C 1-6 It is an alkyl or nitrogen atom protecting group, -COY- is the carbonyl group -NR 1 - Bonded to the nitrogen atom, The polypeptide described in [F-1] or [F-2].

[0101] [F-4]L 3 However, each independently, C 2-10 It is alkylene, where alkylene is hydroxy, C 1-6 Alkoxy, or -NR 2 R 3 It may be substituted with one or more substituents selected from the following: R 1 , R 2 , and R 3 These are, independently, hydrogen atoms and C 1-6 Alkyl or nitrogen atom protecting group, A polypeptide listed in any of [F-1] to [F-3].

[0102] [F-5]D contains multiple nitrogen atoms as branching points, and the branching points are -(C 2-6 Alkylene-CONR 11 -(C 2-6 Alkilen)-, or -(C 2-10 Alkilen) This represents a dendrimer structure linked by R 11 Each of these is independently a hydrogen atom or C 1-6 A polypeptide that is alkyl, as described in any of [F-1] to [F-4].

[0103] [F-6] The polypeptide according to any one of [F-1] to [F-5], wherein D has a polyaminoamine (PAMAM) dendrimer structure in which branching points are linked by -C2H4-CONH-C2H4-.

[0104] [F-7]D is a polypeptide according to any of [F-1] to [F-6], having a generation 1 to 4 polyaminoamine (PAMAM) dendrimer structure. [F-8] All included 13 A polypeptide according to any of [F-1] to [F-7], wherein C is detected as substantially the same signal in nuclear magnetic resonance measurements.

[0105] [F-9] A is independently calculated using Equation 13:

[0106] [ka]

[0107] [In the formula, L 1 C 2-6 It is alkylene, D represents the polyaminoamine (PAMAM) dendrimer structure of generations 1-3. R 2 , and R 3 These are, independently, hydrogen atoms and C 1-6 Selected from alkyl and nitrogen atom protecting groups, n 1 [This represents numbers from 5 to 95] A polypeptide represented by any of the following: [F-1] to [F-8].

[0108] [F-10] 13 A polypeptide described in any of [F-1] to [F-9] for use as a C-MRI contrast agent. [F-11] A polypeptide according to any of [F-1] to [F-10], wherein group A is introduced via a reaction with a group capable of reacting with a sulfur atom introduced into the polypeptide.

[0109] [F-12] A polypeptide according to any of [F-1] to [F-11], wherein the group capable of reacting with a sulfur atom is a group having a double bond or a disulfide group. [F-13] A polypeptide according to any of [F-1] to [F-12], wherein the group capable of reacting with a sulfur atom is a maleimide group or a pyridyl disulfide group.

[0110] [F-14] A polypeptide according to any of [F-1] to [F-13], which is an antibody or its antigen-binding fragment, receptor, ligand, or fusion protein. [F-15] The polypeptide according to any of [F-1] to [F-14], wherein the polypeptide is an antibody selected from IgG or its antigen-binding fragment, a single-chain antibody, and a multispecific antibody or its antigen-binding fragment.

[0111] [G-1] Administer an effective amount of the polypeptide described in any of [E-1]~[F-15] and [E-1]~[F-15] to the target population, and in the target population after administration. 13 This includes performing CMR measurements. 13 C-MR measurement method. [Brief explanation of the drawing]

[0112] [Figure 1] Figure 1 shows the chemical structure of the polyaminoamine (PAMAM) dendrimer (PAMAM(NH2)16, generation 2) of the cystamine core. [Figure 2] Figure 2 shows a scheme illustrating a method for producing a 13C polypeptide using PAMAM(NH2)16 as a starting material. [Figure 3] Figure 3 shows the 13C-NMR measurement results of (methyl-13C)methionine-introduced dendrimers. [Figure 4] Figure 4 shows the 13C-NMR measurement results of (methyl-13C)methionine-introduced dendrimers prepared by 1 to 3 reactions. [Figure 5]Figure 5 is a graph showing the number of (methyl-13C)methionine introduction reactions and the relative signal intensity of 13C derived from methionine. [Figure 6] Figure 6 shows the 1H-NMR measurement results of the (methyl-13C)methionine-introduced dendrimer prepared in Example 2. [Figure 7] Figure 7 shows the 13C-NMR measurement results of the (methyl-13C)methionine-introduced dendrimer prepared in Example 2. [Figure 8] Figure 8 shows the 13C-NMR measurement results of the (methyl-13C)methionine-introduced dendrimer prepared in Example 2. [Figure 9] Figure 9 shows the change in signal intensity of methionine-derived 13C as a result of 13C-NMR measurement of the (methyl-13C)methionine-introduced dendrimer prepared in Example 2. [Figure 10] Figure 10 shows the results of two-dimensional NMR (HSQC) measurements of the (methyl-13C)methionine-introduced dendrimer prepared in Example 2. [Figure 11] Figure 11 shows the results of mass spectrometry of the raw material dendrimer. [Figure 12] Figure 12 shows the results of mass spectrometry of the (methyl-13C)methionine-introduced dendrimer prepared in Example 2. [Figure 13] Figure 13 shows a comparison of the mass spectrometry results of the raw material dendrimer and the (methyl-13C)methionine-introduced dendrimer prepared in Example 2. [Figure 14] Figure 14 shows the 13C-NMR measurement results of (methyl-13C)methionine and (C2-13C)acetic acid-introduced dendrimers prepared in Example 3. [Figure 15] Figure 15 shows the 13C-NMR measurement results of (methyl-13C)methionine and (C2-13C)acetic acid-introduced dendrimers prepared in Example 3. [Figure 16] Figure 16 shows the results of mass spectrometry of the (methyl-13C)methionine-introduced dendrimer and BSA conjugate. [Figure 17] Figure 17 shows the 13C-NMR results of the (methyl-13C)methionine-modified dendrimer and BSA conjugate. [Figure 18] Figure 18 shows the HSQC (two-dimensional NMR of 1H·13C) measurement results of the conjugate of the (methyl-13C)methionine-introduced dendrimer and BSA. [Modes for carrying out the invention]

[0113] In one aspect of the present invention, 13 The functional group X containing C is 13 C-enriched alkyl groups, for example 13 It is a functional group containing a methyl group enriched with C. In one embodiment, 13 Functional group X containing C is -CO 13 CH3, or -S 13 It is a functional group containing CH3. In one aspect, 13 Functional group X containing C is -CO 13 CH3, -NR 1 ―L 2 -S 13 CH3, or -CONR 1 ―L 3 -S 13 It is a functional group containing CH3. In one aspect of the present invention, 13 The degree of enrichment of C is 13 There are no particular limitations as long as it is suitable for use as a C-MRI contrast agent, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0114] In this specification, sulfanyl means a thiol group (-SH) and is synonymous with a sulfhydryl group. In this specification, azide means -N3. In this specification, thiol means a compound having a sulfanyl group.

[0115] In this specification, C 1-6Alkyl refers to a monovalent saturated aliphatic group with 1 to 6 carbon atoms, either linear or branched, and examples include methyl, ethyl, propyl, isopropyl, butyl, 2-butyl, isobutyl, t-butyl, and n-hexyl.

[0116] In this specification, C 2-10 Alkylene refers to a divalent saturated aliphatic group with 2 to 10 carbon atoms in a straight chain or branched structure, with examples including ethylene, n-propylene, isopropylene, and n-butylene.

[0117] In this specification, C 2-6 Alkylene refers to a straight-chain or branched divalent saturated aliphatic group with 2 to 6 carbon atoms, and examples include ethylene, n-propylene, isopropylene, and n-butylene.

[0118] In this specification, C 1-6 Alkoxy is -O(C 1-6 (Alkyl) means C 1-6 Alkyl is defined as already stated. In this specification, the protecting group of carboxyl is not particularly limited, C 1-6 Alkyl, benzyl, C 1-6 Alkoxy C 1-6 Examples of groups include alkyl groups.

[0119] In this specification, the group containing the C double bond is not particularly limited as long as it is a group that is reactive with a sulfanyl group, for example, C 2-6 Examples of alkenyls include ethenyl, allyl, maleimide, acryloyl, and methacryloyl.

[0120] In this specification, the group containing the C triple bond is not particularly limited as long as it is a group that is reactive with azides, for example, C 2-6 Examples of alkynyl compounds include ethinyl and propargyl.

[0121] In this specification, the protecting group for nitrogen atoms is not particularly limited, (C 1-6 Alkyl)carbonyl, (C 1-6Examples of such groups include alkoxycarbonyl and benzyl groups.

[0122] In one aspect of the present invention, dendrimer structure D is a substituted dendrimer. In one aspect of the present invention, dendrimer structure D is a structure that includes a portion of a substituted dendrimer and retains its function as a dendrimer. In one aspect of the present invention, the dendrimer has a diameter between about 1 nm and about 50 nm. In one aspect of the present invention, the diameter of the dendrimer is between about 1 nm and about 20 nm, between about 1 nm and about 10 nm, or between about 1 nm and about 5 nm. In one aspect of the present invention, the diameter of the dendrimer is between about 1 nm and about 2 nm. In one aspect of the present invention, a dendrimer that has formed a conjugate with a polypeptide can have a diameter that is approximately 5 to 15 nm larger than these values ​​compared to a non-conjugated dendrimer. In one aspect of the present invention, the dendrimer has a molecular weight between approximately 500 daltons (Da) and approximately 100,000 Da (for example, between approximately 500 Da and approximately 50,000 Da, or between approximately 1,000 Da and approximately 20,000 Da).

[0123] In one aspect of the present invention, the dendrimers described herein are poly(amidoamine) (PAMAM) dendrimers, polypropylamine (POPAM) dendrimers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) dendrimers, polyethyleneimine dendrimers, polylysine dendrimers, polyester dendrimers, ipticendrimers, aliphatic poly(ether) dendrimers, aromatic polyether dendrimers, or combinations thereof.

[0124] In one aspect of the present invention, dendrimer structure D comprises a PAMAM dendrimer. In one aspect of the present invention, the PAMAM dendrimer comprises a different core having an amidoamine constituent block. In one aspect of the present invention, the PAMAM dendrimer comprises a carboxylic acid and / or amine-terminated group of any generation, including but not limited to PAMA M dendrimers of generation 1, generation 2, generation 3, generation 4, generation 5, generation 6, generation 7, generation 8, generation 9, or generation 10. In one aspect of the present invention, the PAMAM dendrimer is an amino-terminated PAMAM dendrimer of generation 4, generation 5, generation 6, generation 7, or generation 8. In one aspect of the present invention, the group L of dendrimer structure D 1 -X 1 It is introduced into the terminal group of the dendrimer.

[0125] In one embodiment of the present invention, a base X capable of forming a conjugate 1 This is a sulfanyl group (-SH), and a conjugate is formed by reacting this group with a group contained in the polypeptide that is reactive with this group, such as a sulfaryl group or a group containing a double bond, such as a maleimide group. In one embodiment of the present invention, the polypeptide that forms the conjugate is X 1 A group that can react with it has been introduced.

[0126] In one embodiment of the present invention, a base X capable of forming a conjugate 1 The group is capable of click reactions, and examples include an azide group or a group containing a triple bond. In one embodiment of the present invention, the polypeptide that forms the conjugate is X 1A reactive group is introduced, and the compound forming the conjugate is linked to a reactive click chemistry handle contained in the polypeptide (e.g., azide and terminal or strained alkyne, diene and dienophile, thiol and alkene) by a cyclic electronic reaction (e.g., 3+2 cycloaddition or 4+2 cycloaddition). Thus, the linker linking the two contains a five-membered heterocyclic ring resulting from the above reaction. In one embodiment of the present invention, the linker is a divalent group comprising 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, or 1,4-dihydropyridadyl.

[0127] In one aspect of the present invention, dendrimer structure D has a core molecule having at least three functional groups arranged symmetrically or asymmetrically. In one aspect of the present invention, one of the at least three functional groups of the core molecule is group L 1 -X 1 The remaining two or more functional groups contribute to the formation of the dendrimer structure. In one aspect of the present invention, the core portion comprises a chemical structure corresponding to propargylamine, ethylenediamine, triethanolamine, pentaerythritol, azidopropyl(alkyl)amine, hydroxyethyl(alkyl)amine, tetraphenylmethane, trimethoyl chloride, diaminohexane, diaminobutane, cystamine, propylenediamine, lysine, or propyleneamine.

[0128] In one aspect of the present invention, the dendrimer structure D of the compound may include a core portion selected from the group consisting of propargylamine, ethylenediamine, triethanolamine, pentaerythritol, azidopropyl(alkyl)amine, hydroxyethyl(alkyl)amine, tetraphenylmethane, trimethoyl chloride, diaminohexane, diaminobutane, cystamine, and propylenediamine. In one aspect of the present invention, the dendrimer structure D of the compound is a poly(amideamine)(PAMAM) dendrimer having the above-mentioned core portion.

[0129] In one aspect of the present invention, a reactive group (e.g., maleimide, hydrazide, azide, haloacetamide, or alkoxyamine) is introduced into the polypeptide that forms the conjugate, and the reactive group and X 1 The reaction forms a conjugate in which the compound and polypeptide are linked via a linker. In one embodiment of the present invention, the linker is selected from, for example, pyridine disulfide, thiosulfonate, vinyl sulfonate, isocyanate, NHS ester, imide ester, diazine, hydrazine, thiol, carboxylic acid, multipeptide linker, acetylene linker, or cleavable linker.

[0130] In one aspect of the present invention, the polypeptide that forms the conjugate is a target-directed polypeptide. In one embodiment of the present invention, the target-directed polypeptide is an antibody or an antigen-binding fragment selected from receptors, receptor ligands, IgG or its antigen-binding fragment, single-chain antibodies, and multispecific antibodies. In one embodiment of the present invention, the target-directed polypeptide is a molecule such as a compound or polypeptide that can be transported into or across the blood-brain barrier (BBB) ​​of a specific cell type (e.g., liver, lung, kidney, spleen, or muscle). The target-directed polypeptide may be a peptide and may be naturally occurring or produced by chemical synthesis or recombinant gene technology.

[0131] In one aspect of the present invention, the core portion of dendrimer structure D may be any known in the art, including, in the case of PAMAM dendrimers, a selection from the group consisting of propargylamine, ethylenediamine, triethanolamine, pentaerythritol, azidopropyl(alkyl)amine, hydroxyethyl(alkyl)amine, tetraphenylmethane, trimethoyl chloride, diaminohexane, diaminobutane, cystamine, and propylenediamine. The core portion may also be propyleneime, in which case the dendrimer is poly(propyleneamine) (POPAM). Alternatively, the core portion may be lysine, in which case the dendrimer is polylysine. Typically, the core portion may have 1 to 12 branches (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 branches). The core portion is functionalized to form reactive groups for the addition of branch portions (e.g., by reacting with methyl acrylate). One of the branch portions can be attached to the core portion via a functional group. Target-directed peptides and active ingredients can also be attached to the core portion via a functional group, with or without a linker.

[0132] In one aspect of the present invention, dendrimer structure D includes PAMAM or a substructure thereof. The core of PAMAM is cystamine, which reacts with methyl acrylate and then with ethylenediamine to produce generation 0 (G-0) PAMAM (D. A Tomalia et al., Tetrahedron 59, 22, 3799-3813 (26 May 2003)). Sequential reactions create higher generations, which tend to have different properties. Lower generations are generally flexible molecules that do not have a recognizable internal region, while medium-sized (G-3 or G-4) have an internal space that is essentially separated from the dendrimer shell. Very large (G-7 and above) dendrimers are generally closer to solid particles with a very dense surface due to the structure of their shell.

[0133] In one aspect of the present invention, the dendrimer structure D can be synthesized using dendrimer synthesis methods known in the art described herein. For example, the branched portion (X) of the dendrimer コア and X 枝 Parts of the dendrimer can also be purchased from commercial suppliers in layers of varying numbers of branches. There are two common methods used for dendrimer synthesis: branched synthesis and focused synthesis. In branched synthesis, dendrimers are assembled from a polyfunctional core and extended outward by a series of reactions, generally the Michael reaction. Each step of the reaction is generally carried out until completion to prevent errors in the dendrimer that can cause trailing generations (some branches shorter than others). Such impurities can affect the functionality and symmetry of the dendrimer, but they are very difficult to remove by purification because the relative size difference between complete and incomplete dendrimers is very small.

[0134] In one embodiment of the present invention, 13 C-NMR contrast agents are 13 It is used in nuclear magnetic resonance (MRS) diagnostics, which utilize the phenomenon of nuclear magnetic resonance targeting 1C. In one embodiment, MRS diagnostics are performed using an MRI device, MRS (magnetic resonance spectroscopy), or NMR device. The use of these devices can be carried out according to the purpose of the present invention, based on knowledge known to those skilled in the art. Here, nuclear magnetic resonance imaging (MRI) is generally suitable for the analysis of anatomical information. MRS is one of the imaging methods performed with MRI and is generally excellent for the analysis of chemical information of metabolites.

[0135] In one embodiment of the present invention, 13 "C-NMR contrast agent" is 13This technology utilizes the principle of nuclear magnetic resonance (C) phenomena to detect or analyze the dynamics (distribution) and localization of contrast agents within a living organism; detect or diagnose the clinical and pharmacological characteristics of cells, tissues, or organs within a target; detect or diagnose information that serves as an indicator for identifying a disease or condition; detect, identify, or diagnose the shape or location of a disease or condition; confirm the effectiveness of treatment (e.g., confirming the effectiveness of treatment after administration of anticancer drugs); and use it for adjunctive diagnosis during surgical procedures.

[0136] In one embodiment of the present invention, 13 By using a 1C-NMR contrast agent in MRI examination / diagnosis, the image contrast in MRI images can be enhanced, for example, in specific cells, tissues, or organs within the body. In one embodiment of the present invention, 13 By using a 1C-NMR contrast agent for MRS detection / diagnosis, it is possible to alter the waveform pattern measured by the MRS device for specific cells, tissues, or organs within the body.

[0137] MRI devices that use superconducting electromagnets or permanent magnets are known. MRI devices using superconducting electromagnets can generate a strong magnetic field, enabling the creation of detailed and high-contrast images. In the case of MRI devices using superconducting electromagnets, the magnetic field is not limited as long as it is permissible for the imaging target (e.g., a human), but may have magnetic fields of, for example, 10, 9.4, 8, 7, 6, 5, 4, 3, 2, 1.5, 1, or 0.5 Tesla. In one embodiment, from the viewpoint of cost and simplicity, a lower magnetic field is preferable, while from the viewpoint of spatial resolution, a higher magnetic field is preferable.

[0138] Regarding magnetic resonance imaging (MRI), various methods are known to those skilled in the art, including, but not limited to, spin echo (SE), gradient echo (GRE), echo-planar (EPI), fat suppression, and RARE (Rapid Acquisition with Relaxation Enhancement), and all of these can be used for the magnetic resonance diagnostic agent of the present invention. Those skilled in the art will understand that, in light of the objectives and problems of the present invention, an appropriate method can be selected as appropriate to obtain an optimal MRI contrast image. In one embodiment of the present invention, 13 In subjects administered with a 1C-NMR contrast agent, compared to those not administered with the contrast agent, site-specific accumulation sites of the contrast agent or its metabolites (e.g., tumor tissue) were observed. 13 The contrast of the C signal is increased, causing the accumulation area to be output in white (high signal). To further increase this contrast difference, the TR, TE, magnetic field, etc., may be adjusted to optimize the image contrast.

[0139] While not limited to this, in one embodiment, 13 In the accumulation sites of subjects who received C-NMR contrast agent, compared to the case where no contrast agent was administered, 13 The C signal intensity is amplified, for example, by 1.1 to 10 times or more (for example, 1.2 to 5 times or more).

[0140] In one aspect of the present invention, 13 The route of administration of C-NMR contrast agents to the target (living body, etc.) may be systemic or local. While not limited to these, possible routes include oral administration (including sublingual administration), inhalation, direct administration to target tissue or organs via catheter, intravenous administration (including infusion), transdermal administration (such as poultices), suppositories, or parenteral administration via forced enteral nutrition using a nasogastric tube, nasointestinal tube, gastrostomy tube, or jejunostomy tube.

[0141] In one aspect of the present invention, 13The 13C-NMR contrast agent is administered in a dosage form appropriately determined according to the administration route. Examples, but not limited to, include injections, intravenous infusions, tablets, capsules, granules, powders, liquids, aqueous solutions dissolved in syrup, poultices, suppositories, etc.

[0142] In one aspect of the present invention, 13 To prepare a 1C-NMR contrast agent, pharmacologically acceptable carriers, excipients, diluents, additives, disintegrants, binders, coatings, lubricants, gliding agents, flavoring agents, sweeteners, solubilizers, solvents, gelling agents, nutrients, etc. may be added as needed, and furthermore, 13 These may affect the absorption and blood concentration of the 13C-NMR contrast agent, potentially leading to changes in its pharmacokinetics. Examples include water, physiological saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropylcellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin.

[0143] In one aspect of the present invention, 13 The amount, timing, frequency, and duration of administration of the C-NMR contrast agent to the subject may vary depending on the subject's age, weight, the state of the cells, tissues, or organs within the subject to be detected, identified, or diagnosed, or the symptoms or conditions to be detected, identified, or diagnosed in the subject.

[0144] In one aspect of the present invention, 13 The subjects to whom the 13C-NMR contrast agent is administered (applied) are not limited to mammals (humans, non-human mammals (e.g., mice, rats, dogs, cats, rabbits, cattle, horses, sheep, goats, pigs, etc.) or non-mammals (e.g., fish, reptiles, amphibians, or birds)), plants, insects, bacteria, or cells (including cultured cells), tissues, or organs derived therefrom. Alternatively, the subject may be an artificial environment (e.g., an in vitro reaction system). The subject in this invention is preferably a mammal, and particularly preferably a human.

[0145] In one aspect of the present invention, 13 When the subject is a human, the C-NMR contrast agent can be administered in amounts of 0.1 mg to 1000 mg / day per adult, preferably 0.2 mg to 900 mg / day, more preferably 0.3 mg to 600 mg / day, and even more preferably 0.35 mg to 400 mg / day. Generally, local administration can be performed at a lower dose than systemic administration.

[0146] In one aspect of the present invention, 13 Examples of administration frequency for 13C-NMR contrast agents include once to multiple times a day, or continuous administration by intravenous infusion. Generally, in order to use it as a diagnostic agent for accurate detection or diagnosis, it is preferable to administer the next contrast agent as needed, after the administered contrast agent and its resulting metabolites have been completely metabolized, broken down, and excreted in the body.

[0147] In one embodiment of the present invention, 13 The duration of administration of 13C-NMR contrast agents can be determined by pharmacologists and clinicians in the relevant field using known methods, based on various clinical indicators, etc., taking into consideration the symptoms or conditions to be detected or diagnosed in the subject.

[0148] Therefore, in one embodiment of the present invention, 13 After administering a 1C-NMR contrast agent to a subject, the state of the desired cells, body fluids, tissues, or organs within the subject is detected or diagnosed using an MRI or NMR device, etc., at, but not limited to, 1 to 24 hours, preferably 1.5 to 15 hours, and more preferably 2 to 8 hours later. Those skilled in the art will understand that the optimal measurement timing can be appropriately determined in consideration of the time it takes for the contrast agent to reach the desired cells, tissues, or organs within the subject, the accumulation time, and the time it takes for the contrast agent to become saturated (reach its peak) in those desired cells, tissues, or organs. In one embodiment of the present invention, 13 Used as a C-NMR contrast agent, it images cells, tissues, or organs within the target, and the image data (especially with increased contrast) is obtained. 13The accumulation site of the administered contrast agent can be identified by obtaining C-MR image data.

[0149] Therefore, in one embodiment of the present invention, 13 ¹¹C-NMR contrast agents may be used for the detection of the state of cells, bodily fluids, tissues, or organs within a target (in this invention, detection means, for example, detecting as an image (signal intensity in the image) using an MRI device, or detecting as a waveform using an MRS-NMR device) or for diagnosis, etc., utilizing the principle of nuclear magnetic resonance. The present invention may be carried out by appropriately combining any one or more of any embodiments described herein, as long as they do not conflict with the technical specifications.

[0150] In some embodiments, this disclosure provides compositions comprising one or more dendrimer conjugates described herein. In one embodiment of the present invention, the composition is a pharmaceutical composition. A pharmaceutical composition comprising one or more dendrimer conjugates can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate the treatment of the active compound into a pharmaceutically usable preparation. The appropriate formulation is determined according to the selected route of administration. In one embodiment of the present invention, the composition is formulated for parenteral delivery. In one embodiment of the present invention, the composition is formulated for intratumoral injection. In one embodiment of the present invention, the composition can be formulated in sterile saline or buffer solution for injection into the tissue or cells to be treated. The composition can be lyophilized in single-use vials for rehydration immediately before use.

[0151] In one aspect of the present invention, a pharmaceutical composition comprises one or more dendrimer conjugates and one or more pharmaceutically acceptable excipients. Examples of excipients include solvents, diluents, pH modifiers, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, isotonic agents, stabilizers, and combinations thereof. Suitable pharmaceutically acceptable excipients may be selected from materials that are generally recognized as safe (GRAS) and can be administered to a subject without causing undesirable biological side effects or interactions.

[0152] In one aspect of the present invention, pharmaceutically acceptable salts can be prepared by reacting the free acid or base form of a compound with a stoichiometric amount of a suitable base or acid in water or an organic solvent (e.g., a non-aqueous medium, e.g., ether, ethyl acetate, ethanol, isopropanol, or acetonitrile). Pharmaceutically acceptable salts may include inorganic acids, salts of compounds derived from organic acids, alkali metal salts, and alkaline earth metal salts, as well as salts formed by reacting compounds with suitable organic ligands (e.g., quaternary ammonium salts).

[0153] In one aspect of the present invention, the composition is formulated into unit dosage forms for ease of administration and uniformity of dosage. In one aspect of the present invention, a unit dosage form refers to a physically distinct unit of the conjugate suitable for the target being treated. The therapeutically effective dose can first be estimated in a cell culture assay or in an animal model, e.g., mouse, rabbit, dog, or pig. The animal model can also be used to achieve a desired concentration range and route of administration. Such information can be used to determine a useful dose and route for administration in humans. The therapeutic efficacy and toxicity of the conjugate, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal in 50% of the population), can be determined in cell cultures or experimental animals by standard pharmaceutical procedures. The dose-to-therapeutic effect ratio of toxicity is the therapeutic index and can be expressed as the LD50 / ED50 ratio.

[0154] In one aspect of the present invention, the composition is administered topically, for example, by direct injection into the site to be treated. In one aspect of the present invention, the composition is administered by injection, topical application, or otherwise directly to vascular structures on or adjacent to vascular tissue at injury, surgical, or implantation sites. For example, in one aspect of the present invention, the composition is applied topically to vascular tissue exposed during surgical or implantation or transplantation procedures. Pharmaceutical compositions formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), enteral, and topical administration routes are described.

[0155] In one aspect of the present invention, the dendrimer conjugate is formulated for parenteral administration. The phrases “parenteral administration” and “administered parenterally” are terms recognized in the art and include methods of administration other than enteral and topical administration, such as injection, which may include intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. In one aspect of the present invention, the composition is administered parenterally, for example, via subdural, intravenous, intrathecal, intraventricular, intraarterial, intraarticular, synovial, intraamniotic, intraperitoneal, or subcutaneous routes.

[0156] For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Parenteral vehicles (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's glucose, glucose and sodium chloride, Ringer's lactate, and fixative oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and injectable organic esters, such as ethyl oleate. Aqueous carriers include, for example, water, alcohol solutions / aqueous solutions, cyclodextrins, emulsions, or suspensions containing saline and buffered media. Compositions may also be administered as emulsions, such as water-in-oil emulsions. Examples of oils include oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish liver oil, sesame oil, cottonseed oil, and corn oil. Fatty acids suitable for use in parenteral formulations include, for example, oleic acid, stearic acid, isostearic acid, ethyl oleate, and isopropyl myristate.

[0157] In one aspect of the present invention, compositions suitable for parenteral administration may include aqueous and non-aqueous sterile suspensions containing antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Intravenous vehicles may contain fluid and nutritional supplements, as well as electrolyte supplements, such as those based on Ringer's glucose. Generally, water, physiological saline, aqueous glucose and related sugar solutions, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Injectable pharmaceutical carriers for injectable compositions are known in the art.

[0158] The present invention will be illustrated below with examples, but this is not intended to limit the present invention.

[0159] [Example 1] (methyl 13C) Introduction of methionine into dendrimers Cystamin Core Polyaminoamine (PAMAM) Dendrimer (PAMAM(NH2)) 16 , Generation 2) 20% solution (647829 Sigma-Aldrich, 67 mg, dendrimer: 33 mg, 0.01 mmol, terminal NH2: 0.16 mmol), (methyl 13 C) A 5 ml PSB solution was prepared containing methionine (CLM-206-1 Cambridge Isotope Laboratories, Inc., 18 mg / mL, 1 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Code; 1030 WSCD·HCl Peptide Laboratories, Inc., 33 mg / mL solution, 1 mL), and N-hydroxysulfosuccinimide (Sulfo-NHS, H1304 Tokyo Chemical Industry Co., Ltd., 2 mg / mL solution, 1 mL), and the solution was shaken at room temperature for 1 hour.

[0160] Ultrafiltration with Milli-Q water was performed seven times (1 / 3 dilution) using a 1 kDa centrifugal ultrafiltration filter, and impurities and unreacted materials were diluted 1 / 2187 from the stock solution. Regarding the obtained product... 13 The results of the 1C-NMR measurement are shown in Figure 3. Around 13 ppm, (methyl 13 C) Methionine-S 13 A peak originating from CH3 was identified, and this peak could be confirmed even with a data acquisition time of approximately 1 minute.

[0161] The resulting dendrimer is further (methyl 13 C) The reaction was carried out under the same conditions as above to introduce methionine. Products from 1 to 3 reaction cycles 13 The results of the 1C-NMR measurement are shown in Figure 3. By comparing with the peak derived from the dendrimer, it can be seen that by performing the reaction three times, (methyl 13 C) Methionine-S 13 An increase in the intensity of the peak originating from CH3 was confirmed (Figure 5).

[0162] [Example 2] (methyl 13C) Introduction of methionine into dendrimers (methyl 13 C) The introduction reaction of methionine into a dendrimer was carried out under the following conditions: cystamine core polyaminoamine (PAMAM) dendrimer (PAMAM(NH2)) 16 , a 20% solution of generation 2) (167.5 mg, dendrimer: 33 mg, 0.01 mmol, terminal NH2: 0.16 mmol), (methyl 13 C) A 1.2 ml solution of Milli-Q water containing methionine (48 mg / mL, 1 mL), EDC (55 mg / mL solution, 1 mL), and Sulfo-NHS (59 mg / mL solution, 1 mL) was prepared and the solution was shaken at room temperature for 3 days.

[0163] Ultrafiltration with Milli-Q water was performed seven times (1 / 3 dilution) using a 1 kDa centrifugal ultrafiltration filter, and impurities and unreacted materials were diluted from the stock solution to 1 / 2187. Regarding the raw material dendrimers and the resulting product... 1 H-NMR and 13 The results of the 1C-NMR measurements are shown in Figures 6 and 7. 13 The 1C-NMR measurement results showed that (methyl) was present at around 13 ppm. 13 C) Methionine-S 13 Only peaks originating from CH3 were observed, and numerous (methyl) peaks were found. 13 C) It was confirmed that methionine was introduced into the dendrimer.

[0164] Products 13 In 1C NMR measurements, even if the data acquisition time is 2 minutes, methyl 13 C) Methionine-S 13 A peak originating from CH3 was observed (Figure 8). Furthermore, even with a cumulative measurement of 8 times (approximately 30 seconds), -S 13 A peak originating from CH3 was identified (Figure 9). The obtained (methyl 13 C) Methionine-introduced dendrimers were subjected to two-dimensional NMR analysis (HSQC) and mass spectrometry. The results are shown in Figures 10 to 13. Mass spectrometry results, compared with the starting material dendrimers, showed that the product dendrimers contained an average of 6 to 8 (methyl) groups.13 C) It was suspected that methionine had been introduced. The NMR used was a Bruker AVANCE III HD 500 MHz, HSQC, 1 H, 13 The C sequence used the default settings.

[0165] [Example 3] (C2- 13 C) Introduction of acetic acid into dendrimers (methyl) obtained in Example 2 13 C) Methionine-introduced dendrimer (10 mg), (C2- 13 C) Prepare a 0.2 ml solution of Milli-Q water containing acetic acid (3.28 μL, CLM-318-1 Cambridge Isotope Laboratories, Inc.), EDC (1 mL solution at 18 mg / mL), Sulfo-NHS (1 mL solution at 19 mg / mL), and triethylamine for pH adjustment (T0424 Tokyo Chemical Industry Co., Ltd.), and shake the solution at room temperature for 3 days.

[0166] Ultrafiltration with Milli-Q water was performed seven times (1 / 3 dilution) using a 1 kDa centrifugal ultrafiltration filter, and impurities and unreacted materials were diluted to 1 / 2187 from the stock solution. Using a similar method, (C2- 12 C) Acetic acid introduced (methyl 13 C) Methionine-introduced dendrimers were also prepared.

[0167] Regarding the raw material dendrimers and the resulting products 13 The results of the 1C-NMR measurements are shown in Figures 14 and 15. 13 The 1C-NMR measurement results showed that (methyl) was present at around 13 ppm. 13 C) Methionine-S 13 Only peaks originating from CH3 were observed, and numerous (methyl) peaks were found. 13 C) It was confirmed that methionine was introduced into the dendrimer.

[0168] [Example 4] (methyl 13 C) Preparation of methionine-derived dendrimer and polypeptide conjugates 6.7 mg of BSA (A2153 SIGMA-ALDRICH) and 5 mg of 3-sulfo-4-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sodium salt (Sulfo-SMCC, S0883 Tokyo Chemical Industry Co., Ltd.) were each dissolved in 500 μL of Milli-Q water and mixed. After mixing, the mixture was reacted at room temperature for 1 hour. After the reaction, unreacted reagents were removed by ultrafiltration with Milli-Q water three times (1 / 10 dilution) using a 10 kDa centrifugal ultrafiltration filter. The final volume was 100 μL. In 100 μL of BSA solution containing the sulfo-SMCC after this reaction, the disulfide bonds were reduced with Pierce Immobilized TCEP Disulfide Reducing Gel (77712 Thermo Scientific) to bond the amino group. 13 One mg of the 14C-modified dendrimer was mixed and reacted at room temperature for one hour. After the reaction, unreacted reagents were removed by ultrafiltration with Milli-Q water three times (1 / 10 dilution) using a 10 kDa centrifugal ultrafiltration filter. The resulting protein solution was freeze-dried and dissolved in 600 μL of heavy water (Cambridge Isotope Laboratories DLM-4-100) to prepare the sample for NMR.

[0169] 13 The results of the 1C-NMR measurement are shown in Figure 17. 13 C-labeled methionine-introduced dendrimers can be used in as little as 7.5 minutes. 13 It was confirmed that signals originating from the C label could be observed. 13 HSQC of the sample subjected to C-NMR ( 1 H. 13 Figure 18 shows the measurement results of the 2D NMR spectrum (C). 13 The signal observed by 1C NMR is methionine 13 The signal was confirmed to originate from the methyl group at the 1C labeling site.

[0170] Mass spectrometry was performed to confirm the binding of the dendrimer to BSA via the sulfhydryl group. The results are shown in Figure 16. In the left column of Figure 16, an increase in molecular weight was confirmed for the cysteine-introduced BSA prepared as a preliminary experiment (the peak is shifted to the right). In the right column, the binding of the dendrimer to BSA was confirmed using the sulfhydryl group obtained by reducing the cystamine at the center of the dendrimer.

Claims

1. １３ A method for producing a C-MRI contrast agent, １３ C-MRI contrast agents are １３ This polypeptide is labeled by the introduction of C. The above method is at the end １３ The dendrimer structure D has a functional group X containing C, and the group X is capable of forming a conjugate with a polypeptide. １ A compound having this property is reacted with a polypeptide. １３ This includes producing a polypeptide into which a functional group X containing C has been introduced, The method wherein the compound contains 5 to 200 functional groups X.

2. X １ The method according to claim 1, wherein is a sulfanyl, azide, or a group containing a C-C triple bond.

3. The functional group X is １３ CH ３ S - or １３ CH ３ The method according to claim 1, comprising CO-.

4. The polypeptide to be subjected to the reaction is X １ Reactive group X ２ The method according to any one of claims 1 to 3, wherein the polypeptide is introduced.

5. The compound is given by formula 1, formula 2, or formula 3: 【Chemistry 1】 [In the formula, X １ The group is selected from sulfanyl, azide, and groups containing a C-C triple bond. L １ This indicates absence or linker, D represents a dendrimer structure, R １ is a hydrogen atom, or C １－６ Represents alkyl, L ２ Each of these independently represents a linker. L ３ Each of these independently represents a linker. n １ [This represents numbers from 5 to 200] The compound represented by reacts with a polypeptide. １３ The process involves producing a polypeptide into which a functional group X containing C has been introduced. １３ A method for manufacturing C-MRI contrast agents.

6. L １ C ２－１０ The method according to claim 5, comprising alkylene.

7. The compound is represented by formula 1, L ２ However, each is independent of -COY-(C ２－１０ Alkylene) is where alkylene is hydroxy, C １－６ Alkoxy, or -NR ２ R ３ It may be substituted with one or more substituents selected from the following: Y can be directly bonded, -O-, or -NR, independently of each other. ４ - and R １ , R ２ , R ３ , and R ４ These are, independently, hydrogen atoms and C １－６ It is an alkyl or nitrogen atom protecting group, -COY- carbonyl group is -NR １ - Bonded to the nitrogen atom, The method according to claim 5 or 6.

8. The compound is represented by formula 2, L ３ However, each independently, C ２－１０ It is alkylene, where alkylene is hydroxy, C １－６ Alkoxy, or -NR ２ R ３ It may be substituted with one or more substituents selected from the following: R １ , R ２ , and R ３ These are, independently, hydrogen atoms and C １－６ Alkyl or nitrogen atom protecting group, The method according to claim 5 or 6.

9. D contains multiple nitrogen atoms as branching points, and the branching points are - (C ２－６ Alkilen)-CONR １１ - (C ２－６ Alkilen) - or - (C ２－１０ Alkilen) - This represents a dendrimer structure linked by R. １１ Each of these is independently a hydrogen atom or C １－６ The method according to any one of claims 1 to 8, wherein the alkyl group is used.

10. D is at the branching point -C ２ H ４ -CONH-C ２ H ４ The method according to any one of claims 1 to 9, having a polyaminoamine (PAMAM) dendrimer structure linked by -.

11. The method according to any one of claims 1 to 10, wherein D has a generation 1 to 4 polyaminoamine (PAMAM) dendrimer structure.

12. X １ That is sulfanyl, D is a dendrimer structure corresponding to a thiol obtained by reducing the disulfide bond of a dendrimer having a disulfide bond in its core. n １ The method according to any one of claims 1 to 11, wherein is 5 to 95.

13. Functional group X is given by formula 4, formula 5, or formula 6: 【Chemistry 2】 [In the formula, R １ and R ６ These are, independently, a hydrogen atom and C １－６ Selected from alkyl groups, R ２ , and R ３ These are, independently, hydrogen atoms and C １－６ Selected from alkyl and nitrogen atom protecting groups, R ５ These are, independently, hydrogen atoms and C １－６ [Selected from alkyl and carboxyl protecting groups] The method according to any one of claims 1 to 12, as represented by [the above].

14. The method according to any one of claims 1 to 13, wherein the functional group X is represented by formula 3.

15. All functional groups X in the compound are methyl １３ The method according to any one of claims 1 to 14, comprising the structure of C-methionine.

16. All functional groups X １３ The method according to any one of claims 1 to 15, wherein C is detected as substantially the same signal in nuclear magnetic resonance measurements.

17. The compound is, Formula 7: 【Transformation 3】 [In the formula, L １ C ２－６ It is alkylene, X １ It is sulfanyl, D represents a dendrimer structure obtained by reducing the disulfide bond of generation 1-3 polyaminoamine (PAMAM) dendrimers having a disulfide bond in the core portion. R ２ , and R ３ These are, independently, hydrogen atoms and C １－６ Selected from alkyl and nitrogen atom protecting groups, n １ [This represents numbers from 5 to 200] The method according to any one of claims 1 to 16, as represented by [the above].

18. X １ is sulfanyl, X ２ is a group containing a C-C double bond. X １ is Azid, X ２ is a group containing a C-C triple bond, or X ２ is a group containing a C-C triple bond, X ２ The method according to any one of claims 1 to 17, wherein is azide.

19. Polypeptides into which functional group X is introduced are given by formula 10, formula 11, or formula 12: 【Chemistry 4】 [In the formula, R １ , L １ , L ２ , L ３ , D, and n １ This is as already defined. The method according to any one of claims 5 to 18, wherein a conjugate is formed with a group represented by

20. L １ The method according to claim 19, wherein the triazolyl is linked to the polypeptide via 1,2,3-triazolyl, -S-S-, or -S-.

21. The method according to any one of claims 1 to 20, wherein the polypeptide is an antibody or its antigen-binding fragment, receptor, ligand, or fusion protein.

22. The method according to any one of claims 1 to 21, wherein the polypeptide is an antibody or an antigen-binding fragment selected from IgG or its antigen-binding fragment, a single-chain antibody, and a multispecific antibody.

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