Methods of forming radiopaque peptides and medical hydrogels formed therefrom

Abstract

In various aspects, the disclosure relates to methods of forming iodinated peptide compounds, the methods comprising: (a) forming a multifunctional precursor compound comprising (i) residues of an amine-protected peptide comprising two or more amino acid residues having a primary amine-containing pendant group, and (ii) a polyfunctional moiety attached to the amino-protected peptide residue via an amide group or an ester group; (b) subjecting the polyfunctional precursor compound to an amide coupling reaction or an esterification coupling reaction with an iodinated compound to form an iodinated, amine-protected peptide compound comprising n iodinated moieties covalently linked to an amine-protected peptide residue via a residue of a polyfunctional moiety; and (c) deprotecting the iodinated, amine-protected peptide compound to form an iodinated peptide compound.

Description

[0001] Cross-references to related applications This application claims the benefit of US Provisional Patent Application No. 63 / 519,962, filed August 16, 2023, and US Provisional Patent Application No. 63 / 618,654, filed January 8, 2024, the disclosures of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to a method for forming a radiopaque peptide, the use of such a radiopaque peptide as a crosslinking agent for forming a hydrogel, and the hydrogel formed from such a radiopaque peptide. The radiopaque peptide and the hydrogel can be used in, for example, a variety of medical applications. Background Technology

[0003] SpaceOAR® is a rapidly cross-linked hydrogel that polymerizes in vivo within seconds. It is based on a multi-arm polyethylene glycol (PEG) polymer functionalized with succinimide glutarate as the active end group, which then reacts with trilysine to form a cross-link. This product has become a highly successful clinical biomaterial in the treatment of prostate cancer. A further improvement to this structure involves functionalizing a portion of the succinimide glutarate end group with a 2,3,5-triiodobenzamide group, thereby providing radiopacity. This hydrogel, known by the trade name SpaceOAR Vue®, is a radiopaque version of SpaceOAR® for prostate medical applications.

[0004] An alternative strategy for forming iodine-labeled crosslinked hydrogels that provides enhanced radiation impermeability while maintaining the crosslinking density of each polymer molecule is meaningful for medical applications. Summary of the Invention

[0005] In several aspects, this disclosure relates to a method for forming an iodinated peptide compound, the method comprising: (a) forming a multifunctional precursor compound comprising (i) residues of a mine-protected peptide having two or more amino acid residues having primary amine side groups, and (ii) a multifunctional moiety of the mine-protected peptide residues linked by an amide or ester group, the multifunctional moiety further comprising n primary amine groups, n hydroxyl groups, or n carboxyl groups, where n is an integer of 2 or greater; (b) reacting the multifunctional precursor compound with an amine-functional iodinated compound selected from an iodinated moiety comprising an iodinated moiety, a carboxyl-functional iodinated compound comprising an iodinated moiety, and a hydroxy-functional iodinated compound comprising an iodinated moiety. (c) An iodinated compound is subjected to an amide coupling reaction or an esterification coupling reaction to form an iodinated, amino-protected peptide compound comprising n iodinated moieties, the n iodinated moieties being covalently linked to amino-protected peptide residues via multifunctional residues; and (d) the iodinated, amino-protected peptide compound is deprotected to form an iodinated peptide compound.

[0006] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional portion is linked to an amino-protected peptide residue via an amide group, and the multifunctional portion comprises n primary amino groups.

[0007] In some of these embodiments, the multifunctional precursor compound is formed by a method comprising: (i) an amide coupling reaction of a polyamine compound having n+1 primary amino groups with (ii) a carboxyl group at the C-terminus of an amino-protected peptide, wherein the amino-protected peptide comprises two or more amino acid residues having primary amino side groups, and the primary amino groups of the amino-protected peptide are protected from participating in the amide coupling reaction.

[0008] In some of these embodiments, the n primary amino groups of the multifunctional precursor compound undergo an amide coupling reaction with the n carboxyl-functionalized iodide compound to form an iodinated, amino-protected peptide compound.

[0009] In some of these embodiments, the multifunctional moiety is a residue of a polyamine compound having n+1 primary amine groups, one of which is used to form an amide group that links the multifunctional moiety to a peptide residue with amine protection, and n of the n+1 primary amine groups are used for an amide coupling reaction between the multifunctional precursor compound and a carboxyl-functionalized iodinated compound containing an iodinated moiety.

[0010] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional portion is linked to an amino-protected peptide residue via an ester group, and the multifunctional portion contains n hydroxyl groups.

[0011] In some of these embodiments, the multifunctional precursor compound is formed by a method comprising: (i) ester coupling a polyol compound having n+1 hydroxyl groups with (ii) a carboxyl group at the C-terminus of an amino-protected peptide, wherein the amino-protected peptide comprises two or more amino acid residues having primary amine side groups, and the primary amine group of the amino-protected peptide is protected from participating in the ester coupling reaction.

[0012] In some of these embodiments, the n hydroxyl groups of the multifunctional precursor compound undergo an ester coupling reaction with the n carboxyl-functional iodide compound to form an iodinated, amino-protected peptide compound.

[0013] In some of these embodiments, the multifunctional moiety is a residue of a polyol compound having n+1 hydroxyl groups, one of which forms an ester group that links the multifunctional moiety to an amino-protected peptide residue, and n of the n+1 hydroxyl groups are used for an ester coupling reaction between the multifunctional precursor compound and a carboxyl-functionalized iodinated compound containing an iodinated moiety.

[0014] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional portion is linked to an amino-protected peptide residue via an amide group, and the multifunctional portion comprises n carboxyl groups.

[0015] In some of these embodiments, the n carboxyl groups of the polyfunctional precursor compound are ester-coupled with the n hydroxyl-functional iodide compound to form an iodinated, amino-protected peptide compound.

[0016] In some of these embodiments, the n carboxyl groups of the multifunctional precursor compound are subjected to an amide coupling reaction with n amino-functional iodide compounds to form an iodinated, amino-protected peptide compound.

[0017] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional precursor compound is formed by a method comprising: (a) subjecting (i) a polyamine compound having n+1 primary amino groups to (ii) an amide coupling reaction of a carboxyl group at the C-terminus of an amino-protected peptide, the amino-protected peptide comprising two or more amino acid residues having primary amino side groups, the primary amino groups of the amino-protected peptide being protected from participating in the amide coupling reaction, the reaction product having n primary amino groups; and (b) subjecting the n primary amino groups of the reaction product of step (a) to a ring-opening reaction with a cyclic anhydride, thereby forming n carboxyl groups at the sites of the n primary amino groups.

[0018] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional moiety comprises residues of a polyamine compound having n+1 primary amine groups, one of the n+1 primary amine groups being used to form an amide group that links the multifunctional moiety to a peptide residue with protected amine groups, n of the n+1 primary amine groups being used to undergo a ring-opening reaction with a cyclic anhydride to form a multifunctional moiety comprising n carboxyl groups, and the n carboxyl groups being used for an ester coupling reaction between the multifunctional precursor compound and a hydroxy-functionalized iodinated compound comprising an iodinated moiety.

[0019] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional moiety comprises residues of a polyamine compound having n+1 primary amino groups, one of the n+1 primary amino groups being used to form an amide group that links the multifunctional moiety to an amino-protected peptide residue, n of the n+1 primary amino groups being used to undergo a ring-opening reaction with a cyclic anhydride to form a multifunctional moiety comprising n carboxyl groups, and the n carboxyl groups being used for an amide coupling reaction between the multifunctional precursor compound and an amino-functionalized iodinated compound comprising an iodinated moiety.

[0020] In some embodiments that can be used in conjunction with the foregoing aspects and embodiments, the multifunctional precursor compound is formed by a method comprising: (a) reacting (i) a carboxyl-protected polycarboxylamine compound containing an amino group and n protected carboxyl groups protected from amide coupling with (ii) an amide coupling reaction of a carboxyl group at the C-terminus of an amino-protected peptide, the amino-protected peptide comprising two or more amino acid residues having primary amine side groups, the primary amine group of the amino-protected peptide being protected from amide coupling; and (b) deprotecting the n protected carboxyl groups of the reaction product of step (a) without deprotecting the protected primary amine group.

[0021] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional moiety comprises residues of a polycarboxylated amine compound comprising an amino group and n carboxyl groups, the amino group being used to form an amide group that links the multifunctional moiety to a peptide residue protected by the amino group, and the n carboxyl groups being used for an ester coupling reaction between the multifunctional precursor compound and a hydroxy-functionalized iodinated compound comprising an iodinated moiety.

[0022] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional moiety comprises residues of a polycarboxylated amine compound comprising an amino group and n carboxyl groups, the amino group being used to form an amide group that links the multifunctional moiety to an amino-protected peptide residue, and the n carboxyl groups being used for an amide coupling reaction between the multifunctional precursor compound and an amino-functionalized iodinated compound comprising an iodinated moiety.

[0023] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional portion is linked to an amino-protected peptide residue via an ester group, and the multifunctional portion comprises n carboxyl groups.

[0024] In some of these embodiments, the n carboxyl groups of the polyfunctional precursor compound are ester-coupled with the n hydroxyl-functional iodide compound to form an iodinated, amino-protected peptide compound.

[0025] In some of these embodiments, the n carboxyl groups of the multifunctional precursor compound are subjected to an amide coupling reaction with n amino-functional iodide compounds to form an iodinated, amino-protected peptide compound.

[0026] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional precursor compound is formed by a method comprising: (a) ester coupling (i) a polyol compound having n+1 hydroxyl groups with (ii) a carboxyl group at the C-terminus of an amino-protected peptide, the amino-protected peptide comprising two or more amino acid residues having primary amine side groups, the primary amine group of the amino-protected peptide being protected from participating in the amide coupling reaction, the reaction product having n hydroxyl groups; and (b) ring-opening reaction of the n hydroxyl groups of the reaction product of step (a) with a cyclic anhydride, thereby forming n carboxyl groups at the sites of the n hydroxyl groups.

[0027] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional moiety comprises residues of a polyol compound having n+1 hydroxyl groups, one of the n+1 hydroxyl groups being used to form an ester group that links the multifunctional moiety to an amino-protected peptide residue, n of the n+1 hydroxyl groups being used to undergo a ring-opening reaction with a cyclic anhydride to form a multifunctional moiety comprising n carboxyl groups, and the n carboxyl groups being used for an ester coupling reaction between the multifunctional precursor compound and a hydroxy-functionalized iodinated compound comprising an iodinated moiety.

[0028] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional moiety comprises residues of a polyol compound having n+1 hydroxyl groups, one of the n+1 hydroxyl groups being used to form an ester group that links the multifunctional moiety to an amino-protected peptide residue, n of the n+1 hydroxyl groups being used to undergo a ring-opening reaction with a cyclic anhydride to form a multifunctional moiety comprising n carboxyl groups, and the n carboxyl groups being used for an amide coupling reaction between the multifunctional precursor compound and an amino-functionalized iodinated compound comprising an iodinated moiety.

[0029] In some embodiments that can be used in conjunction with the foregoing aspects and embodiments, the multifunctional precursor compound is formed by a method comprising: (a) subjecting (i) a carboxylated hydroxycarboxylic acid compound containing a hydroxyl group and n protected carboxyl groups that are protected from participating in an ester coupling reaction to (ii) an ester coupling reaction of a carboxyl group at the C-terminus of an amino-protected peptide, the amino-protected peptide comprising two or more amino acid residues having primary amine side groups, the primary amine group of the amino-protected peptide being protected from participating in an amide coupling reaction; and (b) deprotecting the n protected carboxyl groups of the reaction product of step (a) without deprotecting the protected primary amine group.

[0030] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional moiety comprises residues of a hydroxycarboxylic acid compound comprising a hydroxyl group and n carboxyl groups, the hydroxyl group being used to form an ester group that links the multifunctional moiety to an amino-protected peptide residue, and the n carboxyl groups being used for an ester coupling reaction between the multifunctional precursor compound and a hydroxyfunctional iodinated compound comprising an iodinated moiety.

[0031] In some embodiments that can be used in conjunction with the above aspects and embodiments, the multifunctional moiety comprises residues of a hydroxycarboxylic acid compound comprising a hydroxyl group and n carboxyl groups, the hydroxyl group being used to form an ester group that links the multifunctional moiety to an amino-protected peptide residue, and the n carboxyl groups being used for an amide coupling reaction between the multifunctional precursor compound and an amino-functionalized iodinated compound comprising an iodinated moiety.

[0032] In some embodiments that can be used in conjunction with the above aspects and implementation schemes, the n iodide moieties comprise one or more iodinated aromatic groups.

[0033] In some embodiments that can be used in conjunction with the foregoing aspects and embodiments, the n iodide moieties comprise one or more iodinated aromatic groups, said iodinated aromatic groups comprising a monocyclic or polycyclic aromatic structure substituted with one or more iodine groups. In some of these embodiments, the monocyclic or polycyclic aromatic structure is further substituted with one or more hydroxyl groups and / or one or more substituents comprising C1-C4-hydroxyalkyl groups. In some of these embodiments, the monocyclic or polycyclic aromatic structure is further substituted with one or more substituents comprising C1-C4-hydroxyalkyl groups, wherein the C1-C4-hydroxyalkyl group comprises an adjacent hydroxyl group, in which case, when the polyfunctional precursor compound reacts with the iodide compound, the adjacent hydroxyl group may comprise an acetal protectant.

[0034] In some embodiments that can be used in conjunction with the foregoing aspects and embodiments, the primary amino group of the peptide includes a protective group selected from the group consisting of tert-butoxycarbonyl, carboxybenzyl, trifluoroacetyl, 6-nitroveratryloxycarbonyl group, and 9-fluorenylmethoxycarbonyl.

[0035] In other aspects of this disclosure, a crosslinking network is provided comprising the following crosslinking reaction products: (a) a radiopaque peptide compound formed according to any of the foregoing aspects and embodiments, and (b) a reactive polymer comprising a portion that reacts with the primary amine group of the radiopaque peptide compound to form a covalent bond.

[0036] In other aspects of this disclosure, a system is provided comprising (a) a first composition comprising a radiopaque peptide compound formed according to any of the foregoing aspects and embodiments, and (b) a second composition comprising a reactive polymer comprising a portion that reacts with a primary amine group of the radiopaque peptide compound, wherein the system is configured to deliver the reactive polymer and the radiopaque peptide compound under conditions such that a covalent crosslink is formed between the reactive polymer and the radiopaque peptide compound.

[0037] The above and other aspects, implementations, features and benefits of this disclosure will become apparent from the following detailed description. Attached Figure Description

[0038] Figure 1A-1D Methods for forming multifunctional precursor compounds according to four embodiments of the present disclosure are illustrated schematically.

[0039] Figure 2 Methods for forming carboxyl-functionalized multifunctional precursor compounds according to three embodiments of the present disclosure are illustrated schematically.

[0040] Figure 2A A method for forming a carboxyl-functionalized multifunctional precursor compound according to another embodiment of the present disclosure is illustrated schematically.

[0041] Figure 3 A method for forming a carboxyl-functionalized multifunctional precursor compound according to another embodiment of the present disclosure is illustrated schematically.

[0042] Figure 4 A method for forming an iodinated peptide compound according to one embodiment of the present disclosure is illustrated schematically.

[0043] Figure 5The illustration schematically depicts a method for forming an amino-functionalized polyiodide compound according to one embodiment of the present disclosure, the amino-functionalized polyiodide compound then reacting with protected trilysine followed by deprotection to form an iodinated peptide compound.

[0044] Figure 6A The diagram schematically illustrates the acetal protection of adjacent hydroxyl groups of iodixanol according to one embodiment of the present disclosure.

[0045] Figure 6B A method for forming an iodinated peptide compound according to one embodiment of the present disclosure is illustrated schematically.

[0046] Figures 7A-7E A method for forming iodinated peptide compounds according to other embodiments of this disclosure is illustrated schematically.

[0047] Figure 8 A delivery device according to one embodiment of the present disclosure is shown.

[0048] Figure 9 A delivery device according to another embodiment of this disclosure is shown. Detailed Implementation

[0049] In several aspects, this disclosure provides a radiopaque peptide comprising a radiopaque portion of one or more types of radiopaque atoms covalently linked to the C-terminus of the peptide.

[0050] The peptides used in this disclosure include peptides containing 2 to 20 amino acid residues, said amino acid residues having a primary amine side group (i.e., an amino acid residue having a -NH2 group, including a guanidinium group), such as lysine, arginine, and / or ornithine amino acid residues (e.g., di-lysine, tri-lysine, tetra-lysine, penta-lysine, hexa-lysine, hepta-lysine, octa-lysine, nona-lysine, deca-lysine, di-ornithine, tri-ornithine, tetra-ornithine, penta-ornithine, hexa-ornithine, hepta-ornithine, octa-ornithine, nona-ornithine, deca-ornithine, di-arginine, tri-arginine, tetra-arginine, penta-arginine, hexa-arginine, hepta-arginine, octa-arginine, nona-arginine, deca-arginine, etc.). The peptides used in this disclosure may also include amino acids other than lysine, arginine, and / or ornithine, particularly those amino acids having a side group that does not react with a carboxyl, amine, or hydroxyl group under amide or ester coupling conditions. Examples of such peptides include glycine, alanine, valine, leucine, isoleucine, and phenylalanine. The total length of the peptides disclosed herein typically ranges from 2 to 20 or more amino acid residues, for example, from 2 to 3 to 4 to 5 to 6 to 8 to 10 to 15 to 20 amino acid residues (i.e., from any two of the aforementioned values).

[0051] The peptides used in the methods of this disclosure include amine-protected peptides, wherein the amine at the N-terminus of the peptide and the primary amine-containing side groups of the amino acid residues of the peptide are protected.

[0052] An iodide portion containing one or more types of iodide atoms includes an iodide portion, which may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more iodine atoms.

[0053] In some embodiments, the iodination moiety comprises one or more iodinated aromatic groups. Examples of iodinated aromatic groups include iodine-substituted monocyclic aromatic groups and iodine-substituted polycyclic aromatic groups, such as phenyl iodide, naphthyl iodide, anthryl iodide, phenanthrene iodide, or tetracenyl iodide. The iodinated aromatic group may be substituted with one, two, three, four, five, six, or more iodine atoms. In some of these embodiments, the aromatic group may also be substituted with one or more hydrophilic groups, for example, one, two, three, four, five, six, or more hydrophilic groups. The hydrophilic group may be a hydroxyl-containing group, selected from, for example, hydroxyl and hydroxyalkyl groups (e.g., hydroxyalkyl groups containing one, two, three, four, etc. carbon atoms).

[0054] Specific examples of the iodination moiety include those comprising one or more monocyclic or polycyclic aromatic structures, which are substituted by (a) one or more iodine groups (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more iodine atoms) and (b) optionally one or more hydroxyl-containing groups, said hydroxyl-containing groups being independently selected from one or more hydroxyl groups and / or one or more C1-C4-hydroxyalkyl groups (e.g., C1-C4-monohydroxyalkyl, C1-C4-dihydroxyalkyl, C1-C4-trihydroxyalkyl, C1-C4-tetrahydroxyalkyl, etc.), said C1-C4-hydroxyalkyl groups being directly or via any suitable linking portion to the monocyclic or polycyclic aromatic structure, said linking portion being, for example, selected from alkyl, ether, ester, amide, amino, carbonate, and combinations thereof.

[0055] In some aspects of this disclosure, radiopaque peptide compounds are formed by a method comprising: (a) forming a multifunctional precursor compound comprising (i) amino-protected peptide residues having two or more amino acid residues having primary amine side groups, and (ii) a multifunctional moiety of the amino-protected peptide residues linked by an amide or ester group, the multifunctional moiety further comprising n primary amine groups, n hydroxyl groups, or n carboxyl groups, wherein n is an integer of 1 or greater (e.g., 2, 3, 4, 5, 6, 7, 8). (a) reacting a multifunctional precursor compound with an iodinated compound selected from an amino-functionalized iodinated compound containing an iodinated moiety, a carboxyl-functionalized iodinated compound containing an iodinated moiety, and a hydroxyl-functionalized iodinated compound containing an iodinated moiety, via an amide coupling reaction or an ester coupling reaction to form an iodinated, amino-protected peptide compound containing n iodinated moieties, the n iodinated moieties being covalently linked to the amino-protected peptide residues; and (c) deprotecting the iodinated, amino-protected peptide compound to form an iodinated peptide compound.

[0056] Examples of protecting groups used in conjunction with amide coupling and ester coupling reactions include tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or (Z), trifluoroacetyl (TFA), 6-nitroveratroloxycarbonyl (Nvoc) and 9-fluorenylmethoxycarbonyl (Fmoc) or allyloxycarbonyl (Alloc); triphenylmethyl (Trt), tert-butyl (t-Bu), etc.

[0057] In some embodiments, the multifunctional precursor compound comprises an amino-protected peptide residue and a multifunctional moiety comprising n primary amino groups linked to the amino-protected peptide residue via amide groups.

[0058] For example, now refer to Figure 1A Amine-protected peptides, specifically tert-butoxycarbonyl-protected trilysine (Boc-protected trilysine) (112a), and polyamine compounds having n+1 primary amino groups, specifically tri(2-aminoethyl)amine having three primary amino groups. (CAS# 4097-89-6) (114a) undergoes an amide coupling reaction to form a multifunctional precursor compound (116a) comprising a Boc-protected trilysine residue and a multifunctional moiety having n primary amine groups, specifically a tri(2-aminoethyl)amine residue having two primary amine groups, wherein the multifunctional moiety is linked to an amine-protected peptide residue via an amide group.

[0059] It should be noted that the various amide coupling and ester coupling reactions described herein can be carried out in the presence of suitable coupling agents, such as carbodiimide coupling agents, such as N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethyl-propyl)carbodiimide (EDC), N-hydroxybenzotriazole (HOBt), BOP reagent and / or other suitable coupling agents.

[0060] As another example, now refer to Figure 1B Amine-protected peptides, specifically Boc-protected trilysine (112a), and polyamine compounds with n+1 primary amino groups, specifically N,N',N'-tetra(2-aminoethyl)-1,2-ethylenediamine with four primary amino groups. (CAS# 4097-90-9) (114b) undergoes an amide coupling reaction to form a multifunctional precursor compound (116b) comprising a Boc-protected trilysine residue and a multifunctional moiety having n primary amine groups, specifically an N,N',N'-tetra(2-aminoethyl)-1,2-ethylenediamine residue having three primary amine groups, wherein the multifunctional moiety is linked to an amine-protected peptide residue via an amide group.

[0061] As another example, now refer to Figure 1C Amine-protected peptides, specifically Boc-protected trilysine (112a), and polyamine compounds with n+1 primary amino groups, specifically ethylenediamine with two primary amino groups. (CAS# 107-15-3) (114c) undergoes an amide coupling reaction to form a monofunctional precursor compound (116c), the monofunctional precursor compound (116c) comprising a Boc-protected trilysine residue and a monofunctional moiety having n primary amino groups, specifically an ethylenediamine residue having one primary amino group, wherein the monofunctional moiety is linked to an amino-protected peptide residue via an amide group.

[0062] Apart from Figure 1A and 1B In addition to tris(2-aminoethyl)amine and N,N',N'-tetra(2-aminoethyl)-1,2-ethylenediamine, this disclosure allows for the combined use of various polyamine compounds, including 3-(2-aminoethyl)pentane-1,5-diamine. (CAS# 460078-00-6), (CAS# 146117-64-8), 1,3,5-tris-(2-aminoethyl)-[1,3,5]triazine-2,4,6-trione, (CAS# 43190-26-7), N,N,N'-Tris(2-aminoethyl)ethylenediamine, (CAS#31295-46-2) and adamantane-1,3,5,7-tetramine, CAS# (16004-77-6).

[0063] Other polyamine compounds can be found in Table 1 below. Table 1. In several embodiments, the polyamine compound does not contain a carboxylic acid group that requires a suitable protection strategy. For example, if the polyamine does contain a carboxylic acid group, it should be converted into, for example, a methyl ester.

[0064] In some embodiments, the multifunctional precursor compound comprises an amino-protected peptide residue and a multifunctional moiety comprising n hydroxyl groups linked to the amino-protected peptide residue via ester groups.

[0065] For example, now refer to Figure 1D Amine-protected peptides, specifically Boc-protected trilysine (112a) and polyols with n+1 hydroxyl groups, specifically triethanolamine with three hydroxyl groups. (CAS# 102-71-6)(114d) undergoes an ester coupling reaction to form a multifunctional precursor compound (116d), the multifunctional precursor compound (116d) comprising a Boc-protected trilysine residue and a multifunctional moiety having n hydroxyl groups, specifically a triethanolamine residue having two hydroxyl groups, wherein the multifunctional moiety is linked to an amino-protected peptide residue via an ester group.

[0066] Apart from Figure 1D In addition to triethanolamine, this disclosure allows for the use of a variety of polyol compounds, including 3-(2-hydroxyethyl)pentane-1,5-diol. (CAS# 53378-75-9), 1-[N,N-bis(2-hydroxyethyl)amino]-2-propanol, (CAS#6712-98-7), S-triazine-1,3,5-triethanol (CAS#4719-04-4), Tris(2-hydroxyethyl) isocyanurate, (CAS# 839-90-7), N,N,N',N'-Tetra(2-hydroxyethyl)ethylenediamine, (CAS# 140-07-8), Miglitol, (CAS# 72432-03-2) and bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane, (CAS# 6976-37-0), etc. Other polyol compounds may be selected from suitable members of the core polyols described below. In several embodiments, the polyol compound does not contain a carboxylic acid group that requires a suitable protection strategy. For example, in the case where the polyol contains a carboxylic acid group, it should be converted to, for example, a methyl ester.

[0067] In some embodiments, the multifunctional precursor compound comprises an amino-protected peptide residue and a multifunctional moiety comprising n carboxyl groups linked to the amino-protected peptide residue via amide groups.

[0068] For example, now refer to Figure 2 The multifunctional compound (116a) (which contains a Boc-protected trilysine residue and a multifunctional moiety having n primary amine or hydroxyl groups, specifically as follows) Figure 1A The tri(2-aminoethyl)amine residue with two primary amino groups formed in the process is combined with cyclic anhydrides, specifically succinic anhydride. (CAS# 108-30-5) (218a) undergoes a ring-opening reaction to form a multifunctional precursor compound (220a) comprising a Boc-protected trilysine residue (represented by TL) and a multifunctional moiety having n carboxyl groups, specifically a tri(2-aminoethyl)amine residue modified by two succinic anhydride residues providing two carboxyl groups, wherein the multifunctional moiety is linked to an amino-protected peptide residue via an amide group.

[0069] As another example, continue to refer to Figure 2 The multifunctional compound (116a) (which contains a Boc-protected trilysine residue and a multifunctional moiety having two amino or hydroxyl groups, specifically a tri(2-aminoethyl)amine residue) and cyclic anhydrides, specifically glutaric anhydride, (CAS# 108-55-4) (218b) undergoes a ring-opening reaction to form a multifunctional precursor compound (220b) comprising a Boc-protected trilysine residue (represented by TL) and a multifunctional moiety having two carboxyl groups, specifically a tri(2-aminoethyl)amine residue modified with two glutaric anhydride residues, wherein the multifunctional moiety is linked to an amino-protected peptide residue via an amide group.

[0070] As another example, further refer to Figure 2 The multifunctional compound (116a) (which contains a Boc-protected trilysine residue and a multifunctional moiety having two amino or hydroxyl groups, specifically a tri(2-aminoethyl)amine residue) and cyclic anhydrides, specifically diethylene glycol anhydrides, (CAS# 4480-83-5) (218c) undergoes a ring-opening reaction to form a multifunctional precursor compound (220c) comprising a Boc-protected trilysine residue (represented by TL) and a multifunctional moiety having two carboxyl groups, specifically a tri(2-aminoethyl)amine residue modified with two diethylene glycol anhydride residues, wherein the multifunctional moiety is linked to an amino-protected peptide residue via an amide group.

[0071] Other cyclic anhydrides are listed in Table 2. These cyclic anhydrides can be iodinated to provide higher radiation impermeability. *Iodide compounds Table 2. As another example and reference Figure 2A ,like Figure 1C A monofunctional compound (116c) formed in the process (which comprises a Boc-protected trilysine residue and a monofunctional moiety having n primary amino groups, specifically an ethylenediamine residue having one primary amino group linked to an amino-protected peptide residue via an amide group) is coupled with a polycarboxylic acid compound, specifically ethylenediaminetetraacetic acid (228) in the presence of a carbodiimide coupling agent (e.g., EDC), to form a polyfunctional precursor compound (230), said polyfunctional precursor compound (230) comprising a Boc-protected trilysine residue and a polyfunctional moiety having n carboxyl groups, specifically an ethylenediamine residue modified with an ethylenediaminetetraacetic acid residue having three carboxyl groups, wherein said polyfunctional moiety is linked to an amino-protected peptide residue via an amide group.

[0072] As another example, refer to Figure 3 Amine-protected peptides, specifically carboxybenzyl-protected trilysine (Cbz-protected trilysine) (312), and protected polycarboxylated amine compounds comprising at least one amino group and n protected carboxyl groups, specifically 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid 1,4,7-tris(1,1-dimethylethyl) ester having one secondary amino group and three Boc-protected carboxyl groups, (CAS# 122555-91-3)(314) undergoes an amide coupling reaction to form a protected intermediate compound (316). The Boc of the protected intermediate compound (316) is then deprotected with a base to form a multifunctional precursor compound (320) comprising a Cbz-protected trilysine residue and a multifunctional moiety having n carboxyl groups, specifically three carboxyl groups, wherein the multifunctional moiety is linked to an amino-protected peptide residue via an amide group.

[0073] Polyamines with carboxylic acid protection (which can then be removed) include: β-alanine N-[3-(1,1-dimethylethoxy)-3-oxopropyl]-N-(2-hydroxyethyl)-1,1-dimethylethyl ester (CAS 831216-48-9), L-glutamic acid di-tert-butyl ester (CAS 16874-06-9), and pimecrolic acid 4-amino-4-[3-(1,1-dimethylethoxy)-3-oxopropyl]-1,7-bis(1,1-dimethylethyl) ester (CAS 136586-99-7).

[0074] In other embodiments, the multifunctional precursor compound comprises an amino-protected peptide residue and a multifunctional moiety linked to the amino-protected peptide residue via an ester group, the multifunctional moiety further comprising n carboxyl groups.

[0075] As an example, multifunctional compounds (e.g.) Figure 1D Compound (116d), comprising a Boc-protected trilysine residue and a multifunctional moiety having n hydroxyl groups, specifically a triethanolamine residue having three hydroxyl groups, can undergo a ring-opening reaction with a cyclic anhydride (e.g., succinic anhydride, glutaric anhydride, or diethylene glycol anhydride) to form a multifunctional precursor compound comprising a Boc-protected trilysine residue and a multifunctional moiety having n carboxyl groups, specifically a triethanolamine residue modified with two glutaric anhydride residues, two succinic anhydride residues, or two diethylene glycol anhydride residues providing two carboxyl groups, wherein the multifunctional moiety is linked to an amino-protected peptide residue via an ester group.

[0076] As another example, amino-protected peptides (e.g., Figure 3 The carboxybenzyl-protected trilysine shown (e.g., Cbz-protected trilysine) (312) can undergo ester coupling with a carboxy-protected hydroxycarboxylic acid compound containing one hydroxyl group and n protected carboxyl groups (e.g., Boc-protected citric acid having one hydroxyl group and three Boc-protected carboxyl groups) to form a protected intermediate compound. Then, similar to Figure 3 The Boc entity of the protected intermediate compound is deprotected with a base to form a multifunctional precursor compound comprising a Cbz-protected trilysine residue and a multifunctional moiety having n carboxyl groups, specifically three carboxyl groups, wherein the multifunctional moiety is linked to an amino-protected peptide residue via an ester group.

[0077] Polyols containing a protected carboxylic acid (which can subsequently be removed) include: 4,10,14-trioxa-7-azahexadecanoic acid 7-(2-hydroxyethyl)-15,15-dimethyl-13-oxo-1,1-dimethylethyl ester (CAS 1415800-34-8) and glycine N-[2-(1,1-dimethylethoxy)-2-oxoethyl]-N-(2-hydroxyethyl)-1,1-dimethylethyl ester (CAS 146432-41-9).

[0078] As previously described, after providing a multifunctional precursor compound (such as any of the compounds described above), the multifunctional precursor compound can undergo an ester coupling reaction or an amide coupling reaction with an amino-functional iodide compound, a carboxyl-functional iodide compound, or a hydroxyl-functional iodide compound to form a protected compound, wherein n iodinated moieties are linked to amino-protected peptide residues, wherein, as previously described, n is an integer of 2 or greater (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater).

[0079] For example, (a) a multifunctional precursor compound containing a peptide residue with protected amino groups and a multifunctional moiety containing n carboxyl groups can undergo an amide coupling reaction with an iodinated compound containing an iodinated moiety containing n amino functional iodinated groups to form a protected compound, wherein the n iodinated moiety is linked to the peptide residue with protected amino groups via amide bonds; (b) a multifunctional precursor compound containing a peptide residue with protected amino groups and a multifunctional moiety containing n carboxyl groups can undergo an ester coupling reaction with an iodinated compound containing an iodinated moiety containing n hydroxy functional iodinated groups to form a protected compound, wherein the n iodinated moiety is linked to the peptide residue with protected amino groups via ester bonds. (c) A multifunctional precursor compound containing a peptide residue with protected amino groups and a multifunctional moiety containing n primary amino groups can undergo an amide coupling reaction with a multifunctional iodide compound containing an iodide moiety containing n carboxyl functional groups to form a protected compound, wherein the n iodide moiety is linked to the peptide residue with protected amino groups via amide bonds; or (d) A multifunctional precursor compound containing a peptide residue with protected amino groups and a multifunctional moiety containing n hydroxyl groups can undergo an ester coupling reaction with a multifunctional iodide compound containing an iodide moiety containing n carboxyl functional groups to form a protected compound, wherein the n iodide moiety is linked to the peptide residue with protected amino groups via ester bonds.

[0080] After forming a protected compound in which n iodide moieties are attached to a peptide residue with a protected amino group, the protecting groups can be removed. For example, Boc-protected groups can be protected by exposure to an acid (e.g., hydrochloric acid or trifluoroacetic acid), and Cbz-protected groups can be protected by using a reducing agent (e.g., a methanol solution of NaBH4 and a catalytic amount of Pd-C), while taking care not to hydrogenate the iodide ring, etc.

[0081] As a specific example, refer to Figure 4 Multifunctional precursor compounds (specifically, those containing a Boc-protected trilysine residue and a multifunctional moiety with n carboxyl groups, specifically two carboxyl groups, such as...) Figure 2 The multifunctional precursor compound (220a) described herein and the n-molecule amino-functional iodinated compound containing the iodination moiety, specifically two molecules of tetraiodothyronine methyl ester (also known as thyroxine methyl ester). (CAS# 32180-11-3) (422) An amide coupling reaction is performed, wherein the carboxyl group of the multifunctional precursor compound (220a) reacts with the amino group of thyroxine methyl ester (422) to form an amide bond. The resulting compound is a protected compound in which n iodinated moieties are linked to amino-protected peptide residues, specifically a protected iodinated peptide compound (424) comprising two thyroxine methyl ester residues and a Boc-protected trilysine residue. In this specific embodiment, the two thyroxine methyl ester residues are linked to the Boc-protected trilysine residue via a bond comprising a polyamine compound residue, specifically a tri(2-aminoethyl)amine residue. The Boc protection is then removed to obtain an iodinated peptide compound (426) comprising two thyroxine methyl ester residues and one trilysine residue. In this specific embodiment, the two thyroxine methyl ester residues are linked to the trilysine residue via a bond comprising a polyamine compound residue, specifically a tri(2-aminoethyl)amine residue.

[0082] Other examples of amino-functional iodinated compounds (several of which are iodinated amino acid esters) include monoiodophenylalanine methyl ester. (CAS# 158686-46-5), Monoiodotyrosine ethyl ester, (CAS# 10051-55-5), diiodotyrosine methyl ester, (CAS# 21959-36-4), Diiodotyrosine ethyl ester, (CAS# 74051-47-1), diiodothyronine methyl ester, (CAS# 203585-45-9), Triiodothyronine methyl ester (also known as T3 methyl ester). (CAS# 3005-96-7), 5-amino-N,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodophthalamide (also known as iohexol-related compounds J). (CAS # 76801-93-9), acetal-protected 5-amino-N,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodophthalamide (acetal-protected iohexol-related compound J). 5-Amino-N,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-isophthalamide (also known as iohexol EP impurity F). (CAS# 1215856-35-1), acetal-protected iohexol EP impurity F, dimethyl 5-amino-2,4,6-triiodo-1,3-phthalate, (CAS#154921-11-6), 2,4,6-Triiodoaniline, (CAS# 24154-37-8) and triiodobenzylamine, ,wait.

[0083] In another example, an amino-functionalized iodide compound can be formed by reacting a polycarboxylated amine compound having a protected amino group and m carboxyl groups (where m is an integer of 2 or greater (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater)) with an amino-functionalized iodide compound containing an iodide moiety. Deprotecting the amino group of the resulting product yields an amino-functionalized iodide compound containing an amino group and m iodide moieties, more specifically, an amino-functionalized iodide compound containing polycarboxylated amine compound residues and m amino-functionalized iodide compound residues. Examples of polycarboxylated amine compounds include glutamic acid (CAS# 56-86-0), 4-amino-4-(2-carboxyethyl)pimelic acid (CAS# 176738-98-0), N-(5-amino-1-carboxypentyl)iminodiacetic acid (CAS# 113231-05-3), glutamyl-glutamic acid (CAS# 3929-61-1), triglutamic acid (CAS# 23684-48-2), or N2,N2-bis(carboxymethyl)lysine (CAS# 129179-17-5).

[0084] exist Figure 5 In the schematic embodiment shown, an amino-protected polycarboxylated amine compound, specifically tert-butoxycarbonyl-protected glutamic acid (Boc-protected glutamic acid) having two carboxylic acid ester groups and one protected amine group (512), undergoes an amide coupling reaction with two molecules of an amino-functionalized iodinated compound containing an iodinated moiety, specifically two molecules of thyroxine methyl ester (514), in the presence of a suitable coupling agent (e.g., 1-ethyl-3-(3-dimethylpropyl)carbodiimide (EDC), to form an amino-protected compound. The amine group is then deprotected with an acid to obtain an amino-functionalized iodinated compound (516) containing two thyroxine methyl ester residues and one glutamic acid residue.

[0085] Then, the amino group of an amino-functionalized iodinated compound containing an amino group and m iodinated moieties can undergo an amide coupling reaction with the carboxyl group of an amino-protected peptide containing two or more amino acid residues with primary amine side groups to form an iodinated, amino-protected peptide compound containing m iodinated moieties, said m iodinated moieties being covalently linked to residues of the amino-protected peptide via residues of a polycarboxylated amine compound. Deprotection of the amino group with an acid yields an iodinated peptide compound containing m iodinated moieties, said m iodinated moieties being covalently linked to residues of a peptide containing two or more amino acid residues with primary amine side groups via residues of a polycarboxylated amine compound.

[0086] For example, turning again Figure 5 The schematic embodiment shown in the diagram involves an amino-functionalized iodinated compound (516) undergoing an amide coupling reaction with an amino-protected trilysine (TL) in the presence of a suitable coupling agent (e.g., EDC) to form a compound comprising two thyroxine methyl ester residues linked to the amino-protected trilysine residues via glutamate residues. Deprotection of the amino group (e.g., by hydrazine ions) yields an iodinated peptide compound (518) comprising two thyroxine methyl ester residues linked to the trilysine residues via glutamate residues.

[0087] Alternatively, the amino group of an amino-functionalized iodide compound containing an amino group and m iodide moieties can undergo an amide coupling reaction with an amino-protected polyfunctional precursor compound as described above, the amino-protected polyfunctional precursor compound comprising (i) an amino-protected peptide residue containing two or more amino acid residues having primary amine side groups, and (ii) a polyfunctional moieties linked to the amino-protected peptide residues via amide or ester groups, the polyfunctional moieties containing n carboxyl groups, where n is an integer of 2 or greater. The amine protection is then removed to yield an iodinated peptide compound containing n×m iodide moieties linked to the peptide residues.

[0088] More specifically, the amino group of an amino-functionalized iodide compound comprising a polycarboxylated amine compound residue and (a) m amino-functionalized iodide compound residues or (b) m hydroxyl-functionalized iodide compound residues can undergo an amide coupling reaction with the carboxyl group of an amino-protected polyfunctional precursor compound, said amino-protected polyfunctional precursor compound comprising (i) an amino-protected peptide residue comprising two or more amino acid residues having primary amine side groups, and (ii) a polyfunctional moiety linked to the amino-protected peptide residue via an amide or ester group, said polyfunctional moiety comprising n carboxyl groups, where n is an integer of 2 or greater. The amino protection is then removed to obtain an iodinated peptide compound comprising n×m iodinated moieties covalently linked to the peptide residue, particularly, the n×m iodinated moieties being linked to the peptide residue via n polycarboxylated amine compound residues and residues of the polyfunctional moieties.

[0089] In a specific instance, multifunctional precursor compounds (e.g., such as...) Figure 2 The multifunctional precursor compounds (220a), (220b), or (220c) described herein, comprising a Boc-protected trilysine residue and a multifunctional moiety having n carboxyl groups, specifically two carboxyl groups, can undergo an amide coupling reaction with n molecules of an amino-functionalized iodide compound (516) containing m thyroxine methyl ester residues, specifically two thyroxine methyl ester residues and one glutamate residue, wherein the amino group of the amino-functionalized iodide compound (516) reacts with each carboxyl group of the multifunctional precursor compound (220a), (220b), or (220c). After coupling, the Boc protection is then removed, providing an iodinated peptide compound comprising n × m iodinated moieties, specifically providing an iodinated peptide compound comprising four thyroxine methyl ester residues and one trilysine residue. In this specific embodiment, the four thyroxine methyl ester residues are linked to the trilysine residues via a bond comprising a polyamine compound residue, specifically a tri(2-aminoethyl)amine residue, two anhydride residues, and two glutamate residues.

[0090] The carboxyl-functionalized iodinated compounds used in this disclosure include triiodobenzoic acid. (CAS# 88-82-4), diatrizoic acid. (CAS# 117-96-4), N-acetyl-3,5-diiodo-L-tyrosine, (CAS# 1027-28-7), N-acetyl-3-diiodo-L-tyrosine, (CAS# 1023-47-8) and N-acetyl-thyroxine, (CAS# 26041-51-0), etc.

[0091] Other carboxyl-functionalized iodinated compounds can be found in Table 3. Table 3. In some embodiments, functional groups in amino-functionalized, carboxyl-functionalized, or hydroxyl-functionalized iodides that are not desired to participate in amide or ester coupling reactions can be protected. As an example, see [reference]. Figure 6A The adjacent hydroxyl groups of iodixanol (615) can be protected with 2,2-dimethoxypropane (617) at room temperature in the presence of p-toluenesulfonic acid (PTSA) to obtain acetal-protected hydroxy-functionalized iodinated compounds, specifically acetal-protected iodixanol (622).

[0092] Acetal-protected hydroxyl-functional iodinated compounds can then be coupled to multifunctional precursor compounds via ester coupling reactions, the multifunctional precursor compounds comprising amino-protected peptide residues and multifunctional moieties containing two or more carboxyl groups, for example... Figure 2 Multifunctional precursor compounds (220a), (220b), (220c), or Figure 3 Multifunctional precursors (320). For example, see reference. Figure 6B , Figure 6A Iodixanol (622), produced in this manner, can be coupled to [a specific product / organization] via ester coupling in the presence of a suitable coupling agent (e.g., N,N'-dicyclohexylcarbodiimide (DCC)). Figure 2 The multifunctional precursor compound (220a) produced in the process is used to form a protected intermediate (624), which is then deprotected (e.g., by hydration with hydrogen ions (H3O)). + This yields an iodinated peptide compound (626) comprising two iodixanol residues and one trilysine residue. In this specific embodiment, the two iodixanol residues are linked to the trilysine residue via a bond comprising a tri(2-aminoethyl)amine residue.

[0093] In the case of iodixanol, it is noted that an alternative is possible, wherein the central hydroxyl group of iodixanol is reacted, for example, with a diamine compound to obtain an amino-functionalized iodide compound having a primary amino group linked to an iodixanol residue by an amide-based bond. This amino-functionalized iodide compound can participate in an amide coupling reaction with a polyfunctional precursor compound comprising an amino-protected peptide residue and a polyfunctional moiety comprising two or more carboxyl groups.

[0094] Combination Figure 7A Another example is described, in which the amino functional groups of an iodinated amino acid compound are protected. Figure 7A In this context, multifunctional precursor compounds, specifically, are... Figure 1AThe description includes a Boc-protected trilysine residue and a multifunctional moiety having n primary amino groups, specifically a multifunctional precursor compound (116a) with two primary amino groups and a tri(2-aminoethyl)amine residue, and n molecules of iodinated amino acid compounds with protected amino groups, specifically two molecules of Boc-protected thyroxine. (CAS# 88404-22-2) (712a) undergoes an amide coupling reaction in which the amino group of the multifunctional precursor compound (116a) reacts with the carboxyl group of a Boc-protected thyroxine (712a) to form an amide bond. The amide coupling reaction shown is carried out in the presence of a carbodiimide coupling agent (e.g., 1-ethyl-3-(3-dimethyl-propyl)carbodiimide (EDC) or N,N'-dicyclohexylcarbodiimide (DCC)) using a catalytic amount of 4-dimethylaminopyridine (DMAP). The resulting compound is a protected compound in which the iodinated moiety of n amino groups is linked to an amino-protected peptide residue, specifically a protected iodinated peptide compound (not shown) comprising two Boc-protected thyroxine residues and a Boc-protected trilysine residue. In this specific embodiment, the two Boc-protected thyroxine residues are linked to the Boc-protected trilysine residue by a bond comprising a polyamine compound residue, specifically a tri(2-aminoethyl)amine residue. The Boc-protection was then removed under acidic conditions to obtain an iodinated peptide compound (714a) containing two thyroxine residues and one trilysine residue. In this specific embodiment, the two thyroxine residues are linked to the trilysine residue via a bond containing a polyamine compound residue, specifically a tri(2-aminoethyl)amine residue.

[0095] Note that this method increases the number of primary amine groups from four to six, which improves water solubility and increases the number of primary amine groups available for future crosslinking. More generally, the reaction between a primary amine-protected iodide compound and a multifunctional precursor compound containing peptide residues with protected amine groups will produce an iodinated peptide compound having primary amine groups in addition to the primary amine groups associated with the peptide residues.

[0096] Now we will combine Figures 7B-7E The specific implementation scheme is described, wherein a carboxyl-functional iodide compound reacts with a multifunctional precursor compound containing a peptide residue with a protected amino group and a multifunctional moiety containing n primary amino groups.

[0097] Now refer to Figure 7B Multifunctional precursor compounds, specifically as follows Figure 1A The compound described herein comprises a Boc-protected trilysine residue and a multifunctional moiety having n primary amino groups, specifically a multifunctional precursor compound (116a) of a tri(2-aminoethyl)amine residue having two primary amino groups and an iodinated amino acid compound having n amino groups protected, specifically two molecules of diatrizoic acid. (712b) An amide coupling reaction is performed in which the amino group of the multifunctional precursor compound (116a) reacts with the carboxyl group of diatrizoic acid (712b) to form an amide bond. The amide coupling reaction shown is carried out in the presence of a carbodiimide coupling agent, specifically EDC. The resulting compound is a protected compound in which n iodinated moieties are linked to a peptide residue with a protected amino group, specifically a protected iodinated peptide compound (not shown) containing two diatrizoic acid residues and a Boc-protected trilysine residue. The Boc-protection is then removed under acidic conditions to give an iodinated peptide compound (714b) containing a diatrizoic acid residue and a trilysine residue. In this specific embodiment, the two diatrizoic acid residues are linked to the trilysine residue via a bond containing a polyamine compound residue, specifically a tri(2-aminoethyl)amine residue.

[0098] Now refer to Figure 7C Multifunctional precursor compounds, specifically as follows Figure 1A The description includes a Boc-protected trilysine residue and a multifunctional moiety with n primary amino groups, specifically a multifunctional precursor compound (116a) with two primary amino groups and a tri(2-aminoethyl)amine residue, and n molecules of iodinated amino acid compounds with protected amino groups, specifically two molecules of tetraiodothyronine. (712c) An amide coupling reaction is performed in which the amino group of the multifunctional precursor compound (116a) reacts with the carboxyl group of tetraiodothyronine (712c) to form an amide bond. The amide coupling reaction shown is carried out in the presence of a carbodiimide coupling agent, specifically EDC. The resulting compound is a protected compound in which n iodinated moieties are linked to a peptide residue with a protected amino group, specifically a protected iodinated peptide compound (not shown) containing two tetraiodothyronine residues and a Boc-protected trilysine residue. The Boc-protection is then removed under acidic conditions to give an iodinated peptide compound (714c) containing a tetraiodothyronine residue and a trilysine residue. In this specific embodiment, the two tetraiodothyronine residues are linked to the trilysine residue via a bond containing a polyamine compound residue, specifically a tri(2-aminoethyl)amine residue.

[0099] Now refer to Figure 7D Multifunctional precursor compounds, specifically as follows Figure 1A The compound described herein comprises a Boc-protected trilysine residue and a multifunctional moiety having n primary amino groups, specifically a multifunctional precursor compound (116a) having two primary amino groups and a tri(2-aminoethyl)amine residue, and n molecules of an iodinated amino acid compound with protected amino groups, specifically two molecules of iodixanol. (712d) An amide coupling reaction is performed in which the amino group of the polyfunctional precursor compound (116a) reacts with the carboxyl group of iodixanol (712d) to form an amide bond. The amide coupling reaction shown is carried out in the presence of a carbodiimide coupling agent, specifically EDC. The resulting compound is a protected compound in which n iodinated moieties are linked to a peptide residue with a protected amino group, specifically a protected iodinated peptide compound (not shown) containing two iodixanol residues and a Boc-protected trilysine residue. The Boc-protection is then removed under acidic conditions to give an iodinated peptide compound (714d) containing an iodixanol residue and a trilysine residue. In this specific embodiment, the two iodixanol residues are linked to the trilysine residue via a bond containing a polyamine compound residue, specifically a tri(2-aminoethyl)amine residue.

[0100] Now refer to Figure 7E Multifunctional precursor compounds, specifically as follows Figure 1A The compound described herein comprises a Boc-protected trilysine residue and a multifunctional moiety having n primary amino groups, specifically a multifunctional precursor compound (116a) having two primary amino groups and a tri(2-aminoethyl)amine residue, and n iodinated amino acid compounds with protected amino groups, specifically two molecules of N-acetyl-thyroxine. (712e) An amide coupling reaction is performed, wherein the amino group of the polyfunctional precursor compound (116a) reacts with the carboxyl group of N-acetyl-thyroxine (712e) to form an amide bond. The amide coupling reaction shown is carried out in the presence of a carbodiimide coupling agent, specifically EDC. The resulting compound is a protected compound in which n iodinated moieties are linked to amino-protected peptide residues, specifically a protected iodinated peptide compound (not shown) containing two N-acetyl-thyroxine residues and a Boc-protected trilysine residue. The Boc protection is then removed under acidic conditions to give an iodinated peptide compound (714e) containing an N-acetyl-thyroxine residue and a trilysine residue. In this specific embodiment, the two N-acetyl-thyroxine residues are linked to the trilysine residue via a bond containing a polyamine compound residue, specifically a tri(2-aminoethyl)amine residue.

[0101] Although not shown, in embodiments in which the carboxyl-functionalized iodide compound has more than one carboxylic acid functional group, a significant excess of iodide can be used during the coupling reaction with the polyfunctional precursor compound to achieve the attachment of only a single carboxyl-functionalized iodide compound to each polyfunctional precursor compound. The product can be further purified by chromatography or, possibly, stepwise crystallization.

[0102] In other aspects of this disclosure, the crosslinked network is formed by reacting (a) a radiopaque peptide compound formed according to this disclosure and (b) a reactive polymer, the radiopaque peptide compound comprising two or more primary amine side groups, and the reactive polymer comprising a portion that reacts with the primary amine groups of the radiopaque peptide.

[0103] In some embodiments, the crosslinked network is a hydrogel. As used herein, a "hydrogel" (also known as a "crosslinked hydrogel") is a crosslinked polymer that, when placed in water, can absorb water but does not dissolve.

[0104] The cross-linked hydrogels of this disclosure can be formed in vivo (e.g., using the delivery device described below), or such cross-linked hydrogels can be formed in vitro and subsequently applied to a subject. The cross-linked hydrogels of this disclosure can be used in a variety of biomedical applications, including implants, medical devices, and pharmaceutical compositions.

[0105] In several embodiments, the cross-linked hydrogel is visible under fluorescence fluoroscopy. The cross-linked hydrogel may have a radiation opacity greater than 100 Hounsfield units (HU), advantageously any value in the range of 100 HU to 250 HU to 500 HU to 750 HU to 1000 HU to 2000 HU or higher (in other words, within the range between any two of the aforementioned values).

[0106] The reactive polymer used in this disclosure includes a reactive multi-arm polymer comprising a plurality of polymer arms connected to a core region, at least a portion of which comprises a hydrophilic polymer segment. One end of the hydrophilic polymer segment is covalently connected to the core region, and the other end of the hydrophilic polymer segment is covalently connected to the reactive portion.

[0107] In some embodiments, at least a portion of the polymer arm comprises a hydrophilic polymer segment having a first end and a second end, a cyclic anhydride residue having a first end and a second end, and a reactive portion covalently attached to the second end of the cyclic anhydride residue, wherein the first end of the hydrophilic polymer segment is covalently attached to a core region, and the first end of the cyclic anhydride residue is covalently attached to the second end of the hydrophilic polymer segment.

[0108] The reactive polymers according to this disclosure include polymers having 3 to 100 arms, for example, any value in the range of 3 to 4 to 5 to 6 to 7 to 8 to 10 to 12 to 15 to 20 to 25 to 50 to 75 to 100 arms (in other words, having a number of arms ranging from any two of the aforementioned values).

[0109] The reactive part includes the part containing electrophilic groups.

[0110] The electrophilic group can be selected from, for example, cyclic imide ester groups (e.g., succinimide ester groups, Maleimide ester group, glutarimide ester group, diethylene glycol imide ester group, phthalimide ester group and bicyclic [2.2.1]hept-5-ene-2,3-dicarboxylic acid imide ester group, ), imidazole ester group, imidazole carboxylic ester group and benzotriazole ester group, etc.

[0111] Electrophilic groups can be attached to hydrophilic polymer segments via any suitable linker, which can be selected from, for example, linkers containing alkyl groups, linkers containing ether groups, linkers containing ester groups, linkers containing amide groups, linkers containing amine groups, linkers containing carbonate groups, or linkers containing combinations of two or more of the aforementioned groups. In some embodiments, the linker comprises a hydrolyzable ester group.

[0112] The hydrophilic polymer segments used in the polymer arm can be selected from a variety of synthetic, natural, or synthetic-natural hybrid hydrophilic polymer segments. Examples of hydrophilic polymer segments include those formed from one or more hydrophilic monomers selected from: C1-C6-epoxides (e.g., ethylene oxide, propylene oxide, tetramethylene oxide, etc.), polar aprotic vinyl monomers (e.g., N-vinylpyrrolidone, acrylamide, N-methacrylamide, dimethacrylamide, N-vinylimidazolium, 4-vinylimidazolium, sodium 4-vinylbenzenesulfonate, etc.), dioxanehexanone, ester monomers (e.g., glycolide, lactide, β-propiolactone, β-butyrolactone, γ-butyrolactone, γ-pentylenesulfonate, etc.). lactones, δ-valerolactone, ε-caprolactone, etc.), oxazoline monomers (e.g., oxazoline and 2-alkyl-2-oxazoline, such as 2-(C1-C6 alkyl)-2-oxazoline, including various isomers such as 2-methyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-n-propyl-2-oxazoline, 2-isopropyl-2-oxazoline, 2-n-butyl-2-oxazoline, 2-isobutyl-2-oxazoline, 2-hexyl-2-oxazoline, etc.), 2-phenyl-2-oxazoline, N-isopropylacrylamide, amino acids and sugars.

[0113] The hydrophilic polymer segments can be selected from, for example, the following polymer segments: polyether segments, including poly(C1-C6-epoxyalkylene) segments, such as poly(ethylene oxide) (PEO) (also known as polyethylene glycol or PEG) segments, poly(propylene oxide) segments, and poly(ethylene oxide-co-propylene oxide) segments; polymer segments formed from one or more polar aprotic vinyl monomers, including poly(N-vinylpyrrolidone) segments, poly(acrylamide) segments, poly(N-methacrylamide) segments, poly(dimethacrylamide) segments, poly(N-vinylimidazolium) segments, poly(4-vinylimidazolium) segments, and poly(sodium 4-vinylbenzenesulfonate) segments; polydioxane segments; polyester segments, including polyglycolic acid segments, polylactic acid segments, poly(lactic acid-co-glycolic acid) segments, and poly(β-propanediol) segments. (Lactone) segments, poly(β-butyrolactone) segments, poly(γ-butyrolactone) segments, poly(γ-valerolactone) segments, poly(δ-valerolactone) segments and poly(ε-caprolactone) segments; polyoxazoline segments, including poly(2-C1-C6-alkyl-2-oxazoline) segments, such as poly(2-methyl-2-oxazoline) segments, poly(2-ethyl-2-oxazoline) segments, poly(2-propyl-2-oxazoline) segments, poly(2-isopropyl-2-oxazoline) segments and poly(2-n-butyl-2-oxazoline) segments, poly(2-phenyl-2-oxazoline) segments, poly(N-isopropylacrylamide) segments, polypeptide segments and polysaccharide segments. Polysaccharide segments include those containing one or more uronic acid substances, such as galacturonic acid, glucuronic acid, and / or iduronic acid. Specific examples of polysaccharide segments include alginic acid, hyaluronic acid, pectin, agar, carrageenan, gellan gum, gum arabic, guar gum, xanthan gum, and carboxymethyl cellulose fractions.

[0114] Polymer segments used in the multi-arm polymers of this disclosure typically contain 2 to 1000 or more monomer units, for example, ranging from 2 to 3 to 4 to 6 to 8 to 10 to 15 to 20 to 25 to 50 to 1000 monomer units (in other words, ranging from any two of the aforementioned values).

[0115] In some embodiments, the core region includes residues of a polyol containing three or more hydroxyl groups for forming the polymer arm. In some advantageous embodiments, the core region includes residues of a polyol containing 3 to 100 hydroxyl groups (e.g., 2 to 3 to 4 to 5 to 6 to 8 to 10 to 15 to 20 to 25 to 50 to 75 to 100 hydroxyl groups).

[0116] Exemplary polyols may be selected from, for example, straight-chain, branched, and cyclic aliphatic polyols (including straight-chain, branched, and cyclic polyhydroxy alkanes), straight-chain, branched, and cyclic polyhydroxy ethers (including polyhydroxy polyethers), straight-chain, branched, and cyclic polyhydroxy alkyl ethers (including polyhydroxy alkyl polyethers), straight-chain, branched, and cyclic sugars and sugar alcohols (e.g., glycerol, mannitol, sorbitol, inositol, xylitol, styraxyl alcohol, threitol, arabinol, erythritol, pentaerythritol, dipentaerythritol, tripentaerythritol, arabinol, hexaglycerol, galactitol, fucose, ribose, arabinose, xylose, etc.). Polyols include threose, rhamnose, galactose, glucose, fructose, sorbitol, mannose, pyranose, atroose, tarose, tagatose, pyranoside, sucrose, lactose, and maltose; polymers of linear, branched, and cyclic sugars and sugar alcohols (defined herein as two or more units) (including oligomers of linear, branched, and cyclic sugars and sugar alcohols (defined herein as two to ten units, including dimers, trimers, tetramers, pentamers, hexamers, heptomers, octamers, nonamerms, and decamers); sugars and sugar alcohols include the aforementioned sugars and sugar alcohols, starch, amylose, dextrin, cyclodextrin, and polyhydroxy crown ethers and polyhydroxyalkyl crown ethers). Exemplary polyols also include aromatic polyols (including 1,1,1-tris(4'-hydroxyphenyl)alkanes, such as 1,1,1-tris(4-hydroxyphenyl)ethane and 2,6-bis(hydroxyalkyl)cresol, etc.).

[0117] Exemplary polyols also include polyhydroxylated polymers. For example, in some embodiments, the core region comprises polyhydroxylated polymer residues, such as polyvinyl alcohol residues, polyallyl alcohol, hydroxyethyl polyacrylate residues, or hydroxyethyl polymethacrylate residues. The length of such polyhydroxylated polymer residues can range, for example, from 3 to 100 monomer units.

[0118] In other embodiments, the core region comprises a silsesquioxane, which is a compound having a cage-like silicon-oxygen core composed of Si-O-Si bonds and tetrahedral Si vertices. -H groups or external organic groups may be covalently attached to the cage-like silicon-oxygen core. In this disclosure, the organic groups comprise polymer arms. Silsesquioxanes used in this disclosure include silsesquioxanes with 6 silicon vertices, silsesquioxanes with 8 silicon vertices, silsesquioxanes with 10 silicon vertices, and silsesquioxanes with 12 silicon vertices, which can be used as the core of 6-arm, 8-arm, 10-arm, and 12-arm polymers, respectively. The silicon-oxygen core is sometimes referred to as a T6, T8, T10, and T12 cage-like silicon-oxygen core (where T = the number of tetrahedral Si vertices). In all cases, each Si atom is bonded to three O atoms, while the oxygen atom is bonded to other Si atoms. Silsesquioxanes include those with the chemical formula [RSiO]. 3 / 2 ] nCompounds, where n is an integer of at least 6, typically 6, 8, 10, or 12 (thus having T6, T8, T... respectively). 10 or T 12 The T8 cage-like siloxane core (R8) is selected from a series of organic functional groups, such as alkyl, aryl, alkoxy, and polymer arms. The T8 cage-like siloxane core has been extensively studied and has the formula [RSiO8]. 3 / 2 ]8, or equivalently R8Si8O 12 The structure is as follows: In this disclosure, the R group includes the polymer arm described herein.

[0119] The reactive multi-arm polymers according to the present invention can be formed from hydroxyl-terminated precursor multi-arm polymers having arms comprising one or more hydroxyl terminal groups. In some of these embodiments, the hydroxyl-terminated precursor multi-arm hydrophilic polymer can react with cyclic anhydrides to form acid-terminated precursor polymers. For example, the terminal hydroxyl groups of the hydrophilic segments can react with cyclic anhydrides (e.g., glutaric anhydride compounds, succinic anhydride compounds, malonic anhydride compounds, adipic anhydride compounds, diethylene glycol anhydride compounds, etc.) to form acid-terminated segments, such as glutaric acid-terminated segments, succinic acid-terminated segments, malonic acid-terminated segments, adipic acid-terminated segments, diethylene glycol-terminated segments, etc.

[0120] The aforementioned cyclic anhydrides can react with hydroxyl-terminated multi-arm hydrophilic precursor polymers under alkaline conditions to form carboxylic acid-terminated precursor polymers containing carboxylic acid end groups, which are attached to the hydrophilic polymer segments via hydrolyzable ester groups. The reactive portion can then be attached to the carboxylic acid-terminated precursor polymer.

[0121] In some embodiments, the electrophilic portion can be attached to a carboxylic acid-terminated precursor polymer. For example, N-hydroxycyclic imide compounds (e.g., N-hydroxysuccinimide, N-hydroxymaleimide, N-hydroxyglutarimide, N-hydroxyphthalimide, or N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide, also known as N-hydroxybicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid imide (HONB), etc.) can be coupled in a suitable coupling agent (e.g., a carbodiimide coupling agent, such as N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-di-di-carbodiimide)). In the presence of methylpropyl carbodiimide (EDC), N-hydroxybenzotriazole (HOBt), BOP reagent, and / or other coupling agents, a carboxylic acid-terminated precursor polymer reacts to form a reactive cyclic imide ester (e.g., succinimide ester group, maleimide ester group, glutarimide ester group, phthalimide ester group, diethylene glycol imide ester group, bicyclic [2.2.1]hept-5-ene-2,3-dicarboxylic acid imide ester group, etc.), which is attached to a hydrophilic polymer segment via a hydrolyzable ester group. In this way, many reactive diester groups can be formed.

[0122] For example, in the specific case of N-hydroxysuccinimide as an N-hydroxycyclic imide compound, exemplary reactive end groups include succinimide malonate group, succinimide glutarate group, succinimide succinate group, succinimide adipate group, and succinimide diethylene glycol ester group, etc. In the specific case of HONB as an N-hydroxy cyclic imide compound, exemplary reactive end groups include bicyclic [2.2.1]hept-5-ene-2,3-dicarboxylic acid imide malonic acid ester group, bicyclic [2.2.1]hept-5-ene-2,3-dicarboxylic acid imide glutarate ester group, bicyclic [2.2.1]hept-5-ene-2,3-dicarboxylic acid imide succinate ester group, bicyclic [2.2.1]hept-5-ene-2,3-dicarboxylic acid imide adipic acid ester group, and bicyclic [2.2.1]hept-5-ene-2,3-dicarboxylic acid imide diglycol ester group, etc. In the specific case where N-hydroxymaleimide is an N-hydroxy cyclic imide compound, exemplary reactive end groups include maleimide malonate groups, maleimide glutarate groups, maleimide succinate groups, maleimide adipate groups, and maleimide diethylene glycol ester groups, etc. In the specific case where N-hydroxyphthalimide is an N-hydroxycyclic imide compound, exemplary reactive end groups include phthalimide malonate group, phthalimide glutarate group, phthalimide succinate group, phthalimide adipate group, and phthalimide diglycol ester group, etc.

[0123] In other aspects of this disclosure, a system is provided comprising (a) a first composition comprising a radiopaque peptide compound as described herein, and (b) a second composition comprising a reactive polymer comprising a reactive portion as described herein, wherein the system is configured to deliver the reactive polymer and the radiopaque peptide compound under conditions such that a covalent crosslink is formed between the reactive polymer and the radiopaque peptide compound.

[0124] The first composition may be a first fluid composition comprising a radiopaque peptide compound or a first dry composition comprising a radiopaque peptide compound, wherein a suitable fluid (e.g., water for injection, saline, etc.) may be added to the first dry composition to form the first fluid composition. In addition to the radiopaque peptide compound, the first composition may also contain additional reagents (including therapeutic agents, imaging agents, colorants, tension modifiers, suspending agents, wetting agents, and pH adjusters as described below).

[0125] The second composition may be a second fluid composition containing a reactive polymer or a second dry composition containing a reactive polymer. A suitable fluid (e.g., water for injection, saline, etc.) may be added to the second dry composition to form the second fluid composition. In addition to the reactive polymer, the second composition may also contain other reagents (including therapeutic agents, imaging agents, colorants, tension modifiers, suspending agents, wetting agents, and pH adjusters as described below).

[0126] In some embodiments, the system is configured to mix a first fluid composition comprising a radiopaque peptide compound with a second fluid comprising a reactive polymer. During mixing of the first and second fluid compositions, the radiopaque peptide compound crosslinks with the reactive polymer to form a crosslinked product. The first and second fluid compositions can be mixed in vivo or in vitro to form a crosslinked hydrogel.

[0127] In some embodiments, the radiopaque peptide compound is initially mixed with the reactive polymer under conditions where crosslinking between the reactive polymer and the radiopaque peptide compound is inhibited (e.g., in some embodiments, an acidic pH). Then, when crosslinking is required, the conditions are changed to increase crosslinking (e.g., in some embodiments, from an acidic pH to an alkaline pH), resulting in crosslinking between the radiopaque peptide compound and the reactive polymer, thereby forming a crosslinked product.

[0128] In some embodiments, the system includes (a) a first composition comprising the radiopaque peptide compound as described above, (b) a second composition comprising the reactive polymer as described above, and (c) a third composition, specifically, an accelerator composition containing an accelerator configured to accelerate the crosslinking reaction between the radiopaque peptide compound and the reactive polymer.

[0129] The first composition may be a first fluid composition comprising a radiopaque peptide compound buffered to an acidic pH or a first dry composition comprising said radiopaque peptide compound, wherein a suitable fluid (e.g., water for injection, saline, acid buffer solution, etc.) may be added to the first dry composition to form a first fluid composition comprising a radiopaque peptide compound buffered to an acidic pH. For example, in some embodiments, the acid buffer composition may include sodium dihydrogen phosphate, etc. The first fluid composition comprising the radiopaque peptide compound may have a pH in the range of, for example, from about 3 to about 5. In addition to the radiopaque peptide compound, the first composition may also contain other agents (including therapeutic agents, imaging agents, colorants, tension modifiers, suspending agents, wetting agents, and pH adjusters as described below).

[0130] The second composition may be a second fluid composition comprising a reactive polymer or a second dry composition comprising a reactive polymer, wherein the fluid composition is formed from the second dry composition, for example by adding a suitable fluid (e.g., water for injection, saline, or a first fluid composition comprising a radiopaque peptide compound buffered to an acidic pH). In addition to the reactive polymer, the second composition may also contain other agents (including therapeutic agents, imaging agents, colorants, tension modifiers, suspending agents, wetting agents, and pH adjusters as described below).

[0131] In one specific embodiment, the first composition is a first fluid composition comprising a radiopaque peptide compound buffered to an acidic pH, and the second composition comprises a dry composition comprising a reactive polymer. The first and second compositions can then be mixed to provide a prepared fluid composition buffered to an acidic pH and comprising the radiopaque peptide compound and the reactive polymer. In one specific example, a syringe containing the first fluid composition comprising the radiopaque peptide compound buffered to an acidic pH can be provided, and a vial containing the dry composition (e.g., powder) comprising the reactive polymer can be provided. The first fluid composition can then be injected into the vial containing the reactive polymer using the syringe to form the prepared fluid composition buffered to an acidic pH and comprising the radiopaque peptide compound and the reactive polymer, which can then be aspirated back into the syringe for administration.

[0132] The accelerator composition can be a liquid accelerator composition buffered to an alkaline pH, or a dry composition containing an alkaline buffer composition. A suitable fluid (e.g., water for injection, saline, etc.) can be added to the dry composition to form a fluid accelerator composition buffered to an alkaline pH. For example, the alkaline buffer composition may include sodium borate and disodium hydrogen phosphate, etc. The fluid accelerator composition may have a pH in the range of, for example, about 9 to about 11. In addition to the above, the fluid accelerator composition may also contain other reagents, including those described below.

[0133] The prepared fluid composition, buffered to an acidic pH and containing a radiopaque peptide compound and a reactive polymer as described above, and the fluid accelerator composition, buffered to an alkaline pH as described above, can be mixed in vivo or in vitro to form a cross-linked hydrogel.

[0134] Other agents used in the compositions described herein include therapeutic agents, imaging agents, colorants, tension modifiers, suspending agents, wetting agents, and pH adjusters.

[0135] Examples of therapeutic agents include antithrombotic agents, anticoagulants, antiplatelet agents, thrombolytic agents, antiproliferative agents, anti-inflammatory agents, proliferation inhibitors, anti-restenosis agents, smooth muscle cell inhibitors, antibiotics, antibacterial agents, analgesics, anesthetics, growth factors, growth factor inhibitors, cell adhesion inhibitors, cell adhesion promoters, anti-angiogenic agents, cytotoxic agents, chemotherapeutic agents, checkpoint inhibitors, immunomodulatory cytokines, T-cell agonists, STING (interferon gene stimulator) agonists, antimetabolites, alkylating agents, microtubule inhibitors, hormones, hormone antagonists, monoclonal antibodies, antimitotic agents, immunosuppressants, tyrosine and serine / threonine kinases, proteasome inhibitors, matrix metalloproteinase inhibitors, Bcl-2 inhibitors, DNA alkylating agents, spindle poisons, poly(DP-ribose) polymerase (PARP) inhibitors, and combinations thereof.

[0136] Examples of imaging agents include (a) fluorescent dyes, such as fluorescein, indocyanine green, or fluorescent proteins (e.g., green, blue, and blue-green fluorescent proteins); (b) contrast agents used in conjunction with magnetic resonance imaging (MRI), including contrast agents containing elements that form paramagnetic ions, such as Gd(III), Mn(II), Fe(III), and compounds containing them (including chelates), such as gadolinium ions chelated with diethylenetriaminepentaacetic acid; and (c) contrast agents used in conjunction with ultrasound imaging, including organic and inorganic echogenic particles (i.e., particles that cause an increase in reflected ultrasound energy) or organic and inorganic echolucent particles. (d) Contrast agents used in conjunction with near-infrared (NIR) imaging, which can be selectively used to impart near-infrared fluorescence to the hydrogel of this disclosure, thereby allowing deep tissue imaging and device labeling, such as NIR-sensitive nanoparticles, such as gold nanoshells, carbon nanotubes (e.g., nanotubes derived with hydroxyl or carboxyl groups, such as partially oxidized carbon nanotubes), dye-containing nanoparticles (e.g., dye-doped nanofibers and dye-encapsulated nanoparticles), and semiconductor quantum dots, etc., and NIR-sensitive dyes (e.g., cyanine dyes). (e) Imageable radioactive isotopes, including 99mTc, 201Th, 51Cr, 67Ga, 68Ga, 111In, 64Cu, 89Zr, 59Fe, 42K, 82Rb, 24Na, 45Ti, 44Sc, 51Cr and 177Lu, and (f) radioactive contrast agents, such as particles of tantalum, tungsten, rhenium, niobium, molybdenum and their alloys, which may be spherical or non-spherical. Other examples of contrast agents include nonionic contrast agents such as iohexol, iodixanol, iofluoxetine, iopamidol, ioxilan, or iopromide; ionic contrast agents such as diatrizoate, iothalamate, methyl diatrizoate, or ioxaglate; and iodized oils, including ethiodized poppyseed oil (available as Lipiodol®).

[0137] Examples of colorants include brilliant blue (e.g., brilliant blue FCF, also known as FD&C blue 1), indigocarmine (also known as FD&C blue 2), indigocarmine lake, FD&C blue 1 lake, and methylene blue (also known as methylene blue chloride), etc.

[0138] Examples of other reagents include tension modifiers such as sugars (e.g., glucose, lactose, etc.), polyols (e.g., glycerol, propylene glycol, mannitol, sorbitol, etc.) and inorganic salts (e.g., potassium chloride, sodium chloride, etc.), suspending agents including various surfactants, wetting agents and polymers (e.g., albumin, PEO, polyvinyl alcohol, block polymers, etc.), and pH adjusters including various buffer solutes.

[0139] In various embodiments, a system is provided that includes one or more delivery devices for delivering a first composition and a second composition to a subject.

[0140] In some embodiments, the system may include a delivery device comprising a first reservoir and a second reservoir, the first reservoir containing a first fluid composition comprising a radiopaque peptide compound as described herein, and the second reservoir containing a second fluid composition comprising a reactive polymer as described herein, wherein the first and second fluid compositions, upon mixing, form a crosslinked product. In some embodiments, the system may include a delivery device comprising a first reservoir and a second reservoir containing a first fluid composition, the first reservoir containing the first fluid composition comprising a radiopaque peptide compound and a reactive polymer and buffered to a first pH, such as the aforementioned prepared fluid composition, and the second reservoir containing a second fluid composition (e.g., a fluid accelerator composition) that, when combined with the first fluid composition, alters the pH of the environment surrounding the radiopaque peptide compound and the reactive polymer (e.g., the aforementioned fluid accelerator composition), resulting in crosslinking between the radiopaque peptide compound and the reactive polymer.

[0141] In either case, during operation, the first fluid composition and the second fluid composition are dispensed from the first reservoir and the second reservoir and mixed, whereby the radiopaque peptide compound and the reactive polymer crosslink with each other to form a crosslinked hydrogel.

[0142] In a particular implementation scheme, and referring to Figure 8The system may include a delivery device 810 comprising a dual-tube syringe, the dual-tube syringe including a first tube 812a having a first tube outlet 814a (the first tube contains a first composition and a first plunger 816a movable within the first tube 812a), and a second tube 812b having a second tube outlet 814b (the second tube 812b contains a second composition and a second plunger 816b movable within the second tube 812b). In some embodiments, the device 810 may further include a mixing section 818 having a first mixing section inlet 818a1 in fluid communication with the first tube outlet 814a, a second mixing section inlet 818b1 in fluid communication with the second tube outlet, and a mixing section outlet 818o.

[0143] In some embodiments, the delivery device may further include a cannula or conduit configured to receive a first fluid composition and a second fluid composition from a first tube and a second tube. For example, the cannula or conduit may be configured to form a fluid connection with the outlet of the mixing section by attaching the cannula or conduit to the outlet of the mixing section (e.g., via a suitable fluid connector such as a Luer connector).

[0144] As another example, the catheter may be a multi-lumen catheter including a first lumen and a second lumen, the proximal end of the first lumen being configured to form a fluid connection with a first tube outlet, and the proximal end of the second lumen being configured to form a fluid connection with a second tube outlet. In some embodiments, the multi-lumen catheter may include a mixing portion having a first mixing portion inlet in fluid communication with the distal end of the first lumen, a second mixing portion inlet in fluid communication with the distal end of the second lumen, and a mixing portion outlet.

[0145] During operation, when the first and second plungers are depressed, a first fluid composition and a second fluid composition are dispensed from the first and second tubes, whereby the first and second fluid compositions mix and the radiopaque peptide compound and reactive polymer are ultimately crosslinked to form a crosslinked hydrogel, which is applied to or within the subject's tissue. For example, the first and second fluid compositions can enter a mixing section from the first and second tubes via a first mixing section inlet and a second mixing section inlet, whereby the first and second fluid compositions mix to form a mixture, which exits the mixing section via a mixing section outlet. In some embodiments, a cannula or catheter is attached to the mixing section outlet, thereby allowing the mixture to be applied to the subject after passing through the cannula or catheter.

[0146] As another example, a first fluid composition may enter the first lumen of a multi-lumen catheter from a first tube outlet, and a second fluid composition may enter the second lumen of the multi-lumen catheter from a second tube outlet. In some embodiments, the first fluid composition and the second fluid composition may enter a mixing section at the distal end of the multi-lumen catheter from the first lumen and the second lumen, respectively, via a first mixing section inlet and a second mixing section inlet, whereby the first fluid composition and the second fluid composition are mixed in the mixing section to form a mixture, which exits the mixing section via a mixing section outlet.

[0147] Regardless of the type of apparatus used to mix the first and second fluid compositions, or how the first and second fluid compositions are mixed, after the mixture of the first and second fluid compositions is formed, the mixture is initially in a fluid state and can be applied to a subject (e.g., a mammal, particularly a human) using a variety of techniques. Alternatively, the first and second fluid compositions can be applied to the subject independently, and the fluid mixture of the first and second fluid compositions is formed inside or on the surface of the subject. In either method, the fluid mixture of the first and second fluid compositions is formed and used in a variety of medical procedures.

[0148] For example, in applications such as the treatment of diseases and cancers, as well as tissue repair and regeneration, the first fluid composition and the second fluid composition, or a fluid mixture thereof, can be injected to provide spacers between tissues; the first fluid composition and the second fluid composition, or a fluid mixture thereof, can be injected (e.g., in the form of blisters) to provide reference markers; the first fluid composition and the second fluid composition, or a fluid mixture thereof, can be injected for tissue enlargement or regeneration; the first fluid composition and the second fluid composition, or a fluid mixture thereof, can be injected as fillers or replacements for soft tissues; the first fluid composition and the second fluid composition, or a fluid mixture thereof, can be injected to provide mechanical support for damaged tissues; the first fluid composition and the second fluid composition, or a fluid mixture thereof, can be injected as scaffolds; the first fluid composition and the second fluid composition, or a fluid mixture thereof, can be injected as embolic compositions; the first fluid composition and the second fluid composition, or a fluid mixture thereof, can be injected as lifting agents for the removal of internal cysts; and / or the first fluid composition and the second fluid composition, or a fluid mixture thereof, can be injected as carriers of therapeutic agents. The first fluid composition and the second fluid composition, or a fluid mixture thereof, can also be injected into the left atrial appendage during a left atrial appendage occlusion procedure. In some embodiments, the first fluid composition and the second fluid composition, or a fluid mixture thereof, may be injected into the left atrial appendage after the introduction of an occlusion device (such as the Watchman® left atrial appendage occlusion device from Boston Scientific Corporation).

[0149] Upon application of the compositions disclosed herein (either as a first fluid composition and a second fluid composition mixed in vivo, or as a fluid mixture of the first fluid composition and the second fluid composition), a cross-linked hydrogel is ultimately formed at the application site.

[0150] After application, the compositions of this disclosure can be imaged using suitable imaging techniques. Typically, the imaging techniques are X-ray based techniques, such as computed tomography or X-ray fluorescence fluoroscopy, or near-IR fluorescence spectroscopy techniques.

[0151] As can be seen from the above, the compositions disclosed herein can be used in a variety of medical procedures, including: procedures for implanting a reference marker comprising a crosslinked product of a first fluid composition and a second fluid composition; procedures for implanting a tissue regeneration scaffold comprising a crosslinked product of a first fluid composition and a second fluid composition; procedures for implanting a tissue support comprising a crosslinked product of a first fluid composition and a second fluid composition; procedures for implanting a building agent comprising a crosslinked product of a first fluid composition and a second fluid composition; procedures for implanting a depot comprising a release therapeutic agent comprising a crosslinked product of a first fluid composition and a second fluid composition; procedures for tissue enlargement comprising implanting a crosslinked product of a first fluid composition and a second fluid composition; and procedures for introducing a crosslinked product of a first fluid composition and a second fluid composition between a first tissue and a second tissue to separate the first tissue from the second tissue.

[0152] The first fluid composition and the second fluid composition, a fluid mixture of the first fluid composition and the second fluid composition, or a crosslinked product of the first fluid composition and the second fluid composition can be injected in combination with a variety of medical procedures including: septal injection between the prostate or vagina and rectum in radiotherapy for rectal cancer; septal injection between the rectum and prostate in radiotherapy for prostate cancer; subcutaneous injection for palliative treatment of prostate cancer; transurethral or submucosal injection for female stress urinary incontinence; intravesical injection for urinary incontinence; intrauterine injection for Asherman's syndrome; submucosal injection for anal incontinence; percutaneous injection for heart failure; intramyocardial injection for heart failure and dilated cardiomyopathy; transendocardial injection for myocardial infarction; intra-articular injection for osteoarthritis; and injections for spinal fusion and spinal, oral, and maxillofacial procedures. Spinal injections for orthopedic trauma surgery; spinal injections for posterolateral lumbar fusion; intradiscal injections for degenerative disc diseases; inter-pancreatic and duodenal injections for pancreatic cancer imaging; resection bed injections for oropharyngeal cancer imaging; peritumoral injections for bladder cancer imaging; submucosal injections for gastrointestinal tumors and polyps; visceral pleural injections for lung biopsies; renal injections for type 2 diabetes and chronic kidney disease; renal cortical injections for chronic kidney disease with congenital abnormalities from the kidneys and urethra; intravitreal injections for neovascular age-related macular degeneration; intratympanic injections for sensorineural hearing loss; and dermal injections to correct wrinkles, creases and folds, signs of facial fat loss, volume reduction, superficial to deep contour defects, correction of depressed skin scars, perioral wrinkles, lip augmentation, facial fat atrophy, and stimulate natural collagen production.

[0153] In the case of in vitro formation, the cross-linked hydrogel can be in any desired form, including plates, cylinders, coatings, or granules. In some embodiments, the cross-linked hydrogel is dried and then granulated into particles of suitable size. Granulation can be performed by any suitable method, such as by grinding (including cryogenic grinding), homogenization, crushing, milling, pulverizing, etc. The particles can be sorted and separated using sieving or other known techniques. The size of the cross-linked hydrogel particles formed using the above and other techniques can vary considerably, for example, with an average size of 50 to 950 micrometers.

[0154] In addition to the cross-linked hydrogels described above, the cross-linked hydrogel compositions according to this disclosure may contain other agents, including therapeutic agents, imaging agents, colorants, tension modifiers, suspending agents, wetting agents, and pH adjusters as described above.

[0155] In various embodiments, a kit is provided that includes one or more delivery devices for delivering a cross-linked hydrogel composition to a subject. Such a system may include one or more of the following: a syringe tubing, which may or may not contain the cross-linked hydrogel composition as described herein; a vial, which may or may not contain the cross-linked hydrogel composition as described herein; a needle; a flexible tube (e.g., adapted for fluid connection of the needle to a syringe); and an injectable liquid, such as water for injection, physiological saline, or phosphate-buffered saline. Whether provided in the form of a syringe, vial, or other reservoir, the cross-linked hydrogel composition may be delivered in a dry form (e.g., powder form) or in a ready-for-injection form, such as an injectable hydrogel form (e.g., a suspension of cross-linked hydrogel particles).

[0156] Figure 9 A syringe 10 is shown, which provides a reservoir for the crosslinked hydrogel composition as described above. The syringe 10 may include a tube 12, a plunger 14, and one or more stoppers 16. The tube 12 may include, for example, a Luer adapter (or other suitable adapter / connector) at its distal end 18 for attachment to an injection needle 50 via a flexible conduit 29. The proximal end of the conduit 29 may include a suitable connector 20 for receiving the tube 12. In other instances, the tube 12 may be directly connected to the injection needle 50. The syringe tube 12 can be used as a reservoir for containing the crosslinked hydrogel composition 15 for injection via the needle 50.

[0157] The cross-linked hydrogel compositions described herein can be used for a variety of purposes.

[0158] For example, in applications such as the treatment of diseases and cancers, as well as the repair and regeneration of tissues, cross-linked hydrogel compositions can be injected to provide spacers between tissues, cross-linked hydrogel compositions (e.g., in the form of bubbles) can be injected to provide reference markers, cross-linked hydrogel compositions can be injected for tissue enlargement or regeneration, cross-linked hydrogel compositions can be injected as fillers or substitutes for soft tissues, cross-linked hydrogel compositions can be injected to provide mechanical support for damaged tissues, cross-linked hydrogel compositions can be injected as scaffolds, and / or cross-linked hydrogel compositions can be injected as carriers of therapeutic agents.

[0159] After application, the cross-linked hydrogel composition of this disclosure can be imaged using suitable imaging techniques.

[0160] As described above, the cross-linked hydrogel composition of this disclosure can be used in a variety of medical procedures, including: procedures for implanting a reference marker comprising cross-linked hydrogel, procedures for implanting a tissue regeneration scaffold comprising cross-linked hydrogel, procedures for implanting a tissue support comprising cross-linked hydrogel, procedures for implanting a tissue filler comprising cross-linked hydrogel, procedures for implanting a therapeutic reservoir comprising cross-linked hydrogel, procedures for tissue enlargement including implantation of cross-linked hydrogel, and procedures for introducing cross-linked hydrogel between a first tissue and a second tissue to separate the first tissue from the second tissue.

[0161] The cross-linked hydrogel composition can be injected in conjunction with a variety of medical procedures, including: injection as a septum between the prostate or vagina and rectum in radiotherapy for rectal cancer; injection as a septum between the rectum and prostate in radiotherapy for prostate cancer; subcutaneous injection for palliative treatment of prostate cancer; transurethral or submucosal injection for female stress urinary incontinence; intravesical injection for urinary incontinence; intrauterine injection for Asherman's syndrome; submucosal injection for anal incontinence; percutaneous injection for heart failure; intramyocardial injection for heart failure and dilated cardiomyopathy; transendocardial injection for myocardial infarction; intra-articular injection for osteoarthritis; spinal injection for spinal fusion and spinal, maxillofacial, and orthopedic trauma surgery; and spinal injection for posterolateral lumbar fusion. It is used for intradiscal injection in degenerative disc diseases, injection between the pancreas and duodenum for pancreatic cancer imaging, resection bed injection for oropharyngeal cancer imaging, peritumoral injection for bladder cancer imaging, submucosal injection for gastrointestinal tumors and polyps, visceral pleural injection for lung biopsy, renal injection for type 2 diabetes and chronic kidney disease, renal cortical injection for chronic kidney disease with congenital abnormalities from the kidney and urethra, intravitreal injection for neovascular age-related macular degeneration, intratympanic injection for sensorineural hearing loss, and dermal injection to correct wrinkles, creases and folds, signs of facial fat loss, volume reduction, superficial to deep contour defects, correction of depressed skin scars, perioral wrinkles, lip augmentation, facial fat atrophy, and to stimulate natural collagen production.

[0162] The cross-linked hydrogel compositions according to this disclosure include smooth compositions for medical applications, compositions for the release of therapeutic agents (e.g., by including one or more therapeutic agents in the matrix of the cross-linked hydrogel), and implants (which may be formed in vitro or in vivo) (e.g., compositions used as tissue markers, compositions acting as spacers to reduce the side effects of off-target radiation therapy, cosmetic compositions, etc.).

Claims

1. A method for forming an iodinated peptide compound, comprising: (a) forming a multifunctional precursor compound comprising (i) an amino-protected peptide residue comprising two or more amino acid residues having primary amine side groups, and (ii) a multifunctional moiety linked to the amino-protected peptide residue via an amide or ester group, the multifunctional moiety further comprising n primary amine groups, n hydroxyl groups, or n carboxyl groups, where n is an integer of 1 or greater; (b) subjecting the multifunctional precursor compound to an amide coupling or ester coupling reaction with an iodinated compound selected from an amino-functionalized iodinated compound containing an iodinated moiety, a carboxyl-functionalized iodinated compound containing an iodinated moiety, and a hydroxyl-functionalized iodinated compound containing an iodinated moiety, to form an iodinated, amino-protected peptide compound comprising n iodinated moieties, the n iodinated moieties being covalently linked to the amino-protected peptide residue via residues of the multifunctional moiety; and (c) deprotecting the iodinated, amino-protected peptide compound to form the iodinated peptide compound.

2. The method of claim 1, wherein the multifunctional portion is linked to the amino-protected peptide residue via an amide group, and wherein the multifunctional portion comprises n primary amino groups.

3. The method of claim 2, wherein the multifunctional precursor compound is formed by comprising: (i) an amide coupling reaction of a polyamine compound having n+1 primary amine groups with (ii) a carboxyl group at the C-terminus of an amino-protected peptide, wherein the amino-protected peptide comprises two or more amino acid residues having primary amine side groups.

4. The method according to any one of claims 2-3, wherein the n primary amino groups of the polyfunctional precursor compound are subjected to an amide coupling reaction with the n carboxyl-functional iodide compounds to form the iodinated, amino-protected peptide compound.

5. The method of claim 1, wherein the multifunctional portion is linked to the amino-protected peptide residue via an ester group, and wherein the multifunctional portion comprises n hydroxyl groups.

6. The method of claim 5, wherein the polyfunctional precursor compound is formed by a method comprising ester coupling (i) a polyol compound having n+1 hydroxyl groups with (ii) a carboxyl group at the C-terminus of an amino-protected peptide, the amino-protected peptide comprising two or more amino acid residues having primary amine side groups.

7. The method according to any one of claims 5-6, wherein the n hydroxyl groups of the polyfunctional precursor compound undergo an ester coupling reaction with the n carboxyl-functional iodide compounds to form the iodinated, amino-protected peptide compound.

8. The method of claim 1, wherein the multifunctional portion is linked to the amino-protected peptide residue via an amide group, and wherein the multifunctional portion comprises n carboxyl groups.

9. The method of claim 8, wherein the polyfunctional precursor compound is formed by comprising: (a) amide coupling (i) a polyamine compound having n+1 primary amine groups with (ii) a carboxyl group at the C-terminus of an amino-protected peptide, the amino-protected peptide comprising two or more amino acid residues having primary amine side groups, the reaction product having n primary amine groups; and (b) ring-opening reaction of the n primary amine groups of the reaction product of step (a) with a cyclic anhydride to form n carboxyl groups at the sites of the n primary amine groups.

10. The method of claim 8, wherein the polyfunctional precursor compound is formed by comprising: (a) ester coupling (i) a polyol compound having n+1 hydroxyl groups with (ii) a carboxyl group at the C-terminus of an amino-protected peptide, the amino-protected peptide comprising two or more amino acid residues having primary amine side groups, the reaction product having n hydroxyl groups; and (b) ring-opening reaction of the n hydroxyl groups of the reaction product of step (a) with a cyclic anhydride to form n carboxyl groups at the sites of the n hydroxyl groups.

11. The method of claim 8, wherein the multifunctional precursor compound is formed by comprising: (a) reacting (i) a carboxyl-protected polycarboxylamine compound comprising at least one amino group and n protected carboxyl groups protected from amide coupling with (ii) an amide coupling reaction of a carboxyl group at the C-terminus of an amino-protected peptide comprising two or more amino acid residues having primary amine side groups; and (b) deprotecting the n protected carboxyl groups of the reaction product of step (a) without deprotecting the protected amino groups.

12. The method of claim 8, wherein the multifunctional precursor compound is formed by comprising: (a) esterifying (i) a carboxylated hydroxycarboxylic acid compound comprising a hydroxyl group and n protected carboxyl groups that are protected from ester coupling with (ii) a carboxyl group at the C-terminus of an amino-protected peptide, the amino-protected peptide comprising two or more amino acid residues having primary amine side groups; and (b) deprotecting the n protected carboxyl groups of the reaction product of step (a) without deprotecting the protected amine groups.

13. The method according to any one of claims 8-12, wherein the n carboxyl groups of the polyfunctional precursor compound undergo an ester coupling reaction with the n hydroxyl-functional iodide compounds to form the iodinated, amino-protected peptide compound, or wherein the n carboxyl groups of the polyfunctional precursor compound undergo an amide coupling reaction with the n amino-functional iodide compounds to form the iodinated, amino-protected peptide compound.

14. The method according to any one of claims 1-13, wherein each of the n iodinated portions comprises one or more iodinated aromatic groups.

15. The method according to any one of claims 1-14, wherein the two or more amino acids having primary amine side groups are selected from lysine, ornithine, arginine, and any combination of two or all three of lysine, ornithine, and arginine.

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