Cross-linking agent and medical hydrogel formed therefrom

Abstract

In some aspects, the disclosure relates to methods comprising (a) performing ring-opening polymerization of one or more types of amino acid N-carboxylic acid anhydride (NCA) monomers in the presence of an initiator compound to produce an intermediate peptide compound, the monomers comprising at least one type of protected amino acid NCA monomer having a protected pendant amine group, the intermediate peptide compound comprises an amino acid chain having a protected side chain amine group covalently linked to the initiator residue, and (b) deprotecting the intermediate peptide compound to form a final peptide compound comprising an amino acid chain having a side chain amine group covalently linked to the initiator residue.

Description

[0001] Cross-references to related applications This application claims the benefit of US Provisional Patent Application No. 63 / 515,513, filed on July 25, 2023, the disclosure of which is incorporated herein by reference. Technical Field

[0002] This disclosure relates to methods for forming peptides from amino acid N-carboxyanhydride monomers using various initiators, including methods for forming radiopaque peptides using radiopaque initiators. This disclosure also relates to the use of such peptides as crosslinking agents for forming hydrogels, and hydrogels formed from such peptides. The crosslinking agent and the hydrogel can be used in, for example, various 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] However, although the current synthetic routes for forming trilysine crosslinking agents are sufficient to produce the desired product, Figure 1 The process shown is quite lengthy because it involves five steps. Furthermore, for cases where additional lysine units may be needed (e.g., in the production of tetralysine, pentalysine, hexalysine, etc.), two additional steps are required for each added lysine.

[0005] For these and other reasons, there is a need for alternative strategies for forming trilysine and for forming iodine-labeled crosslinked hydrogels that provide enhanced radiation impermeability while maintaining the crosslinking density of each polymer molecule. Summary of the Invention

[0006] In some aspects, this disclosure relates to a method comprising: (a) performing ring-opening polymerization of one or more types of amino acid N-carboxylic anhydride (NCA) monomers in the presence of an initiator compound to produce an intermediate peptide compound, said monomer comprising at least one type of protected amino acid NCA monomer having a protected pendant amine group, said intermediate peptide compound comprising an amino acid chain having a protected pendant amine group covalently linked to an initiator residue; and (b) deprotecting the intermediate peptide compound to form a final peptide compound comprising an amino acid chain having a side-chain amine group covalently linked to an initiator residue.

[0007] In some embodiments, the method further includes separating the final peptide compound according to its molecular weight to provide a final peptide compound having amino acid chains of equal length.

[0008] In some embodiments that can be used in conjunction with the above aspects and embodiments, the molar ratio of amino acid N-carboxylic anhydride (NCA) monomer to initiator compound ranges from 2:1 to 100:1.

[0009] In some embodiments that can be used in conjunction with the above aspects and implementation schemes, the length of the amino acid chain ranges from 2 to 50 amino acids.

[0010] In some embodiments that can be used in conjunction with the above aspects and implementation schemes, the protected side chain amine group is a protected primary amine group.

[0011] In some embodiments that can be used in conjunction with the above aspects and implementation schemes, the protected side chain amine group is a protected alkyl amine group.

[0012] In some embodiments that can be used in conjunction with the above aspects and embodiments, the protected side chain amine group includes a protective group selected from the following: tert-butoxycarbonyl, carboxybenzyl, trifluoroacetyl, 6-nitroveratryloxycarbonyl group and 9-fluorenylmethoxycarbonyl.

[0013] In some embodiments that can be used in conjunction with the above aspects and embodiments, at least one type of protected amino acid NCA monomer includes protected lysine NCA monomer and / or protected ornithine NCA monomer.

[0014] In some embodiments that can be used in conjunction with the above aspects and implementation methods, the initiator contains a primary amine group or an aliphatic hydroxyl group.

[0015] In some embodiments that can be used in conjunction with the above aspects and implementation schemes, the initiator includes an amine protected by a trimethylsilyl group.

[0016] In some embodiments that can be used in conjunction with the above aspects and embodiments, the initiator comprises (a) a primary amino or aliphatic group and (b) an iodinated aromatic group.

[0017] In some embodiments that can be used in conjunction with the above aspects and embodiments, the initiator comprises (a) a primary amine or aliphatic group that is linked to (b) an iodinated aromatic group via a linear linker or a multi-arm linker.

[0018] In some embodiments that can be used in conjunction with the above aspects and implementation schemes, the initiator includes iodinated amino acid esters.

[0019] In some embodiments that can be used in conjunction with the above aspects and implementation schemes, the initiator includes a metal catalyst.

[0020] In some embodiments that can be used in conjunction with the above aspects and embodiments, one or more types of amino acid N-carboxylic anhydride (NCA) monomers include a single type of protected amino acid NCA monomer.

[0021] In some embodiments that can be used in conjunction with the above aspects and embodiments, one or more types of amino acid N-carboxylic anhydride (NCA) monomers include at least one type of iodinated amino acid NCA monomer.

[0022] In some respects, this disclosure relates to the production of a final peptide compound by means of methods according to any of the foregoing aspects and embodiments.

[0023] In some aspects, this disclosure relates to a system for forming a hydrogel, the system comprising (a) a final peptide compound produced by a method according to any of the foregoing aspects and embodiments, and (b) a reactive multi-arm polymer comprising a plurality of hydrophilic polymer arms having reactive end groups that react with the amino groups of the final peptide compound.

[0024] In some implementations, the system also includes a delivery device.

[0025] In some aspects, this disclosure relates to a reaction product comprising: (a) a final peptide compound produced by a method according to any of the foregoing aspects and embodiments, and (b) a reactive multiarm polymer comprising a plurality of hydrophilic polymer arms having reactive end groups that react with the amino groups of the final peptide compound.

[0026] In some respects, this disclosure relates to treatment methods that include administering such a reaction product to a subject.

[0027] In some aspects, this disclosure relates to a treatment method comprising administering to a subject a mixture comprising: a final peptide compound produced by a method according to any of the foregoing aspects and embodiments, and a reactive multi-arm polymer comprising a plurality of hydrophilic polymer arms having reactive end groups that react with the amino groups of the final peptide compound, wherein the final peptide compound and the reactive multi-arm polymer are crosslinked after administration.

[0028] In some embodiments, the treatment method includes administering to a subject a first fluid composition comprising a final peptide compound and a reactive polymer, and a second fluid composition comprising a promoter that accelerates covalent crosslinking formation. In some embodiments, a binaural syringe is used to deliver the first and second fluid compositions.

[0029] The above and other aspects, implementations, features and benefits of this disclosure will become apparent from the following detailed description. Attached Figure Description

[0030] Figure 1 An existing scheme for the formation of trilysine is illustrated schematically.

[0031] Figure 2 A process for forming a peptide compound according to one embodiment of the present disclosure is illustrated schematically.

[0032] Figure 3A A process for forming a peptide compound according to one embodiment of the present disclosure is illustrated schematically.

[0033] Figure 3B A schematic illustration shows an embodiment of the present disclosure for forming Figure 3A The process of initiating agents.

[0034] Figure 4A A process for forming a peptide compound according to one embodiment of the present disclosure is illustrated schematically.

[0035] Figure 4B A schematic illustration shows an embodiment of the present disclosure for forming Figure 4A The process of initiating agents.

[0036] Figure 5A A process for forming a peptide compound according to one embodiment of the present disclosure is illustrated schematically.

[0037] Figure 5B A schematic illustration shows an embodiment of the present disclosure for forming Figure 5A The process of initiating agents.

[0038] Figure 6A process for forming a peptide compound according to one embodiment of the present disclosure is illustrated schematically.

[0039] Figure 7 A delivery device according to one embodiment of the present disclosure is shown.

[0040] Figure 8 A delivery device according to another embodiment of this disclosure is shown. Detailed Implementation

[0041] In some aspects of this disclosure, peptide compounds are formed by ring-opening polymerization of amino acid N-carboxylic anhydride (NCA) using a suitable initiator, said initiator being incorporated into the peptide chain produced by polymerization. Thus, peptide compounds according to this disclosure comprise initiator residues and an amino acid chain covalently linked to the initiator residues. The initiator residues include amine-containing initiator residues and hydroxyl-containing residues. The length of the amino acid chain can range from 2 to 50 amino acids, typically from 3 to 10 amino acids. The amino acid chain according to this disclosure comprises amino acids having primary amine side groups. In specific examples, the primary amine side group is an aminoalkyl group (e.g., C1-C6-aminoalkyl, including aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, and 6-aminohexyl, and their isomers). The length of the amino acid chain is determined by the molar ratio of the amino acid NCA monomer to the initiator compound. Typically, the molar ratio of the amino acid N-carboxylic anhydride (NCA) monomer to the initiator compound ranges from 2:1 to 50:1, more typically from 3:1 to 10:1.

[0042] In some implementations, amino acid NCA polymerization is performed using an amine-containing initiator based on a nucleophilic ring-opening chain growth process (in which the polymer grows linearly with monomer conversion). In this process, when the amine reacts with the NCA monomer, the NCA ring opens, releasing carbon dioxide and forming a molecule with a new primary amine terminal group, which can be used for further reaction with another NCA monomer. As previously described, the initiator used in this process is incorporated into the resulting peptide chain. Further information can be found, for example, in Carmen M. González-Henríquez, et al., “Strategies to Fabricate Polypeptide-Based Structures via Ring-Opening Polymerization of N-Carboxyanhydrides.” Polymers (Basel). November 2017; 9(11): 551.

[0043] Initiators include those that have unmodified amines or silyl-protected amines, such as trimethylsilyl-protected amines.

[0044] In this disclosure, amino acid NCA monomers containing protected amine side groups are used. Examples of such amino acid NCA monomers include amino-protected lysine NCA monomers (where the protected group is 4-aminobutyl) and amino-protected ornithine NCA monomers (where the protected group is 3-aminopropyl), etc. In the case of lysine, various protective chemicals have been reported, wherein the protecting groups include tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or (Z), trifluoroacetyl (TFA), 6-nitroveratroloxycarbonyl (Nvoc), and 9-fluorenylmethoxycarbonyl (Fmoc). Ibid., cite Hernández, JR; Klok, HA, “Synthesis and ring-opening (co) polymerization of L-lysine N-carboxyanhydrides containing labile side-chain protective groups.” J. Polym. Sci. Part A 2003, 41, 1167–1187.

[0045] Specific examples of amino acid NCA monomers with protected amino groups include lysine (Boc)-NCA. (CAS# 33043-60-6), Ornithine (Boc)-NCA, (CAS# 96165-58-1), Lysine (Z)-NCA (also known as Lysine (Cbz)-NCA), (CAS# 1676-86-4), Ornithine (Z)-NCA (also known as Ornithine (Cbz)-NCA). (CAS# 13296-21-4) and lysine (TFA)-NCA, (CAS# 42267-27-6), etc.

[0046] In this disclosure, a variety of primary and secondary amine initiators can be used. In some embodiments, an amine iodide initiator can be used to provide radiation impermeability to the resulting peptide. In some of these embodiments, the amine iodide initiator is a compound comprising a primary or secondary amine group, more typically a primary amine group, and one or more iodinated aromatic groups.

[0047] Examples of iodinated aromatic groups include iodine-substituted monocyclic aromatic groups and iodine-substituted polycyclic aromatic groups, such as iodine-substituted phenyl, iodine-substituted naphthyl, iodine-substituted anthraceneyl, iodine-substituted phenanthryl, and iodine-substituted tetraphenyl. The aromatic group can be substituted with one, two, three, four, five, six, or more iodine atoms. In some embodiments, the aromatic group can also be substituted with one or more hydrophilic groups; for example, the aromatic group can be substituted with one, two, three, four, five, six, or more hydrophilic groups. The one or more hydrophilic groups can include, for example, one or more of the following groups: hydroxyl, hydroxyalkyl (e.g., C1-C4-hydroxyalkyl containing one, two, three, or four carbon atoms and containing one, two, three, or four or more hydroxyl groups), and ester (e.g., C2-C6-ester containing two, three, four, five, six, etc. carbons), etc. One or more hydrophilic groups may be attached to an aromatic group directly or through any suitable linker, which may be selected from, for example, alkyl (e.g., alkyl containing one, two, three, four, etc.), amide, amino, ether, ester, or carbonate groups.

[0048] Specific examples of iodinated amine initiators used in this disclosure include 5-amino-N,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodophthalamide. (CAS# 76801-93-9) (A well-known biocompatible radiocontrast agent precursor, also known as iohexol-related compound J), TMS-iohexol J, 5-Amino-N1,N3-bis(2,3-dihydroxypropyl)-2,4-diiodophthalamide (CAS# 1215856-35-1), Dimethyl 5-amino-2,4,6-triiodo-1,3-phthalate, (CAS# 154921-11-6), And bis-diazotate (w / y-linker) .

[0049] Examples of iodinated amine initiators also include iodinated amino acid esters, such as C1-C5-alkyl esters of iodinated amino acids, preferably methyl esters of iodinated amino acids. Specific examples include C1-C5-alkyl esters of any of the following iodinated amino acids. After polymerization, the C1-C5-alkyl ester can be converted to the corresponding carboxylic acid if desired.

[0050] As used herein, an "amino acid" is an organic compound that comprises an amino group (-NH2), a carboxylic acid group (-COOH), and side groups specific to each amino acid. Depending on the surrounding pH, the amino group may be positively charged (-NH3). + ) and / or the carboxylic acid group may be negatively charged (-COO) - Iodinated amino acids are amino acids whose side groups contain one or more iodine atoms.

[0051] In various embodiments, the side groups of the iodinated amino acid include one, two, three, four, five, six, seven, eight, or more iodinated aromatic groups. One or more iodinated aromatic groups may be directly attached to the remainder of the amino acid, attached to the remainder of the amino acid via a suitable linker selected from, for example, alkyl groups (e.g., C1-C4-alkyl groups containing one, two, three, four, etc.), amide groups, amino groups, ether groups, ester groups, or carbonate groups, or attached via a suitable linker to another iodinated aromatic group selected from, for example, alkyl groups (e.g., C1-C4-alkyl groups containing one, two, three, four, etc.), amide groups, amino groups, ether groups, ester groups, or carbonate groups. Examples of iodinated aromatic groups include iodine-substituted monocyclic aromatic groups and iodine-substituted polycyclic aromatic groups, such as those listed above.

[0052] Examples of iodinated amino acid esters include iodinated α-amino acid esters, iodinated β-amino acid esters, iodinated γ-amino acid esters, iodinated δ-amino acid esters, and iodinated ε-amino acid esters.

[0053] Specific examples of iodinated amino acid ester initiators include: monoiodophenylalanine methyl ester, Monoiodotyrosine methyl ester diiodotyrosine methyl ester , Triiodothyronine methyl ester (also known as T3 methyl ester). T4 methyl thyroxine (also known as thyroxine methyl ester or T4 methyl ester). thyroxine methyl ester And bisthyroxine methyl ester (w / multi-arm connector). And 6-iodo-L-DOPA methyl ester, etc. Although methyl esters are shown, higher alkyl esters can be used.

[0054] Now refer to Figure 2 The description includes specific examples of procedures according to this disclosure, wherein radiopaque peptide compounds are formed by the ring-opening polymerization of protected amino acid NCA. Figure 2In the ring-opening polymerization step, thyroxine methyl ester (210) is used as an iodide initiator, and lysine (Cbz)-NCA (212) is used as an NCA monomer to form a protected radiopaque peptide compound (214) comprising a Cbz-protected trilysine amino acid chain covalently linked to thyroxine methyl ester residues via an amide bond. In a subsequent step, the protected radiopaque peptide compound (214) is deprotected by hydrogenation in acetic acid to form a final radiopaque peptide compound (216) comprising a trilysine amino acid chain covalently linked to thyroxine methyl ester residues via an amide bond.

[0055] Because it needs to form a tripeptide, therefore... Figure 2 The ring-opening polymerization uses a monomer to initiator ratio of approximately 3:1. For tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, etc., the corresponding monomer to initiator ratios are typically approximately 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc. It should be understood that the peptide synthesis schemes disclosed herein are relative to... Figure 1 The main advantage of this peptide synthesis scheme is that peptides of increased length can be formed by increasing the monomer-to-initiator ratio without requiring any additional synthetic steps. As discussed further below, the peptide products of this disclosure have the potential to be used as cross-linking agents due to the presence of primary amine groups. The synthetic procedure described in this disclosure allows for the adjustment of the number of primary amine cross-linking groups without requiring additional synthetic steps that would otherwise increase the cost of cross-linking agents.

[0056] It should be noted that the products of the ring-opening polymerization process described herein will be statistical products, meaning that peptides of varying lengths will be produced. For example, in the case of the lysine-thyroxine conjugates described above, the majority of the product is a trilysine-thyroxine conjugate; however, other conjugates (including dilysine-thyroxine and tetralysine-thyroxine conjugates) are also expected. Therefore, in various embodiments, the products of the ring-opening polymerization process described herein can be subjected to further purification steps to separate the desired peptide product (in this case, the trilysine product) from products of varying lengths (which will have lower or higher molecular weights). Techniques for separating compounds based on their molecular weight include high-performance liquid chromatography (HPLC), simulated moving bed chromatography, ion exchange separation, and membrane filtration, among others.

[0057] It is also noted that the alkyl ester products formed in this paper can be converted into carboxylic acid products by hydrolysis.

[0058] Figure 3AAnother example of the procedure according to this disclosure is shown, wherein bisthyroxine methyl ester (w / multi-arm linker) (310) is used as an iodide initiator, and lysine (Cbz)-NCA (312) is used as an NCA monomer in the ring-opening polymerization step to form a protected radiopaque peptide compound (314) comprising a Cbz protected trilysine amino acid chain covalently linked to the bisthyroxine residues via amide bonds. A glutamic acid multi-arm linker is shown as an example herein, but any linear linker or multi-arm linker may be used. In a subsequent step, the protected radiopaque peptide compound (314) is deprotected by hydrogenation in acetic acid to form a final radiopaque peptide compound (316) comprising a trilysine amino acid chain covalently linked to the bisthyroxine methyl ester residues via amide bonds.

[0059] Figure 3B The present disclosure illustrates the method for forming Figure 3A The procedure for the dithyroxine methyl ester compound (310). For example... Figure 3B As shown, thyroxine methyl ester (302), 1-ethyl-3-(3-dimethyl-propyl)carbodiimide (EDC) coupling agent, t-BOC-protected α-glutamic acid (304), and a catalytic amount of 4-dimethylaminopyridine (DMAP) were dissolved in dimethylformamide (DMF). The reaction mixture was stirred at 50°C for 24 hours. After the coupling reaction, the solvent was removed under vacuum, and the t-BOC-protected product (306) was dissolved in ethyl acetate and washed with brine. The ethyl acetate was then removed under vacuum. The t-BOC-protected product (306) was dissolved in methanol and deprotected by acidification with HCl. The solution was stirred overnight at room temperature. After the reaction, the solvent was removed under vacuum to give dithyroxine methyl ester (w / multi-arm linker) (310).

[0060] Figure 4A Another example of the procedure according to this disclosure is shown, wherein a bidiazotate (w / multi-arm linker) (410) is used as an amine iodide initiator, and lysine (Cbz)-NCA (412) is used as an NCA monomer in the ring-opening polymerization step to form a protected radiopaque peptide compound (414) comprising a Cbz-protected trilysine amino acid chain covalently linked to the bidiazotate residues via amide bonds. In a subsequent step, the protected radiopaque peptide compound (414) is deprotected by hydrogenation in acetic acid to form a final radiopaque peptide compound (416) comprising a trilysine amino acid chain covalently linked to a dimethyl ester residue via amide bonds.

[0061] Figure 4B The present disclosure illustrates the method for forming Figure 4A The procedure for the didiazotate compound (410). For example... Figure 4BAs shown, diatrizoate (402), t-boc-protected tri-2-aminoethylamine (404), EDC coupling agent, and a catalytic amount of DMAP were dissolved in DMF. The reaction mixture was stirred at 50 °C for 24 hours. After the coupling reaction, the solvent was removed under vacuum, and the t-boc-protected product (not shown) was dissolved in ethyl acetate and washed with brine. The ethyl acetate was then removed under vacuum. The t-boc-protected product was dissolved in methanol and acidified with HCl. The solution was stirred overnight at room temperature. After the reaction, the solvent was removed under vacuum to give bisdiatrizoate (w / multi-arm linker) (410).

[0062] Figure 5A Another example of the procedure according to this disclosure is shown, wherein acetal-protected iodixanol (w / multi-arm linker) (510) is used as an iodide initiator, and lysine (Cbz)-NCA (512) is used as an NCA monomer in the ring-opening polymerization step to form a radiopaque acetal-protected and Cbz-protected radiopaque peptide compound (514). In a subsequent step, the radiopaque acetal-protected and Cbz-protected radiopaque peptide compound (514) is deprotected to form a final radiopaque trilysine product (516), wherein the trilysine oligomer is linked to the iodixanol residue via an amide bond.

[0063] Figure 5B This disclosure illustrates a method that can be used to form Figure 4A The procedure for the diiodixanol compound. For example... Figure 4B As shown, acetal-protected iodixanol (502), t-boc-protected 3-[(2-aminoethyl)(2-carboxyethyl)amino]propionic acid dihydrochloride (504) (CAS: 141702-92-3 of the parent compound), and EDC coupling agent and a catalytic amount of DMAP were dissolved in DMF. The reaction mixture was stirred at 50°C for 24 hours. After the coupling reaction, the solvent was removed under vacuum, and the product (not shown) was dissolved in ethyl acetate and washed with brine. Then, the ethyl acetate was removed under vacuum. The product was dissolved in methanol and acidified with HCl. The solution was stirred overnight at room temperature. After the reaction, the solvent was removed under vacuum to give bis(iodixanol) (w / multi-arm linker) compound (516), which is the deprotected form of bis(iodixanol) (w / multi-arm linker) compound (510).

[0064] In other embodiments of this disclosure, the polymerization of amino acid NCA is initiated by a hydroxyl-containing initiator. The polymerization is carried out via acid catalysis followed by base quenching and polymerization, wherein the acid catalyst is methanesulfonic acid. Base quenching can be performed using N-ethyldiisopropylamine or triethylamine.

[0065] In this disclosure, a variety of hydroxyl-containing initiators can be used. In some embodiments, an iodinated hydroxyl-containing initiator can be used to provide transmissivity linearity for the resulting peptide. In some of these embodiments, the hydroxyl-containing initiator is a compound comprising a hydroxyl group and 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 those listed above. In some embodiments, one or more hydroxyl groups are present in the hydroxyl-containing cyclic substituent of the iodine-substituted monocyclic aromatic group or the iodine-substituted polycyclic aromatic group. For example, the iodine-substituted monocyclic aromatic group and / or the iodine-substituted polycyclic aromatic group can be substituted with one or more C1-C6-hydroxyalkyl groups. When two or more hydroxyl groups are present on the initiator, branched peptides can be formed.

[0066] A specific example of a hydroxyl-containing initiator is iodixanol (which is a well-known biocompatible radiocontrast agent precursor). wait.

[0067] Figure 6 Another example of the procedure according to this disclosure is shown, wherein acetal-protected iodixanol (610; CAS: 192449-65-3, synthesized from parent iodixanol) is used as an iodinated hydroxyl-containing initiator, and lysine (Cbz)-NCA (612) is used as the NCA monomer in the ring-opening polymerization step to form a radiopaque acetal-protected and Cbz-protected radiopaque peptide compound (614). In a subsequent step, the radiopaque acetal-protected and Cbz-protected radiopaque peptide compound (614) is deprotected by acetal deprotection (treatment with iodine chloride, followed by sodium hydroxide treatment) and Cbz deprotection (treatment with Pd(OAc)2 and a hydrogen source or mercaptoethanol) to form a final radiopaque trilysine product (616), wherein the trilysine oligomer is linked to the iodixanol residues via ester bonds.

[0068] 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 provide a primary amine group attached to iodixanol via an amide-based linker.

[0069] Note that although the Cbz protection of lysine NCA is shown in Figure 12-6, other types of protection can be used, including the protective groups described above. See, for example, Brian V. Falcone et al., “Synthesis of bis-Phenylalanine, A Novel Eight-Membered Cyclic Dipeptide” Synthetic Communications, 38: 411–418, 2008, which describes Boc deprotection in the presence of aryl iodides, particularly scheme 1, where Boc deprotection is accomplished under condition a, FMOC protection is performed under condition b, and the deprotected amine is exposed to 4N HCl in dioxane.

[0070] It should also be noted that although this article specifically describes the iodine group, other radiopaque halogen groups, including bromine, may also be used.

[0071] It should also be noted that although the initiators described above are iodinated initiators, non-iodinated initiators can also be used in this disclosure. Specific examples of non-iodinated initiators include ammonium chloride, hexamethyldisilazane (CAS 999-97-3), which, upon deprotection by a fluoride source, yields trilysine and other aliphatic amines, or trimethylsilyl-protected aliphatic amines. Furthermore, transition metal initiators can be used to polymerize NCA monomers. These transition metal complexes can include cobalt, nickel, etc., and can be removed after polymerization by precipitation or dialysis.

[0072] In other embodiments, radiation impermeability can be introduced into the final product by using iodinated amino acid NCA derivatives in ring-opening synthesis. For example, in some embodiments, protected iodinated phenylalanine NCA or protected iodinated tyrosine NCA can be used in ring-opening synthesis. These NCAs can be used to form statistical copolymers, gradient copolymers, or block copolymers with NCA derivatives containing protected amines.

[0073] In some aspects of this disclosure, crosslinked hydrogels are provided that comprise the following crosslinking reaction products: (a) a peptide compound according to this disclosure, said peptide compound comprising initiator residues and an amino acid chain comprising a plurality of amino acids having primary amine side groups, and (b) a reactive polymer comprising a reactive moiety.

[0074] Such cross-linked hydrogels can be formed in vivo (e.g., using the delivery device described below), or they can be formed in vitro and subsequently applied to a subject. Such cross-linked hydrogels can be used in a variety of biomedical applications, including implants, medical devices, and pharmaceutical compositions.

[0075] In some 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).

[0076] 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.

[0077] 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.

[0078] 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).

[0079] The reactive part includes the part containing electrophilic groups.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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-hexanone segments; and polyester segments, including polyglycolic acid segments. Polylactic acid segments, poly(lactic acid-co-glycolic acid) segments, poly(β-propiolactone) 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, 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.

[0084] The polymer segments used in the multi-arm polymers of this disclosure typically contain 10 to 1000 monomer units or more.

[0085] 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.

[0086] 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, hexamethylenetetramine, galactitol, fucose, ribose, arabinose, xylose, etc. Lysol, rhamnose, galactose, glucose, fructose, sorbitol, mannose, pyranose, azoose, 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.).

[0087] Exemplary polyols also include polyhydroxylated polymers. For example, in some embodiments, the core region comprises polyhydroxylated polymer residues, such as poly(vinyl alcohol) residues, poly(allyl alcohol), polyhydroxyethyl acrylate residues, or polyhydroxyethyl methacrylate residues. The length of such polyhydroxylated polymer residues can range, for example, from 3 to 100 monomer units.

[0088] 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, wherein n is an integer of at least 6, typically 6, 8, 10, or 12 (thus having T6, T8, T10, or T12 cage-like siloxane cores respectively), wherein R can be selected from a range of organic functional groups, such as alkyl, aryl, alkoxy, and polymer arms. T8 cage-like siloxane cores have been extensively studied, with the formula [RSiO]. 3 / 2 ]8, or equivalently R8Si8O 12 The structure is as follows: In this disclosure, the R group includes the polymer arm described herein.

[0089] 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.

[0090] The aforementioned cyclic anhydrides can react with hydroxyl-terminated precursor multi-arm hydrophilic polymers under alkaline conditions to form carboxylic acid-terminated precursor polymers containing carboxylic acid end groups, which are connected to the hydrophilic polymer segments via hydrolyzable ester groups.

[0091] The reactive portion can then be attached to a carboxylic acid-terminated precursor polymer.

[0092] 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.

[0093] 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-hydroxycyclic 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 group, bicyclic [2.2.1]hept-5-ene-2,3-dicarboxylic acid imide succinate group, bicyclic [2.2.1]hept-5-ene-2,3-dicarboxylic acid imide adipate 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.

[0094] In some aspects of this disclosure, a system is provided comprising (a) a first composition comprising a 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 peptide compound under conditions such that a covalent crosslink is formed between the reactive polymer and the peptide compound.

[0095] The first composition may be a first fluid composition comprising a peptide compound or a first dry composition comprising a 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 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).

[0096] 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 additional reagents (including therapeutic agents, imaging agents, colorants, tension modifiers, suspending agents, wetting agents, and pH adjusters as described below).

[0097] In some embodiments, the system is configured to mix a first fluid composition comprising a peptide compound with a second fluid comprising a reactive polymer. During mixing of the first and second fluid compositions, the peptide compound crosslinks with the reactive polymer to form a crosslinked product. The system can mix the first and second fluid compositions in vivo or in vitro to form a crosslinked hydrogel.

[0098] In some embodiments, the peptide compound is initially mixed with the reactive polymer under conditions where crosslinking between the peptide compound and the reactive polymer 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 peptide compound and the reactive polymer, thereby forming a crosslinked product.

[0099] In some embodiments, the system includes (a) a first composition comprising a peptide compound as described above, (b) a second composition comprising a 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 peptide compound and the reactive polymer.

[0100] The first composition may be a first fluid composition comprising a peptide compound buffered to an acidic pH or a first dry composition comprising said 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 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 peptide compound may have a pH in the range of, for example, about 3 to about 5. In addition to the 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).

[0101] 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 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).

[0102] In one particular embodiment, the first composition is a first fluid composition comprising a peptide compound buffered to an acidic pH, and the second composition comprises a dry composition comprising a reactive polymer. The first composition and the second composition can then be mixed to provide a preparative fluid composition buffered to an acidic pH and comprising the peptide compound and the reactive polymer. In one specific example, a syringe containing the first fluid composition comprising the peptide compound buffered to an acidic pH can be provided, and a vial containing the dry composition comprising the reactive polymer (e.g., powder) can be provided. The first fluid composition can then be injected into the vial containing the reactive polymer using the syringe to form a preparative fluid composition buffered to an acidic pH and comprising the peptide compound and the reactive polymer, which can then be withdrawn back into the syringe for administration.

[0103] 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.

[0104] The preparative fluid composition containing peptide compounds and reactive polymers buffered to an acidic pH 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.

[0105] Other agents used in the compositions described herein include therapeutic agents, imaging agents, colorants, tension modifiers, suspending agents, wetting agents, and pH adjusters.

[0106] 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.

[0107] 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, metrizoate, or ioxaglate; and iodized oils, including ethiodized poppyseed oil (available as Lipiodol®).

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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 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, the first reservoir containing a first fluid composition comprising a peptide compound and a reactive polymer and buffered to an acidic pH, such as the aforementioned prepared fluid composition, and the second reservoir containing a second fluid composition, such as the aforementioned fluid accelerator composition.

[0112] 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 peptide compound and the reactive polymer crosslink with each other to form a crosslinked hydrogel.

[0113] In a particular implementation scheme, and referring to Figure 7 The system may include a delivery device 710 comprising a dual-tube syringe, the dual-tube syringe including a first tube 712a having a first tube outlet 714a (the first tube contains a first composition and a first plunger 716a movable within the first tube 712a), and a second tube 712b having a second tube outlet 714b (the second tube 712b contains a second composition and a second plunger 716b movable within the second tube 712b). In some embodiments, the device 710 may further include a mixing section 718 having a first mixing section inlet 718a1 in fluid communication with the first tube outlet 714a, a second mixing section inlet 718b1 in fluid communication with the second tube outlet, and a mixing section outlet 718o.

[0114] 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).

[0115] 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.

[0116] 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 ultimately crosslink to form a crosslinked hydrogel, which is then applied to or within the subject's tissue. For example, the first and second fluid compositions may 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.

[0117] 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.

[0118] 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.

[0119] For example, in applications such as the treatment of diseases and cancers, as well as the repair and regeneration of tissues, 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 substitutes 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; and / or the first fluid composition and the second fluid composition or a fluid mixture thereof can be injected as carriers of therapeutic agents.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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 provided 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).

[0127] Figure 8 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.

[0128] The cross-linked hydrogel compositions described herein can be used for a variety of purposes.

[0129] 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.

[0130] After application, the cross-linked hydrogel composition of this disclosure can be imaged using suitable imaging techniques.

[0131] 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.

[0132] 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.

[0133] 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 comprising: (a) performing ring-opening polymerization of one or more types of amino acid N-carboxylic anhydride (NCA) monomers in the presence of an initiator compound to produce an intermediate peptide compound, said amino acid N-carboxylic anhydride (NCA) monomer comprising at least one type of protected amino acid NCA monomer having protected side chain amine groups, said intermediate peptide compound comprising an amino acid chain having protected side chain amine groups covalently linked to an initiator residue; and (b) deprotecting said intermediate peptide compound to form a final peptide compound comprising an amino acid chain having side chain amine groups covalently linked to said initiator residue.

2. The method of claim 1, further comprising separating the final peptide compound according to molecular weight to provide a final peptide compound having amino acid chains of equal length.

3. The method according to any one of claims 1-2, wherein the molar ratio of the amino acid N-carboxylic anhydride (NCA) monomer to the initiator compound ranges from 2:1 to 100:

1.

4. The method according to any one of claims 1-3, wherein the length of the amino acid chain ranges from 2 to 50 amino acids.

5. The method according to any one of claims 1-4, wherein the protected side chain amine group is a protected primary amine group.

6. The method according to any one of claims 1-4, wherein the protected side chain amine group is a protected alkyl amine group.

7. The method according to any one of claims 1-6, wherein the protected side chain amine group comprises a protective group selected from the group consisting of tert-butoxycarbonyl, carboxybenzyl, trifluoroacetyl, 6-nitroveratroloxycarbonyl, and 9-fluorenylmethoxycarbonyl.

8. The method according to any one of claims 1-7, wherein the at least one type of protected amino acid NCA monomer comprises a protected lysine NCA monomer and / or a protected ornithine NCA monomer.

9. The method according to any one of claims 1-8, wherein the initiator comprises a primary amino group or an aliphatic hydroxyl group.

10. The method according to any one of claims 1-8, wherein the initiator comprises an amine protected by a trimethylsilyl group.

11. The method according to any one of claims 1-8, wherein the initiator comprises (a) a primary amino or aliphatic group and (b) an iodinated aromatic group.

12. The method according to any one of claims 1-8, wherein the initiator comprises (a) a primary amino group or an aliphatic group, which is connected to (b) an iodinated aromatic group via a linear linker or a multi-arm linker.

13. The method according to any one of claims 1-8, wherein the initiator comprises an iodinated amino acid ester.

14. The method according to any one of claims 1-13, wherein the one or more types of amino acid N-carboxylic anhydride (NCA) monomers comprise a single type of protected amino acid NCA monomer.

15. The method according to any one of claims 1-13, wherein the one or more types of amino acid N-carboxylic anhydride (NCA) monomers comprise at least one type of iodinated amino acid NCA monomer.

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