Conjugate compounds and methods for producing conjugate compounds

Abstract

The purpose of the present invention is to provide a conjugate compound capable of inhibiting complement activation. The conjugate compound contains: a polymer (A) which has a constitutional unit derived from a monomer (a) having two or more hydroxyl groups in the molecule and having 2-10 carbon atoms constituting the side chain among the carbon atoms of the constitutional unit; and a component (B) containing at least one selected from the group consisting of an amino acid, a polypeptide, a protein, a nucleoside, a nucleotide, and a nucleic acid.

Description

[Technical Field]

[0001] The present invention relates to a conjugate compound and a method for producing the conjugate compound. The conjugate compound of the present invention can be suitably used as a pharmaceutical additive or a pharmaceutical. [Background technology]

[0002] In recent years, active efforts have been made to develop drug delivery system (DDS)-based formulations. Polyethylene glycol (PEG) is widely used in recent DDS formulations to improve blood (body) residence time and formulation stability. For example, PEG-modified liposomes, which are liposomes or polymeric micelles modified with PEG, are used as drug carriers as long-term blood-retention liposomes, and formulations such as Doxil (registered trademark) in which doxorubicin is encapsulated are being used clinically. PEG has a simple backbone structure that makes it highly flexible. It also has the property of being able to hydrate many water molecules, so by modifying drug particles or carriers with PEG, a thick hydration layer is formed on the particle surface. This hydration layer is known to suppress interactions with serum proteins and cells, resulting in a significant extension of the drug's residence time in the blood (body) (stealth).

[0003] For example, Patent Document 1 describes a method for producing a PEGylated lactoferrin complex in which linear polyethylene glycol (PEG) or a modified product thereof is covalently bonded to lactoferrin via an amide bond, which method is characterized by comprising a step of reacting a reaction solution containing lactoferrin and a linear PEG derivative having a paranitrophenyl group under conditions in which an amide group is formed between the paranitrophenyl group and lactoferrin, thereby maintaining the important biological activity of lactoferrin based on its iron-binding ability, and because the bond with the linear PEG derivative confers resistance to proteases such as pepsin, it has a long lifespan in the body and can exert its biological activity for a long period of time in the body.

[0004] Patent Document 2 describes that polyethylene glycol-modified human interferon α2b (IFN-α2b) having the following structure, obtained by linking polyethylene glycol (YPEG) having a Y-branched structure to human interferon α2b (IFN-α2b), is used in the production of a pharmaceutical composition for treating viral infections such as hepatitis C.

[0005] [ka]

[0006] (wherein Pa and Pb are the same or different polyethylene glycols, j is an integer of 1 to 12, Ri is hydrogen, a substituted or unsubstituted C1-12 alkyl group, a substituted aralkyl group, an aryl group, or a heteroalkyl group, X1 and X2 are each independently a linking group, and X1 is (CH2) n and X2 is (CH2) n , (CH2) n OCO, (CH2) n NHCO, (CH2) n CO, where n is an integer of 1 to 10, and YPEG is linked to IFN-α2b via an amide bond with the ε-amino group in the side chain of the lysine of hIFN-α2b corresponding to position 134 of SEQ ID NO: 1. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2009 / 113743 [Patent Document 2] Special Publication No. 2010-538022 Summary of the Invention

[0008] However, in recent years, it has been reported that PEGylated drugs modified with PEG can cause harmful immune reactions due to unintended complement activation in the body, and that frequent administration reduces their pharmacological effect. Therefore, there has been a strong demand for alternative compounds to PEGylated drugs that can inhibit complement activation.

[0009] The inventors have conducted research in light of the above-mentioned problems and have discovered a conjugate compound as an alternative to PEGylated pharmaceuticals, which comprises a polymer (A) having a structural unit derived from a monomer (a) which has two or more hydroxyl groups and in which the number of carbon atoms constituting the side chain among the carbon atoms of the structural unit is 2 to 10, and a component (B) containing at least one member selected from the group consisting of amino acids, polypeptides, proteins, nucleosides, nucleotides and nucleic acids, and have completed the present invention. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a photograph of the gel obtained by subjecting conjugate 1 described in Example 1 to polyacrylamide electrophoresis (SDS-PAGE) and then staining the gel. [Figure 2] 1 shows the results of evaluating the complement activation of conjugates 1 to 4 described in Example 1, a conjugate in which maleimide PEG is used instead of polymer 1 in Example 1, and BSA. [Figure 3] 1 shows the results of analyzing conjugate 2 described in Example 1 by size exclusion chromatography. [Figure 4]1 shows the results of a nucleic acid degradation test for conjugate 5 described in Example 2 and an unmodified oligonucleic acid. [Figure 5] 1 shows the results of evaluating complement activation of conjugates 5 and 6 described in Example 2 and a conjugate in which maleimide PEG was used instead of polymer 1 in Example 1. [Figure 6] 1 shows the results of a cytotoxicity test using polymer 1, polymer 2, conjugates 1 and 4 described in Example 1, conjugate 5 described in Example 2, and conjugate 7 described in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0011] The conjugate compound of the present disclosure is characterized by being formed from a polymer (A) having structural units derived from a monomer (a) that has two or more hydroxyl groups and in which the number of carbon atoms constituting the side chain among the carbon atoms of the structural unit is 2 to 10, and a component (B) containing at least one member selected from the group consisting of amino acids, polypeptides, proteins, nucleosides, nucleotides, and nucleic acids. The conjugate compound of the present disclosure provides a novel conjugate compound modified with a compound other than PEG.

[0012] The conjugate compound of the present disclosure can be obtained by synthesizing a polymer (A) having a functional group introduced at its terminal and then reacting the polymer (A) with a component (B) containing at least one selected from the group consisting of amino acids, polypeptides, proteins, nucleosides, nucleotides, and nucleic acids. The functional group of the polymer (A) having a functional group introduced at its terminal may be introduced by a polymerization reaction, or may be introduced by further reaction with a compound having a functional group after polymerization. One feature of the conjugate compound of the present disclosure is that the polymer (A) is bonded to the component (B) containing at least one selected from the group consisting of amino acids, polypeptides, proteins, nucleosides, nucleotides, and nucleic acids through a chemoselective reaction.

[0013] Preferred embodiments of the present invention will be described below. However, the present invention is not limited to the following embodiments. Furthermore, a combination of two or more of the individual preferred embodiments of the present invention described below is also a preferred embodiment of the present invention. In this specification, the range "X to Y" means "X or more and Y or less," and "weight" and "mass" are treated as synonyms. Furthermore, in this specification, "(meth)acrylate" means acrylate or methacrylate, and "(meth)acrylic" means acrylic or methacrylic, and acrylate and methacrylate may be used alone or in combination. Furthermore, unless otherwise specified, measurements of operations and physical properties are performed at room temperature (20 to 25°C) and a relative humidity of 40 to 50%.

[0014] <Polymer (A)> In the present disclosure, a structural unit derived from a monomer (a) having two or more hydroxyl groups and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit refers to a unit that is formed by polymerizing one of the polymerizable unsaturated double bonds of a monomer (a) having two or more hydroxyl groups and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit, and that forms a part of a polymer as a result of opening the polymerizable unsaturated double bond. The structural unit derived from monomer (a) may be a structural unit formed by a different manufacturing method, as long as it has the same structure as the structural unit formed by polymerization of monomer (a) as described above. In this specification, unless otherwise specified, a structural unit derived from monomer (a) refers to a structural unit derived from one molecule of monomer (a), and does not refer to the entire structural unit contained in the polymer. Furthermore, a structural unit derived from monomer (b) described below is interpreted in the same way as the structural unit derived from monomer (a) above.

[0015] The monomer (a) of the present disclosure, which has two or more hydroxyl groups and in which the number of carbon atoms constituting the side chain among the carbon atoms of the constituent unit is 2 to 10, is preferably a vinyl monomer, more preferably a (meth)acrylic monomer. Furthermore, the monomer (a) may be a monofunctional monomer or a polyfunctional monomer, but preferably contains a monofunctional monomer, and more preferably consists of a monofunctional monomer.

[0016] The number of hydroxyl groups contained in the molecule of the monomer (a) is two or more, for example, 2 to 8, 2 to 6, or 2 to 4 hydroxyl groups.

[0017] As the monomer (a) of the present disclosure, (meth)acrylates such as glycerin mono(meth)acrylate (also known as 2,3-dihydroxypropyl(meth)acrylate), 1,2-dihydroxyethyl(meth)acrylate, 2,2-dihydroxyethyl(meth)acrylate, dihydroxybutyl(meth)acrylate, trimethylolpropane mono(meth)acrylate, pentaerythritol mono(meth)acrylate, dipentaerythritol mono(meth)acrylate, etc. Among these, it is preferable that the monomer (a) contains glycerin monoacrylate (GLMA) and / or glycerin monomethacrylate (GLMMA) in view of industrial availability, high reactivity, high binding affinity between the resulting polymer and component (B), and complement activation inhibitory effect. For example, the content of glycerin monoacrylate (GLMA) and / or glycerin monomethacrylate (GLMMA) in the monomer (a) is, in order of preference, 20 parts by mass or more (up to 100 parts by mass), 40 parts by mass or more, 60 parts by mass or more, 80 parts by mass or more, 90 parts by mass or more, and 95 parts by mass or more. By polymerizing these monomers (a), the ethylenic double bond contained in the monomer (a) is cleaved to generate a structural unit. Note that only one type of monomer (a) may be used alone, or two or more types may be used in combination.

[0018] Furthermore, among the carbon atoms of the structural unit derived from monomer (a), the number of carbon atoms constituting the side chain is 2 to 10, but in this specification, the term "side chain" refers to the portion other than the main chain. "The number of carbon atoms constituting the side chain is 2 to 10" refers to the (total) number of carbon atoms in the entire side chain (all (four) groups bonded to carbon atoms in the main chain). The term "main chain" refers to the chain of continuously bonded carbon atoms in a polymer formed by linking structural units, that has the largest number of carbon atoms. As mentioned above, among the carbon atoms of the structural unit derived from monomer (a), the number of carbon atoms constituting the side chain is 2 to 10, but this number is preferably 3 to 8, and more preferably 4 to 6. Furthermore, the side chain possessed by the structural unit derived from monomer (a) may be an unsubstituted or substituted alkyl group having 2 to 10 carbon atoms, and the substituent may be a hydroxyl group, and the substituent may contain two or more hydroxyl groups.

[0019] The constituent unit derived from the monomer (a) of the present disclosure preferably includes a constituent unit represented by the following general formula (5).

[0020] [ka]

[0021] In general formula (5), R1 represents a hydrogen atom or a methyl group, and X represents -C(=O)-O-, -C(=O)-NH-, -O-, -CHO-, or -CHCHO-, preferably -C(=O)-O-. The content of the structural unit represented by general formula (5) among the structural units of monomer (a) is, for example, in order of preference, 20 parts by mass or more (upper limit 100 parts by mass), 40 parts by mass or more, 60 parts by mass or more, 80 parts by mass or more, 90 parts by mass or more, and 95 parts by mass or more.

[0022] Among the structural units represented by the general formula (5) above, those in which R1 is a hydrogen atom and X is -C(=O)-O- are derived from glycerin monoacrylate (GLMA) as the monomer (a).Furthermore, among the structural units represented by the chemical formula (1) above, those in which R1 is a methyl group and X is -C(=O)-O- are derived from glycerin monomethacrylate (GLMMA) as the monomer (a).

[0023] The content of the structural unit derived from the monomer (a) in 100 parts by mass of the polymer (A) of the present disclosure is preferably 5 parts by mass or more (upper limit 100 parts by mass), more preferably 20 parts by mass or more, even more preferably 40 parts by mass or more, and may be 60, 80, 90, 95 parts by mass or more, or even 100 parts by mass.

[0024] When the polymer (A) of the present disclosure contains a structural unit other than the monomer (a), the structural unit other than the monomer (a) may be derived from any radically polymerizable monomer (hereinafter, a monomer that becomes a structural unit other than the monomer (a) by copolymerization is also referred to as "monomer (b)"). When the polymer (A) contains a structural unit derived from the monomer (b), the proportion of the structural unit derived from the monomer (b) in 100 parts by mass of the polymer (A) is, for example, 99 parts by mass or less, preferably 80 parts by mass or less, more preferably 50 parts by mass or less, even more preferably 40 parts by mass or less, still more preferably 20 parts by mass or less, and particularly preferably 10 parts by mass or less.

[0025] Examples of the monomer (b) include, other than the monomer (a), hydroxyl group-containing (meth)acrylates, polyoxyalkylene group-containing monomers, alkoxyalkyl (meth)acrylates, vinyl monomers, cyclic compounds, etc. These monomers (b) may also be used alone or in combination of two or more.

[0026] Examples of the hydroxyl group-containing (meth)acrylate include hydroxyalkyl (meth)acrylates in which the hydroxyalkyl group has 2 to 4 carbon atoms, such as 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate.

[0027] Examples of the polyoxyalkylene group-containing unsaturated monomer include a monomer represented by the following general formula (6).

[0028] [ka]

[0029] In general formula (6), R 2 , R 3 and R 4 each independently represents a hydrogen atom or a methyl group, and R 5 represents an alkylene group having 2 to 18 carbon atoms, and R 6 represents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms, Y represents an alkylene group having 1 to 5 carbon atoms, a -CO- group, or R 2 R 4 C=CR 3 When the - group is a vinyl group, it represents a direct bond, and m represents -(R 5 O)-groups and represents a number of 1 to 300. 5 O) m There are two or more types of R 5 When the R 5 O may be bonded in any of random, block or alternating forms.

[0030] In general formula (6), R 6 is a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms. 6Of these, a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms is more preferred, a hydrogen atom or a hydrocarbon group having 1 to 3 carbon atoms is even more preferred, and a hydrogen atom or a hydrocarbon group having 1 or 2 carbon atoms is even more preferred. Of the hydrocarbon groups, an alkyl group or alkenyl group is preferred, an alkyl group having 1 to 20 carbon atoms is more preferred, an alkyl group having 1 to 10 carbon atoms is even more preferred, and an alkyl group having 1 to 3 carbon atoms is even more preferred.

[0031] In the general formula (6), the formula: -R 5 The oxyalkylene group represented by O- is an oxyalkylene group having 2 to 18 carbon atoms. Examples of the oxyalkylene group include an oxyethylene group, an oxypropylene group, an oxybutylene group, an oxyisobutylene group, an oxy-1-butene group, and an oxy-2-butene group. Among these oxyalkylene groups, an oxyalkylene group having 2 to 8 carbon atoms is preferred, an oxyalkylene group having 2 to 4 carbon atoms such as an oxyethylene group, an oxypropylene group, or an oxybutylene group is more preferred, and an oxyethylene group is even more preferred.

[0032] In the general formula (6), m is a group represented by the formula: -R 5 The average number of moles of oxyalkylene groups added is represented by O-. The average number of moles added means the average number of moles of oxyalkylene groups per mole of the polyoxyalkylene group-containing unsaturated monomer. The lower limit of m is preferably 2 or more, more preferably 4 or more, and even more preferably 8 or more. The upper limit of m is preferably 100 or less, more preferably 50 or less. X is an alkylene group having 1 to 5 carbon atoms, a -CO- group, or R 2 R 4 C=CR 3 When the - group is a vinyl group, it represents a direct bond. Among these groups, the -CO- group is preferred. Examples of polyoxyalkylene group-containing unsaturated monomers include unsaturated alcohol polyalkylene glycol adducts, polyalkylene glycol ester-based monomers, and (alkoxy)polyalkylene glycol monomaleic acid esters.

[0033] The unsaturated alcohol polyalkylene glycol adduct is a compound that polyalkylene glycol chain is added to the alcohol having unsaturated group.As the unsaturated alcohol polyalkylene glycol adduct, for example, polyethylene glycol monovinyl ether, polyethylene glycol monoallyl ether, polyethylene glycol mono (2-methyl-2-propenyl) ether, polyethylene glycol mono (2-butenyl) ether, polyethylene glycol mono (3-methyl-3-butenyl) ether, polyethylene glycol mono (3-methyl-2-butenyl) ether, polyethylene glycol mono (2-methyl-3-butenyl) ether, polyethylene glycol mono (2-methyl-2-butenyl) ether, polyethylene glycol mono (1,1-dimethyl-2-propenyl) ether, polyethylene polypropylene glycol mono (3-methyl-3-butenyl) ether, methoxypolyethylene glycol mono (3-methyl-3-butenyl) ether etc. can be mentioned.

[0034] The polyalkylene glycol ester monomer is a monomer in which an unsaturated group and a polyalkylene glycol chain are bonded via an ester bond.

[0035] The polyalkylene glycol ester monomer is preferably, for example, an ester of an alkoxypolyalkylene glycol in which 1 to 300 moles of an oxyalkylene group having 2 to 18 carbon atoms is added to an alcohol, and (meth)acrylic acid. Among the alkoxypolyalkylene glycols, those containing an oxyethylene group as the main component are preferred. Examples of the alcohol include aliphatic alcohols having 1 to 30 carbon atoms such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-hexanol, 2-hexanol, 3-hexanol, octanol, 2-ethyl-1-hexanol, nonyl alcohol, lauryl alcohol, cetyl alcohol, and stearyl alcohol; alicyclic alcohols having 3 to 30 carbon atoms such as cyclohexanol; and unsaturated alcohols having 3 to 30 carbon atoms such as (meth)allyl alcohol, 3-buten-1-ol, and 3-methyl-3-buten-1-ol. Examples of the esterified products include methoxypolyethylene glycol mono(meth)acrylate, methoxy(polyethylene glycol polypropylene glycol) mono(meth)acrylate, methoxy(polyethylene glycol polybutylene glycol) mono(meth)acrylate, methoxy(polyethylene glycol polypropylene glycol polybutylene glycol) mono(meth)acrylate, etc. Among the polyalkylene glycol ester monomers, for example, (alkoxy)polyalkylene glycol mono(meth)acrylates such as methoxypolyethylene glycol monomethacrylate are preferred.

[0036] Examples of the alkoxyalkyl (meth)acrylate include alkoxyalkyl (meth)acrylates in which the alkoxy group has 1 to 4 carbon atoms and the alkyl group has 1 to 4 carbon atoms, such as methoxymethyl (meth)acrylate, methoxyethyl (meth)acrylate, methoxypropyl (meth)acrylate, ethoxymethyl (meth)acrylate, ethoxyethyl (meth)acrylate, and ethoxypropyl (meth)acrylate. These alkoxyalkyl (meth)acrylates may be used alone or in combination of two or more.

[0037] Examples of the vinyl monomer include (meth)acrylic acid, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, tert-butyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, n-lauryl (meth)acrylate, n-stearyl (meth)acrylate, diaminomethyl (meth)acrylate, diaminoethyl (meth)acrylate, dimethylamino (meth)acrylate, diethylamino (meth)acrylate, glycidyl (meth)acrylate, styrene, aziridines, 2-(meth)acryloyloxymethyl phosphorylcholine, 2-(meth)acryloyloxyethyl phosphorylcholine, tetrahydrofurfuryl (meth)acrylate, isopropylacrylamide, vinyl alcohol, vinylformamide, vinylisobutylacrylamide, (meth)acrylamide, dimethylacrylamide, vinylacetamide, and N-vinylpyrrolidone.

[0038] Examples of the alkylene oxide include alkylene oxides having 2 to 4 carbon atoms, such as ethylene oxide and propylene oxide.

[0039] Examples of the alkoxy polyoxyalkylene glycol include polyethylene glycol, polypropylene glycol, methoxypolyethylene glycol, ethoxypolyethylene glycol, methoxypolypropylene glycol, and ethoxypolypropylene glycol, which have an alkoxy group having 1 to 4 carbon atoms and an oxyalkylene group having 1 to 4 carbon atoms, and the number of moles of the oxyalkylene group added is 2 to 30.

[0040] Examples of the cyclic compound include lactides such as L-lactide, lactones such as ε-caprolactone, trimethyl carbonate, cyclic amino acids, and morpholine-2,5-dione.

[0041] When the polymer (A) is composed of a polymer containing a structural unit derived from the monomer (a), the polymers constituting the polymer may have a structure of a block copolymer obtained by bonding the same or different types of polymers together.

[0042] The number average molecular weight (Mn) of the polymer (A) of the present disclosure is preferably 1,000 or more, more preferably 2,000 or more, even more preferably 3,000 or more, for example, 6,000 or more, from the viewpoint of improving the enzyme resistance of the conjugate compound. Furthermore, the number average molecular weight (Mn) of the polymer is preferably 50,000 or less, more preferably 30,000 or less, even more preferably 15,000 or less, from the viewpoint of excretion from the body. The number average molecular weight (Mn) of the polymer refers to the value measured in accordance with the Mn measurement method for the polymers obtained in Production Examples 1 to 4 in the Examples described below. The number average molecular weight (Mn) of the polymer (A) of the present disclosure is preferably 1,000 to 50,000, and more preferably 3,000 to 30,000.

[0043] The polydispersity (value of [weight average molecular weight (Mw) / number average molecular weight (Mn)]) of the polymer (A) of the present disclosure is preferably 1.00 to 5.00, more preferably 1.00 to 3.00, even more preferably 1.00 to 2.00, even more preferably 1.00 to 1.50, and even more preferably 1.00 to 1.30, from the viewpoint of the uniformity of the molecular weight of the conjugate compound. The weight average molecular weight (Mw) value means the value measured based on the above-mentioned measurement method and measurement conditions for Mn.

[0044] <Ingredient (B)> The polymer (A) of the present disclosure preferably forms a conjugate compound with a component (B) containing at least one selected from the group consisting of amino acids, peptides, proteins, nucleosides, nucleotides, and nucleic acids.

[0045] The amino acids of the present disclosure include organic compounds containing both amino and carboxyl groups and their salts, and include not only natural amino acids (L-amino acids) but also unnatural amino acids (D-amino acids, modified amino acids, amino acid derivatives, etc.).

[0046] Specific amino acids include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, tryptophan, proline, and valine. Other amino acids include, but are not limited to, argininosuccinic acid, citrulline, cysteine ​​sulfinic acid, 3,4-dihydroxyphenylalanine, homocysteine, homoserine, ornithine, carnitine, selenocysteine, selenomethionine, 3-monoiodotyrosine, 3,5-diiodotyrosine, 3,5,5'-triiodothyronine, and 3,3',5,5'-tetraiodothyronine.

[0047] Modified amino acid refers to an amino acid (e.g., an N-alkyl amino acid, an N-acyl amino acid, or an N-methyl amino acid) modified by addition, deletion, substitution, or a combination thereof of at least one atom. Examples of modified amino acids include, but are not limited to, amino acid derivatives such as trimethylglycine, N-methyl-glycine, and N-methyl-alanine.

[0048] Other unnatural amino acids include D-amino acids, hydroxylysine, dehydroalanine, pyrrolidine, 2-aminoisobutyric acid, gamma-aminobutyric acid, 5-hydroxytryptophan, S-adenosylmethionine, S-adenosylhomocysteine, 4-hydroxyproline, N-Cbz-protected amino acids, 2,4-diaminobutyric acid, homoarginine, norleucine, N-methylaminobutyric acid, naphthylalanine, phenylglycine, beta-phenylproline, tert-leucine, and 4-aminocyclohexyl. These include, but are not limited to, alanine, N-methyl-norleucine, 3,4-dehydroproline, N,N-dimethylaminoglycine, N-methylaminoglycine, 4-aminopiperidine-4-carboxylic acid, 6-aminocaproic acid, trans-4-(aminomethyl)-cyclohexanecarboxylic acid, 2-, 3-, and 4-(aminomethyl)-benzoic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclopropanecarboxylic acid, and 2-benzyl-5-aminopentanoic acid.

[0049] The peptide of the present disclosure refers to a compound in which amino acids are peptide-bonded, and examples thereof include compounds in which 2 to 50 amino acids are peptide-bonded. Examples of the above peptides or proteins described below include peptide derivatives, peptide aptamers, antibodies such as immunoglobulins, antibody fragments, antibody derivatives, peptide-nucleic acids (PNAs), hormones such as interleukins, lymphokines, and cytokines, enzymes, growth factors, etc.

[0050] The term "protein" as used herein refers to a compound in which amino acids are linked by peptide bonds, and includes compounds with 50 or more amino acids bonded together. Specific examples include enzymes, hormones, cytokines, and antibodies. The amino acids constituting the peptides or proteins of the present disclosure include not only natural amino acids but also unnatural amino acids. The primary structure of the peptides or proteins of the present disclosure is a linear or cyclic structure, and may contain both structures simultaneously. It may form a secondary structure that is either helical or sheet-like, and may also form a three-dimensional structure such as a tertiary or quaternary structure. As used herein, peptide or protein derivatives include peptides and proteins containing amino acid derivatives, as well as protein hydrolysates obtained by partially hydrolyzing peptides or proteins with acids, alkalis, or enzymes, and derivatives thereof such as cationized, acylated, alkyl-esterified, and siliconized products.

[0051] The molecular weight of the protein of the present disclosure is preferably 1,000 or more, more preferably 3,000 or more, even more preferably 5,000 or more, and is preferably 4,000,000 or less, more preferably 1,000,000 or less, even more preferably 500,000 or less. The molecular weight of a protein can be measured by known methods, for example, SDS-PAGE or mass spectrometry (MALDI TOF MS).

[0052] Nucleosides of the present disclosure include, but are not limited to, ribonucleosides and deoxyribonucleosides, and may be artificially modified nucleosides.

[0053] The nucleotide of the present disclosure is exemplified by, but not limited to, a compound in which one to three phosphates are bound to the above-mentioned nucleoside. Artificially modified nucleotides are also acceptable.

[0054] Nucleic acids of the present disclosure include, but are not limited to, DNA and RNA. Examples of nucleic acids of the present disclosure include oligonucleotides (e.g., those having a length of 2 to 100 bases), gapmers, ribozymes, aptamers, artificial nucleic acids, etc. The oligonucleotides may be siRNA, miRNA, aptamers, CpG oligos, or antisense DNA / RNA.

[0055] The nucleoside of the present disclosure is not particularly limited, but is preferably a compound in which a base such as a purine base, a pyrimidine base, nicotinamide, or dimethylisoalloxazine is bound to a sugar. Examples of nucleosides of the present disclosure include, but are not limited to, adenosine, deoxyadenosine, guanosine, deoxyguanosine, 5-methyluridine, thymidine, uridine, methylpseudouridine, pseudouridine, deoxyuridine, cytidine, and deoxycytidine.

[0056] Nucleotides of the present disclosure include natural and non-natural nucleotides. Natural nucleotides include deoxyribonucleotides having a base of any of adenine, guanine, cytosine, and thymine, and ribonucleotides having a base of any of adenine, guanine, cytosine, and uracil. Non-natural nucleotides include artificial nucleotides having properties and / or structures similar to those of natural nucleotides, as well as non-natural nucleosides or artificial nucleotides containing non-natural bases having properties and / or structures similar to those of natural nucleosides or natural bases that are components of natural nucleotides. Examples of non-natural nucleosides include abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, and nucleosides with other sugar modifications. Nucleosides with substituted pentasaccharides (2'-O-methylribose, 2'-deoxy-2'-fluororibose, 3'-O-methylribose, 1',2'-deoxyribose), arabinose, substituted arabinose sugars, substituted hexasaccharides, and alpha-anomeric sugar modifications are also included. Non-natural nucleotides also include nucleotides containing artificially constructed base analogs or artificially chemically modified bases (modified bases). Examples of base analogs include the 2-oxo(1H)-pyridin-3-yl group, 5-substituted-2-oxo(1H)-pyridin-3-yl group, 2-amino-6-(2-thiazolyl)purin-9-yl group, and 2-amino-6-(2-oxazolyl)purin-9-yl group.

[0057] Modified bases include, for example, modified pyrimidines (e.g., 5-hydroxycytosine, 5-fluorouracil, 4-thiouracil), modified purines (e.g., 6-methyladenine, 6-thioguanosine), and other heterocyclic bases. Chemically modified nucleic acids and nucleic acid analogs, such as methylphosphonate DNA / RNA, phosphorothioate DNA / RNA, phosphoramidate DNA / RNA, and 2'-O-methyl DNA / RNA, may also be included. Nucleic acid analogs are artificially constructed compounds with structures and / or properties similar to those of natural nucleic acids, such as peptide nucleic acids (PNAs), phosphate-containing peptide nucleic acids (PHONAs), bridged nucleic acids (BNAs / LNAs), and morpholino nucleic acids.

[0058] Nucleic acids of the present disclosure include polymers in which nucleotides are linked by phosphodiester linkages, methylphosphonate linkages, methylthiophosphonate linkages, phosphoromorpholidate linkages, phosphoropiperazidate linkages, phosphoroamidate linkages, phosphorothioate linkages, or phosphorodithioate linkages, etc.

[0059] The DNA of the present disclosure may be in a form in which a base selected from adenine, guanine, cytosine, and thymine is bound to deoxyribose rings linked via phosphodiester bonds, and these may have a substituent.

[0060] The DNA disclosed herein may be single-stranded DNA, double-stranded DNA, or a DNA-RNA hybrid, and specific examples include genomic DNA, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, DNA oligonucleotides, DNA polynucleotides, aDNA, plasmids, antisense DNA oligonucleotides, aptamers, decoys, and viral DNA.

[0061] The RNA of the present disclosure may be in a form in which a base selected from adenine, guanine, cytosine, and uracil is bound to a ribose ring linked via a phosphodiester bond, and these may have a substituent.

[0062] The RNA of the present disclosure may be single-stranded RNA, double-stranded RNA, or a DNA-RNA hybrid, and specific examples include RNA oligonucleotides, messenger RNA (mRNA), immunostimulatory RNA, small interfering RNA (siRNA), antisense RNA, microRNA (miRNA), small nuclear RNA (snRNA), small hairpin (sh)RNA, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), viral RNA (vRNA), aptamers, and ribozymes. The mRNA of the present disclosure may contain pseudouridine or N instead of uridine with uracil attached to the ribose ring. 1 Artificial mRNAs using modified nucleic acids such as -methylpseudouridine and other derivatives may also be used. The RNA is preferably an oligonucleotide having 15 to 50 nucleotide units, more preferably an oligonucleotide having 20 to 30 linked nucleotide units. The microRNA (miRNA) of the present disclosure can be a miRNA consisting of 17 to 25 linked nucleotide units. The siRNA can, for example, contain 16 to 30 nucleotide units and have a double-stranded region. In another embodiment, the nucleic acid is an immunostimulatory oligonucleotide, a decoy oligonucleotide, a supermir, a miRNA mimic, or a miRNA inhibitor. A supermir refers to a single-stranded, double-stranded, or partially double-stranded oligomer or polymer of RNA or deoxyribonucleic acid (DNA), or both, or modified versions thereof, that has a nucleotide sequence substantially identical to that of a miRNA and is antisense to its target. A miRNA mimic represents a group of molecules that can be used to mimic the gene silencing ability of one or more miRNAs. Thus, the term "miRNA mimic" refers to a synthetic non-coding RNA that can enter the RNAi pathway and regulate gene expression (i.e., the miRNA is not obtained by purification from an endogenous miRNA source).

[0063] The gapmer of the present disclosure refers to an antisense nucleic acid in which artificial nucleic acids are arranged on both ends, and is expected to have the effect of enhancing the activity of the antisense nucleic acid.

[0064] The aptamer of the present disclosure is a nucleic acid molecule that binds to a target protein and is a single-stranded DNA or single-stranded RNA.

[0065] The molecular weight of the nucleic acid of the present disclosure is preferably 300 or more, more preferably 1,000 or more, even more preferably 2,000 or more, and is preferably 4,000,000 or less, more preferably 3,000,000 or less, even more preferably 2,000,000 or less, and may be 1,500,000 or less. The molecular weight of a nucleic acid can be measured by known methods, such as agarose electrophoresis, SDS-PAGE, HPLC-MS, and MALDI-TOFMS.

[0066] <Conjugate compounds> The mass ratio of polymer (A) to component (B) in the conjugate compound of the present disclosure is not particularly limited and may be appropriately set depending on the types of polymer (A) and component (B), molecular weight, balance of hydrophilicity and hydrophobicity, delivery site, desired blood retention time, and the like. From the viewpoint of efficient binding, however, polymer (A) / component (B) is preferably 1 / 999 to 499 / 1, more preferably 1 / 499 to 150 / 1, even more preferably 1 / 499 to 99 / 1, and even more preferably 1 / 199 to 49 / 1.

[0067] In the conjugate compound of the present disclosure, the polymer (A) and the component (B) are preferably bonded via a divalent linking group, from the viewpoint of improving the heat resistance of the conjugate compound. Specific examples of the divalent linking group of the present disclosure include -S-, -SC(=S)-, -SC(=S)-S-, -SC(=S)-N(-Ra)-, -SC(=S)-O-, -S-Rb-C(=O)-O-, -S-Rb-C(=O)-N(-Ra)-, -S-Rb-O-, -S-Rb-OC(=O)-, -O-, -OC(=O)-, -N(-Ra)-C(=O)-, and divalent linking groups containing these. Here, Ra is a hydrogen atom or a hydrocarbon group having 1 to 30 carbon atoms. Rb is a hydrocarbon group having 1 to 30 carbon atoms. Note that a residue obtained by removing one hydrogen atom and one hydrocarbon group having 8 or more carbon atoms from a lipid (including modified lipids) is also one preferred form of the divalent linking group. The divalent linking group preferably has a molecular weight of 5,000 or less, more preferably 2,000 or less, and even more preferably 1,000 or less.

[0068] From the viewpoint of bond stability, the conjugate compound of the present disclosure preferably has at least one bond represented by general formula (1) to general formula (4), and more preferably has at least one bond represented by general formula (1) or general formula (2). Note that the bond represented by general formula (1) is, for example, a bond formed by a combination of a thiol group and a maleimide group, the bond represented by general formula (2) is, for example, a bond formed by a combination of an -NH2 group (amino group) and a maleimide group, the bond represented by general formula (3) is, for example, a bond formed by a combination of an -NH2 group (amino group) and a succinimide group (referring to an N-hydroxysuccinimide (NHS) ester group; the same applies hereinafter), and the bond represented by general formula (4) is, for example, a bond formed by a combination of a thiol group and a succinimide group.

[0069] [ka]

[0070] [ka]

[0071] [ka]

[0072] [ka]

[0073] (In Formulas 1 to 4, the dashed lines indicate bonding to the polymer (A) and the component (B), respectively.) From the viewpoint of improving the heat resistance of the conjugate compound, it is preferable that the terminal functional group of polymer (A) and / or component (B) has at least one selected from the group consisting of an azide group, a thiol group, an amino group, an alkynyl group, a maleimide group, a succinimide group, a leaving group (such as a halogen), and a disulfide group. The conjugate compound of the present disclosure preferably contains a bond formed by a combination of an azide group-alkynyl group, a thiol group-disulfide group, or a thiol group or an amino group with a maleimide group, a succinimide group, or a leaving group. A bond formed by a combination of a thiol group-disulfide group, a thiol group or an amino group with a maleimide group or a succinimide group is more preferable, and a bond formed by a combination of a thiol group-maleimide group or an amino group-succinimide group is even more preferable.

[0074] The conjugate compound of the present disclosure may be in the form of a powder, dispersion, solution, or paste, but is preferably in the form of a powder from the viewpoint of ease of storage.

[0075] <Method for producing polymer (A)> The polymer (A) of the present disclosure can be obtained by polymerizing a monomer composition containing a monomer (a) and, if necessary, a monomer (b). Examples of methods for polymerizing the monomer composition include living radical polymerization methods, such as radical polymerization, atom transfer radical polymerization, and reversible addition-fragmentation chain transfer (RAFT) polymerization, as well as ionic polymerization, ring-opening polymerization, coordination polymerization, and polycondensation. However, the present invention is not limited to these examples.

[0076] A solvent may be used when polymerizing the monomer composition. Examples of the solvent include aromatic solvents such as benzene, toluene, and xylene; alcoholic solvents such as methanol, ethanol, isopropanol, n-butanol, and tert-butanol; halogen-containing solvents such as dichloroethane and dichloromethane; ether solvents such as diethyl ether, propylene glycol methyl ether, dipropylene glycol methyl ether, ethyl cellosolve, and butyl cellosolve; ester solvents such as ethyl acetate, butyl acetate, and cellosolve acetate; ketone solvents such as acetone, methyl ethyl ketone, methyl isobutyl ketone, and diacetone alcohol; and amide solvents such as dimethylformamide. Among these, alcoholic solvents are preferred from the viewpoint of reactivity. These solvents may be used alone or in combination of two or more. The amount of solvent may be appropriately determined taking into consideration the polymerization conditions, the composition of the monomer composition, the concentration of the resulting polymer, and the like.

[0077] When polymerizing the monomer composition, a chain transfer agent can be used to adjust the molecular weight of the polymer or to introduce a functional group such as a hydrocarbon group or an amino group. Examples of chain transfer agents include hydrophilic thiol-based chain transfer agents such as alkali metal thioacetates, such as sodium thioacetate and potassium thioacetate, cysteine, cysteamine, mercaptoethanol, thioglycerol, thioglycolic acid, mercaptopropionic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, thioacetic acid, thiomalic acid, 2-mercaptoethanesulfonic acid, and sodium and potassium salts thereof; primary alcohols, such as 2-aminopropan-1-ol; secondary alcohols, such as isopropanol; phosphorous acid, hypophosphorous acid, and salts thereof (e.g., sodium hypophosphite, potassium hypophosphite, etc.); sulfurous acid, hydrogen sulfite, dithionous acid, metabisulfite, and salts thereof (e.g., sodium sulfite, sodium hydrogen sulfite, etc.); Non-thiol chain transfer agents such as sodium dithionite, sodium metabisulfite, potassium sulfite, potassium bisulfite, potassium dithionite, and potassium metabisulfite; and hydrophobic thiol chain transfer agents such as butanethiol, octanethiol, decanethiol, dodecanethiol, hexadecanethiol, octadecanethiol, thiocholesterol, cyclohexyl mercaptan, thiophenol, octyl thioglycolate, octyl 2-mercaptopropionate, octyl 3-mercaptopropionate, 2-ethylhexyl mercaptopropionate, 2-mercaptoethyl octanoate, 1,8-dimercapto-3,6-dioxaoctane, decanetrithiol, and dodecyl mercaptan. Furthermore, when performing reversible addition-fragmentation chain transfer (RAFT) polymerization, a reversible addition-fragmentation chain transfer (RAFT) agent must be used as the chain transfer agent. Such RAFT agents include 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 2-cyano-2-propyl benzothioate, 2-cyano-2-propyl dodecyl trithiocarbonate, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid, cyanomethyl dodecyl thiocarbonate, cyanomethylmethyl(phenyl)carbamothioate, bis(thiobenzoyl)disulfide, bis(dodecylsulfanylthiocarbonyl)disulfide, and the like.These chain transfer agents may be used alone or in combination of two or more. The amount of the chain transfer agent is not particularly limited and may be appropriately set depending on the types of monomers contained in the monomer composition, polymerization conditions such as polymerization temperature, the molecular weight of the target polymer, etc. However, when obtaining a polymer having a number average molecular weight of several thousand to several tens of thousands, the amount of the chain transfer agent is preferably 0.1 to 20 parts by mass, more preferably 0.5 to 15 parts by mass, per 100 parts by mass of the monomer.

[0078] When polymerizing the monomer composition, a polymerization initiator can be used. Examples of the polymerization initiator include azoisobutyronitrile, 2,2'-azobis(4-dimethoxy-2,4-dimethylvaleronitrile), 4,4'-azobis(4-cyanopentanoic acid), 2,2'-azobis[2-methyl-N-[1,1-bis(hydroxymethyl)-2-hydroxyethyl]propionamide], and 2,2'-azobis[N-(2-hydroxyethyl)-2-methoxypropaneamide]. amide], 2,2'-azobis(2-methyl-2-propenylpropanamide), 2,2'-bis(2-imidazolin-2-yl)[2,2'-azobispropane] dihydrochloride, 2,2'-azobis(propane-2-carboxamidine) dihydrochloride, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine], 2,2'-azobis[2-[1-(2-hydroxyethyl) Radical polymerization initiators such as 2-(2-imidazolin-2-yl)propane dihydrochloride, 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), tert-butylperoxy-2-ethylhexanoate, 2,2'-azobis(isobutyronitrile), benzoyl peroxide, di-tert-butyl peroxide, cyclohexanone peroxide, and acetylacetone peroxide; living radical polymerization initiators such as bromomethylbenzene, 1-(bromomethyl)-4-methylbenzene, ethyl 2-bromoisobutyrate, hydroxyethyl 2-bromoisobutyrate, bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide, 10-undecenyl 2-bromoisobutyrate, and 4-(1-bromoethyl)benzoic acid. These polymerization initiators may be used alone or in combination of two or more.

[0079] The amount of the polymerization initiator may be appropriately set depending on the desired physical properties of the resulting polymer, but typically, the amount of the polymerization initiator per 100 parts by mass of the monomer is preferably 0.001 to 20 parts by mass, more preferably 0.005 to 10 parts by mass.

[0080] The polymerization conditions for polymerizing the monomer composition may be appropriately set depending on the polymerization method, and are not particularly limited. The polymerization temperature is preferably room temperature to 200°C, more preferably 40 to 140°C. The atmosphere for polymerizing the monomer composition is preferably an inert gas such as nitrogen gas or argon gas. The reaction time may be appropriately set so as to complete the polymerization reaction of the monomers.

[0081] As described above, a polymer can be obtained by preferably polymerizing the monomer composition. The obtained polymer may be used as polymer (A) as is, but it is preferable that the polymer has a functional group at its end when forming a conjugate with component (B). By having a functional group at the end, it can be easily linked to component (B) via the functional group.

[0082] The functional group that the polymer (A) of the present disclosure may have is preferably an anionic functional group, a cationic functional group, a nonionic functional group, or an amphoteric functional group. The functional group is preferably a reactive functional group. Suitable reactive functional groups include an -SH group, a group represented by the formula -COOM (where M represents a hydrogen atom or an alkali metal atom), a hydroxyl group, an allyl group, an epoxy group, an aldehyde group, an -NH2 group (amino group), and a CONH- group. Examples of M include alkali metal atoms such as sodium and potassium. Among these, a preferred reactive functional group is an -NH2 group (amino group) in terms of ease of forming a conjugate with component (B) and ease of forming a bond with a linker when synthesizing a conjugate having a linker between polymer (A) and component (B). When the polymer has a functional group at its terminal, the number of such functional groups is not particularly limited, but is preferably 1 to 6, more preferably 1 to 4, and even more preferably 1 to 2.

[0083] To introduce a functional group into the terminal of the polymer (A) of the present disclosure, a functional group-containing compound for introducing a functional group into the polymer can be used. Examples of functional group-containing compounds for introducing a functional group into the terminal of the polymer include alkali metal thioacetates such as sodium thioacetate and potassium thioacetate, cysteine, cysteamine, mercaptoethanol, thioglycerol, thioglycolic acid, mercaptopropionic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, thioacetic acid, thiomalic acid, 2-mercaptoethanesulfonic acid, and their sodium and potassium salts; thiol-based chain transfer agents such as 16-amino-1-hexadecanethiol hydroxychloride; 4,4'-azobis(4-cyanopentanoic acid), 2,2'-azobis[2-methyl-N-[1,1-bis(hydroxymethyl)-2-hydroxyethoxy]-2-methyl ... Examples of the polymerization initiator include a polymerization initiator having a functional group introduced therein, such as 2,2'-azobis[N-(2-hydroxyethyl)-2-methoxypropanamide], 2,2'-azobis(2-methyl-2-propenylpropanamide), 2,2'-bis(2-imidazolin-2-yl)[2,2'-azobispropane] dihydrochloride, 2,2'-azobis(propane-2-carboxamidine) dihydrochloride, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine], 2,2'-azobis[2-[1-(2-hydroxyethyl)-2-imidazolin-2-yl]propane dihydrochloride, cyclohexanone peroxide, and acetylacetone peroxide. These functional group-containing compounds may be used alone or in combination of two or more. The functional group-containing compounds described above include those corresponding to the chain transfer agents and polymerization initiators described above. The functional group-containing compounds corresponding to the chain transfer agents and polymerization initiators may be used for only one of the purposes of the chain transfer agent or polymerization initiator and the functional group-containing compound, or may be used for both purposes.

[0084] When a living polymerization initiator is used as the polymerization initiator, a functional group may be introduced into the terminal of a polymer prepared using the living polymerization initiator by reacting a functional group-containing compound with a halogen atom present at the terminal of the polymer. Examples of functional group-containing compounds that can react with such halogen atoms to introduce a functional group into the terminal of the polymer include amine compounds such as ethylenediamine and propyldiamine, dithiol compounds such as ethanedithiol, propanedithiol, and hexadecanedithiol, allyl mercaptan, and thiol compounds such as cysteine, cysteamine, mercaptoethanol, thioglycerol, thioglycolic acid, mercaptopropionic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, thioacetic acid, thiomalic acid, 2-mercaptoethanesulfonic acid, and sodium and potassium salts thereof.

[0085] The amount of the functional group-containing compound for introducing a functional group into the terminal of the polymer (A) of the present disclosure is not particularly limited and may be appropriately set depending on the types of monomers (structural units) constituting the polymer, polymerization conditions such as polymerization temperature, the target molecular weight of the polymer, etc. When obtaining a polymer having a number average molecular weight of several thousand to several tens of thousands, the amount of the chain transfer agent is preferably 0.1 to 20 parts by mass, more preferably 0.5 to 15 parts by mass, per 100 parts by mass of the monomer. Examples of methods for introducing a functional group into the terminal of the polymer (A) include: (1) A method of obtaining a polymer by polymerizing a monomer composition in the presence of a polymerization initiator having the functional group introduced therein; (2) A method of obtaining a polymer by polymerizing a monomer composition in the presence of a chain transfer agent having the functional group introduced therein. (3) A method in which halogen atoms present at the terminals of a polymer are reacted with a compound containing a functional group. However, the present invention is not limited to these examples.

[0086] Furthermore, when forming a conjugate compound with component (B), the polymer (A) of the present disclosure preferably has a functional group derived from a maleimide structure or a succinimide structure (also referred to as a maleimide group or a succinimide group) at its terminal, in addition to or instead of the above-mentioned functional group. This structure can function as a linker structure between the polymer (A) and component (B). The presence of this functional group facilitates chemoselective bonding with component (B). For example, the maleimide structure reacts with thiol groups, and the succinimide structure reacts with amino groups, resulting in good binding to proteins such as antibodies and compounds with terminal thiol groups.

[0087] In order to introduce a functional group derived from a maleimide structure or a succinimide structure into the terminal of a polymer, (1) A method of obtaining a polymer by polymerizing a monomer composition in the presence of a chain transfer agent having a maleimide group or a succinimide group, or a RAFT agent such as 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester; (2) A method of reacting a functional group (e.g., an amino group) present at the end of a polymer with a succinimide group (N-hydroxysuccinimide (NHS) ester group) in a maleimide group-containing compound (e.g., N-succinimidyl 4-maleimidobutyrate). However, the present invention is not limited to these examples.

[0088] <Method for producing conjugate compounds> The conjugate compound of the present disclosure is preferably obtained by a step of reacting a polymer (A) having a structural unit derived from a monomer (a) having two or more hydroxyl groups and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit, with a component (B) containing any one selected from the group consisting of amino acids, polypeptides, proteins, nucleosides, nucleotides, and nucleic acids, preferably in a mass ratio of polymer (A) / component (B) of 1 / 999 to 499 / 1. The monomers and physical properties used in the polymer (A) and the compound used in the component (B) are as described above.

[0089] The polymer (A) of the present disclosure preferably has a functional group at its terminal, and more preferably has a maleimide structure or a succinimide structure. Component (B) of the present disclosure preferably has a functional group in its molecule when forming a conjugate compound. Such a functional group may be a functional group inherent in component (B), such as when component (B) has an amino group or a thiol group (e.g., a cysteine ​​residue in a protein) in its molecule, or the functional group may be introduced into an amino acid, polypeptide, protein, nucleoside, nucleotide, or nucleic acid. Methods for introducing functional groups into amino acids, polypeptides, proteins, nucleosides, nucleotides, or nucleic acids are known. For example, thiol groups may be introduced into nucleic acids by first introducing a disulfide bond into the nucleic acid and then reducing the introduced disulfide bond using a reducing agent such as dithiothreitol, 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to introduce (generate) the thiol group. Furthermore, thiol groups may be introduced (generated) by reducing disulfide bonds in proteins using a reducing agent such as dithiothreitol, 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Examples of the terminal functional group of component (B) include an azide group, a thiol group, an amino group, an alkynyl group, a maleimide group, a succinimide group, a leaving group (such as a halogen), and a disulfide group. From the viewpoints of universality in bio-related molecules and ease of material availability and synthesis, a thiol group, an amino group, and a disulfide group are more preferred, and a thiol group and an amino group are even more preferred. In a preferred embodiment, component (B) is at least one selected from the group consisting of an amino acid, a polypeptide, a protein, a protein having a thiol group introduced therein, a nucleoside having a thiol group introduced therein, a nucleotide having a thiol group introduced therein, and a nucleic acid having a thiol group introduced therein.

[0090] The conjugate compound of the present disclosure is preferably obtained by reacting a terminal functional group of polymer (A) with at least one selected from the group consisting of an azide group, a thiol group, an amino group, an alkynyl group, a maleimide group, a succinimide group, and a leaving group of component (B). In one preferred embodiment of the production method, polymer (A) has a maleimide structure or a succinimide structure at its terminal, component (B) has at least one selected from the group consisting of an azide group, a thiol group, an amino group, an alkynyl group, a maleimide group, a succinimide group, and a leaving group, and the maleimide structure or succinimide structure is reacted with at least one selected from the group consisting of an azide group, a thiol group, an amino group, an alkynyl group, a maleimide group, a succinimide group, and a leaving group. Another preferred embodiment of the production method is (1) a polymer (A) having a maleimide structure at its terminal, a component (B) having a thiol group, and reacting the maleimide structure with the thiol group, or (2) a polymer (A) having a succinimide structure at its terminal, a component (B) having an amino group, and reacting the succinimide structure with the amino group.

[0091] The reaction may be carried out in a buffer solution. The reaction is preferably carried out at around room temperature to maintain the activity of component (B), for example, at 5 to 40°C, or 20 to 30°C. The reaction time is appropriately set taking into consideration the reactivity of polymer (A) and component (B), and is, for example, 30 minutes to 10 days.

[0092] <Applications of conjugate compounds> The conjugate compound of the present disclosure is highly biocompatible and therefore suitable for medical applications. That is, according to another aspect of the present invention, a medicament containing the conjugate compound of the present disclosure is provided. The medicament may consist of the conjugate compound of the present disclosure, or may be a composition further containing other components. Examples of other components include water, saline, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, carboxymethylcellulose sodium salt, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, phosphate-buffered saline, biodegradable polymers, serum-free media, surfactants acceptable as pharmaceutical additives, and physiological pH buffer solutions acceptable in vivo. These additives may be used alone or in combination of two or more.

[0093] Furthermore, the conjugate compound of the present disclosure or a composition containing the conjugate compound is preferably used as a liquid formulation, a solid formulation, or a gel formulation. The liquid formulation of the present disclosure preferably contains, in addition to the conjugate compound of the present disclosure, water, saline, phosphate buffered saline, citrate-phosphate buffer, or the like. The solid formulation of the present disclosure preferably contains, in addition to the conjugate compound of the present disclosure, an excipient such as mannitol, xylitol, maltodextrin, sodium carboxymethylcellulose, polyethylene glycol, agar, or lactose.

[0094] In addition to the conjugate compound of the present disclosure, the gel formulation of the present disclosure preferably contains a neutralized anionic polymer such as polyacrylic acid, a thickener such as carboxypolymethylene and carboxymethylcellulose, Pemulen, a polymeric emulsifier, or polycarbophil, a lower alcohol such as ethanol or isopropanol, and water. The conjugate compound of the present disclosure or a composition containing the conjugate compound can be suitably used as a pharmaceutical additive when it does not contain a medicinal ingredient. Examples of pharmaceutical additives include carriers for holding medicines, etc. As a method for holding medicines, etc. in the conjugate compound or medical resin composition according to the present disclosure, it is preferable to use a component (B) of the present disclosure that is itself an active pharmaceutical ingredient.

[0095] Furthermore, the conjugate compound or a pharmaceutical composition containing the conjugate compound can be used for either in vitro or in vivo testing. Furthermore, the method of administering the conjugate compound to a living body is preferably parenteral administration, i.e., intraarticular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, or intramuscular administration. Intravenous or intraperitoneal administration of the (pharmaceutical) composition can also be performed by bolus injection. The conjugate compounds of the present disclosure or pharmaceuticals containing the conjugate compounds are expected to be pharmaceuticals with good retention in the blood, since they suppress complement activation and do not induce unintended harmful immune responses. [Example]

[0096] [Measurement of average molecular weight of polymer] The number average molecular weights of the polymers produced in Production Examples 1 to 4 described below were measured by gel permeation chromatography (GPC) under the following measurement conditions. [Measurement conditions for number average molecular weight of polymer] Measuring equipment: Tosoh Corporation, product number: HLC-8320GPC Molecular weight column: Tosoh Corporation, product number: TSKgel SuperAWM-H and SuperAW2500, two columns connected in series Eluent: 10mmol / L lithium bromide added dimethylformamide Standard material for calibration curve: Polystyrene Preparation of measurement solution: Dissolve the polymer in dimethylformamide to prepare a solution with a polymer concentration of 0.2% by mass, and use the filtrate after filtering the solution.

[0097] [Example of production of terminal functional group-introduced compound (polymer)] (Production Example 1) A test tube with a sidearm was charged with 1.0 g of glycerin monoacrylate, 0.046 g of 16-amino-1-hexadecanethiol hydroxychloride, 0.015 g of 2,2'-azobis(isobutyronitrile), 3.2 mL of ethanol, and 0.8 mL of butanol. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at 80°C for 2 hours. The supernatant was removed from the resulting reaction solution, yielding polyglycerin monoacrylate containing terminal amino (ammonium) groups. The number-average molecular weight of the resulting compound (polymer) was 8,000. Then, 0.640 g of the obtained polyglycerol monoacrylate containing an amino group (ammonium group) at the end, 0.643 g of N-succinimidyl 4-maleimidobutyrate, 9 mL of dimethyl sulfoxide, and 1 mL of triethylamine acetate solution (2 mol / L, pH 7.0) were placed in a branched test tube. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at room temperature for 23 hours. The resulting reaction solution was diluted 5-fold with water and then purified by gel filtration (Cytiva PD-MidiTrap). TM G-25) to obtain polyglycerol monoacrylate (polymer 1) having a maleimide structure introduced at the end. The number-average molecular weight of the obtained compound (polymer 1) was 9,200, and the polydispersity index Mw / Mn was 1.96.

[0098] (Production Example 2) A Schlenk flask equipped with a three-way stopcock was charged with 1.0 g of glycerin monomethacrylate, 0.022 g of N-hydroxysuccinimidyl 4-cyano-4-(phenylcarbonothioylthio)pentanoate, 0.016 g of 2,2'-azobis(2,4-dimethylvaleronitrile), 0.8 g of ethanol, and 0.2 g of n-butanol. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at 50°C for 30 minutes. The resulting reaction solution was added dropwise to diethyl ether for purification and dried under reduced pressure to obtain polyglycerin monomethacrylate (polymer 2) with succinimide groups introduced at its termini. The resulting compound (polymer 2) had a number-average molecular weight of 25,000 and a polydispersity index (Mw / Mn) of 1.73.

[0099] (Production Example 3) A test tube with a sidearm was charged with 1.0 g of glycerin monomethacrylate, 0.046 g of 16-amino-1-hexadecanethiol hydroxychloride, 0.015 g of 2,2'-azobis(isobutyronitrile), 3.2 mL of ethanol, and 0.8 mL of butanol. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at 80°C for 2 hours. The supernatant was removed from the resulting reaction mixture, yielding polyglycerin monomethacrylate containing terminal amino (ammonium) groups. The number-average molecular weight of the resulting compound (polymer) was 8,500. Then, 0.630 g of the obtained polyglycerol monomethacrylate containing an amino group (ammonium group) at the end, 0.643 g of N-succinimidyl 4-maleimidobutyrate, 9 mL of dimethyl sulfoxide, and 1 mL of triethylamine acetate solution (2 mol / L, pH 7.0) were placed in a branched test tube. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at room temperature for 23 hours. The resulting reaction solution was diluted 5-fold with water and then purified by gel filtration (Cytiva PD-MidiTrap). TM G-25) to obtain polyglycerol monomethacrylate (polymer 3) having a maleimide structure introduced at the end. The number-average molecular weight of the obtained compound (polymer 3) was 10,500, and the polydispersity index Mw / Mn was 1.98.

[0100] (Production Example 4) A Schlenk flask equipped with a three-way stopcock was charged with 1.0 g of glycerin monoacrylate, 0.022 g of N-hydroxysuccinimidyl 4-cyano-4-(phenylcarbonothioylthio)pentanoate, 0.016 g of 2,2'-azobis(2,4-dimethylvaleronitrile), 0.8 g of ethanol, and 0.2 g of n-butanol. The atmosphere inside the tube was then replaced with nitrogen, and the mixture was stirred at 50°C for 30 minutes. The resulting reaction solution was added dropwise to diethyl ether for purification and dried under reduced pressure to obtain polyglycerin monoacrylate (polymer 4) with succinimide groups introduced at the termini. The resulting compound (polymer 4) had a number-average molecular weight of 22,000 and a polydispersity index (Mw / Mn) of 1.89.

[0101] Example 1: Preparation of polymer-protein conjugates Conjugate buffer A (pH 7.2) was prepared by dissolving 0.52 g of sodium dihydrogen phosphate dihydrate, 2.41 g of disodium hydrogen phosphate dodecahydrate, 8.77 g of sodium chloride, and 3.73 g of EDTA 2Na in 1 L of ultrapure water. 41.5 mg of bovine serum albumin (Fujifilm Wako Pure Chemical Industries, Ltd.) and 66 mg of polymer 1 prepared in Preparation Example 1 were dissolved in 1 mL of this conjugate buffer A. After dissolution, 0.1 mL of each solution was added to a test tube, mixed, and incubated at 25°C for 1 hour to obtain the reaction product. After incubation, the reaction product was concentrated using an ultrafiltration unit (Amicon Ultra 50K device, Merck Co., Ltd.), diluted with PBS, and concentrated again using the ultrafiltration unit. This procedure was repeated three times to remove unreacted polymer 1, yielding conjugate 1 (PGLMA-Mal-BSA), in which the maleimide group of polymer 1 was linked to the thiol group of BSA.

[0102] Furthermore, conjugates were prepared using the same method as above for polymers 2, 3, and 4 prepared in Production Examples 2, 3, and 4, respectively. Conjugate 2 (PGLMMA-Suc-BSA) was prepared by bonding the succinimide group of polymer 2 to the amino group of BSA. Conjugate 3 (PGLMMA-Mal-BSA) was prepared by bonding the maleimide group of polymer 3 to the thiol group of BSA. Conjugate 4 (PGLMMA-Suc-BSA) was prepared by bonding the succinimide group of polymer 4 to the amino group of BSA.

[0103] [Polyacrylamide gel electrophoresis (SDS-PAGE)] Ultrafiltered conjugate 1 (PGLMA-Mal-BSA) was diluted with an appropriate amount of ultrapure water. 20 μL of the diluted solution was mixed with 20 μL of sample treatment buffer (sample buffer (containing 3-mercapto-1,2-propanediol) (x2), Fujifilm Wako Pure Chemical Corporation) and heated at 95°C for 10 minutes. The heated sample was applied to a precast gel (ehr-T10L e-Pagel HR 10%, ATTO Corporation) and subjected to SDS-PAGE. Multicolor Protein Ladder (10-315 kDa, Nippon Gene Co., Ltd.) was used as a molecular weight marker. After electrophoresis, the gel was stained with Quick CBB Plus (Fujifilm Wako Pure Chemical Corporation), destained as appropriate, and photographed using a gel imaging system (GEL Doc Go, Bio-Rad Laboratories, Inc.). As a result, as shown in Figure 1, a single band was visible for BSA alone, whereas for PGLMA-Mal-BSA, the band spread toward the top of the gel, confirming that the terminally functionalized PGLMA and BSA had bound to each other to form a conjugate.

[0104] [Complement activation assessment (1)] Using a microspectrophotometer (NanoDrop ND-1000, Thermo Fisher Scientific), ultrafiltered conjugates 1 to 4 were diluted with PBS so that the 280 nm wavelength reading was 100. Separately, a conjugate (PEG-BSA) prepared as described in the "Preparation of Protein Conjugates" section, except that maleimide PEG (SUNBRIGHT ME-100MA, NOF Corporation) was used instead of polymer 1, and 100 mg of BSA were dissolved in 1 mL of PBS. Forty μL of each solution was mixed with 160 μL of human serum (purchased from Tennessee Blood Service) and incubated at 37°C for 1 hour. After incubation, 5 μL of 50 mM EDTA solution was added to 50 μL of the mixture to terminate the reaction. Complement activation was assessed for each mixture using an enzyme immunoassay kit (MicroVue™ SC5b-9 Plus EIA, Quidel Corporation). Complement concentration was measured according to the manufacturer's protocol using a plate reader (SH-9000, Corona Electric Co., Ltd.) A reaction mixture in which PBS was reacted with human serum was used as a negative control, and the ratio of the measured value to the measured value for each reaction mixture was calculated using the following formula. (Ratio to negative control) = (measured value for each sample) ÷ (measured value for negative control) As a result, as shown in Figure 2, it was found that conjugates 1 to 4 inhibited complement activation compared to BSA, whereas PEG-BSA induced stronger complement activation than BSA alone.

[0105] [Analysis of reaction products of polymer 2 and protein] The conjugate of polymer 2 and protein (conjugate 2) prepared in Example 1 was analyzed by size exclusion chromatography under the following measurement conditions. [HPLC analysis conditions for the reaction product of polymer 2 and protein] Measurement equipment: Shimadzu HPLC (column oven: CTO-20AC, pump: LC-20AD, degasser: DGU-20A, autosampler: SIL-20AC HT, detector: ELSD-LTII) Analytical column: Size exclusion chromatography column (TSKgel G3000SWXL, Tosoh Corporation) Eluent: 20 mM acetic acid-ammonium acetate (pH 4.00) Preparation of measurement solution: Dissolve only the conjugate or BSA in the eluent to prepare a solution with a solute concentration of 0.1% by mass, and use the filtrate after filtering the solution. Conjugate 2 (PGLMMA-Suc-BSA) was analyzed under the above analytical conditions. As shown in Figure 3, the PGLMMA-Suc-BSA peak appeared earlier than the BSA peak alone. This indicates that BSA was polymerized, demonstrating the formation of a conjugate.

[0106] Example 2: Preparation of a conjugate of a polymer and an oligonucleic acid The nucleic acids used were oligonucleotides (synthesis requested from Hokkaido System Science Co., Ltd.) with the base sequence (5'-d(TAGCACCATGGTTT)-3') based on a known paper (PSE Deder, RJ DeVine, JM Dagle, JA Walder (1991) Antisense Research and Development, 1(2), 141-51) and oligonucleotides (synthesis requested from Hokkaido System Science Co., Ltd.) with 3-(propyldisulfanyl)propan-1-ol introduced at the 3' end. First, nucleic acid with terminal 3-(propyldisulfanyl)propan-1-ol was dissolved in ultrapure water to prepare a 44.8 μM oligonucleotide solution (320 μL, 14.3 nmol). 160 μL (16.0 μmol) of 0.1 M DTT solution (15.5 mg of (±)-dithiothreitol dissolved in 1.0 mL of ultrapure water) was added to the solution, and the mixture was left to stand at room temperature (approximately 25°C) for 30 minutes to obtain a reaction solution containing oligonucleotides with terminal thiol groups. Approximately 1 mL of ethyl acetate was added to the reaction solution containing the terminal thiol oligonucleotide, followed by shaking vigorously and removing the ethyl acetate layer. This washing procedure was repeated five times to obtain a terminal thiol oligonucleotide solution. To the obtained terminal thiol oligonucleic acid solution, polymer 1 solution prepared by dissolving 7.8 mg of the polymer prepared in Production Example 1 in 320 μL of ultrapure water was added (polymer 1: oligonucleic acid = 130:1 (mass ratio)), and after mixing, the mixture was left to stand at room temperature (approximately 25°C) for 7 days to obtain a reaction product. This reaction product was purified under the conditions described in the section [Purification of reaction product of polymer 1 and oligonucleic acid] to obtain a solution containing conjugate 5 (PGLMA-Mal-nucleic acid) in which the maleimide group of polymer 1 and the thiol group of the nucleic acid are bonded. The obtained solution containing PGLMA-Mal-nucleic acid was illustrated. TM Nap TM -10 Columns Sephadex TM After desalting using G-25 DNA Grade (Cytiva), the product was lyophilized and dissolved in 300 μL of ultrapure water to obtain a PGLMA-Mal-nucleic acid solution (23.5 μM).

[0107] Furthermore, for Polymer 3 prepared in Production Example 3, a conjugate with an oligonucleic acid was prepared in the same manner as for Polymer 1, except that the working weight and liquid volume were reduced to one-tenth of the original amount, to obtain a reaction product. Each of the resulting reaction products was purified using an ion exchange spin column (Vivapure (registered trademark) Q Mini H, SARTORIUS). The purification method was in accordance with the manufacturer's protocol, and 40 μL of an ultrapure aqueous solution containing Conjugate 6 (PGLMMA-Mal-nucleic acid) was obtained.

[0108] [Purification of the reaction product of polymer 1 and oligonucleic acid] The reaction product produced in the reaction between polymer 1 and oligonucleic acid described in Example 2 was purified by ion exchange chromatography under the following purification conditions. [Conditions for purification of the reaction product of polymer 1 and oligonucleotide] Measurement equipment: Cytiva AKTA avant150 Purification column: Cytiva BioPro SmartSep Q20 250 x 6.0 mm ID Column temperature: room temperature (approximately 25°C) Eluent: (Solution A) 20 mM phosphate buffer (pH 8.0), 15% acetonitrile, (Solution B) 20 mM phosphate buffer (pH 8.0), 0.75 M sodium bromide, 15% acetonitrile Elution conditions: Equilibration (14 mL of 100% solution A) → Sample loading (1 mL) → Column cleaning (7 mL of 100% solution A) → Elution (7 mL of 100% solution A is run, followed by 21 mL of solution B, increasing the concentration from 15% to 75%) ·Flow velocity: 150cm / h Detection method: UV (wavelength: 260 nm) [Quantitative analysis of conjugates of polymer 1 and oligonucleotides and unmodified oligonucleotides] Quantitative analysis of PGLMA-Mal-nucleic acid and unmodified nucleic acid in the nucleic acid degradation test described below was carried out by ion exchange chromatography under the following analytical conditions: [Analysis conditions for conjugates of polymer 1 and oligonucleic acid] Measurement equipment: Agilent Technologies 1260 Infinity II Analytical column: BioPro IEX SmartSep Q20 Ion Exchange Screening Kit manufactured by YMC Co., Ltd. Column temperature: room temperature (approximately 25°C) Eluent: (Solution A) 20 mM phosphate buffer (pH 8.0), 15% acetonitrile, (Solution B) 20 mM phosphate buffer (pH 8.0), 0.75 M sodium bromide, 15% acetonitrile Eluent gradient: 0-4.00 min (0-15% B solution), 4.00-10.00 min (15-100% B solution), 10.00-12.00 min (100% B solution), 12.00-12.10 min (100-0% B solution), 12.10-15.00 min (0% B solution) ·Flow rate: 0.5mL / min Detection method: UV (wavelength: 260 nm) Preparation of measurement solution: After each reaction time, collect the reaction solution and heat it at 80°C for 15 minutes to inactivate Exonuclease I. Add 85 μL of ultrapure water to 15 μL of the inactivated reaction solution to use as the measurement solution.

[0109] [Analysis conditions for unmodified oligonucleotides] Measurement equipment: Cytiva ACQUITY UPLC H-Class Analytical column: Tosoh Corporation TSKgel Super-Octyl 2.0 mm ID x 10 cm Column temperature: 60℃ Eluent: (Solution A) 100 mM triethylamine acetate buffer (pH 7.0), (Solution B) purified water:acetonitrile = 1:1 Eluent gradient: 0-10.00 min (2-30% B), 10.00-10.50 min (30-80% B), 10.50-15.00 min (80% B), 15.00-15.50 min (80-2% B), 15.50-20.00 min (2% B) ·Flow rate: 0.2mL / min Detection method: UV (wavelength: 270 nm) Preparation of measurement solution: The solution used for measurement is treated in the same manner as in the section [Conditions for analysis of conjugates of polymer 1 and oligonucleic acid].

[0110] [Nucleic acid degradation test] A test tube was charged with 42.5 μL (1.00 nmol) of the PGLMA-Mal-nucleic acid solution obtained in the section [Preparation of a conjugate of polymer 1 and oligonucleotide], 5 μL of 10× buffer solution (670 mM glycine-KOH (pH 9.5), 10 mM DTT, 67 mM MgCl), 2.5 μL of ultrapure water, and 0.5 μL of Exonuclease I (Takara Bio Inc.) (final concentration of Exonuclease I in the mixed solution: 0.05 Unit / μL), and the mixture was allowed to stand at room temperature (approximately 25°C). As a control, 10 μL (1.00 nmol) of a solution prepared by dissolving the unmodified nucleic acid (5'-d(TAGCACCATGGTTT)-3') used in the section "Preparation of Conjugates of Polymer 1 and Nucleic Acid" in ultrapure water to a concentration of 100 μM was added to a test tube, 5 μL of 10x buffer solution, 34.5 μL of ultrapure water, and 0.5 μL of Exonuclease I (Takara Bio Inc.) (final Exonuclease I concentration of 0.05 Unit / μL). The mixture was mixed and allowed to stand at room temperature (approximately 25°C). The reaction mixture was sampled 0, 10, and 20 minutes after the start of the reaction and analyzed under the analytical conditions specified in the section "Analysis Conditions for Conjugates of Polymer 1 and Oligonucleotide" or "Analysis Conditions for Unmodified Oligonucleotide." The residual nucleic acid percentage was calculated using the following formula: (Residual rate) [%] = (Area of ​​PGLMA-Mal-nucleic acid or unmodified nucleic acid after each reaction time%) ÷ (Area of ​​PGLMA-Mal-nucleic acid or unmodified nucleic acid at the start of the reaction%) × 100 As a result, as shown in Figure 4, nucleic acid degradation was observed over time in the unmodified nucleic acid, but no significant nucleic acid degradation was observed in the PGLMA-Mal-nucleic acid.

[0111] [Complement activation assessment (2)] Using a microspectrophotometer (NanoDrop ND-1000, Thermo Fisher Scientific), the resulting conjugates 5 and 6 were each diluted with PBS so that the 260 nm reading was 100. Separately, a conjugate (PEG-nucleic acid) prepared as described in the section "Preparation of Polymer-Oligonucleotide Conjugates," except that maleimide PEG (SUNBRIGHT ME-100MA, NOF Corporation) was used instead of polymer 3, and the oligonucleotide (base sequence (5'-d(TAGCACCATGGTTT)-3')) described in Example 2 were dissolved in 20 μL of PBS using the method described above to obtain a 260 nm reading of 100. Four μL of each solution was mixed with 16 μL of human serum (purchased from Tennessee Blood Service) and incubated at 37°C for 1 hour. To 15 μL of the incubated mixture, 1.5 μL of 50 mM EDTA solution was added to terminate the reaction. Complement activation was assessed for each mixture using an enzyme immunoassay kit (MicroVue™ SC5b-9 Plus EIA, Quidel Corporation). Complement concentration was measured according to the manufacturer's protocol using a plate reader (SH-9000, Corona Electric Co., Ltd.). A reaction mixture in which PBS was reacted with human serum was used as a negative control, and the ratio of the measured value to the measured value for each reaction mixture was calculated using the following formula. (Ratio to negative control) = (measured value for each sample) ÷ (measured value for negative control) As a result, as shown in Figure 5, no significant difference was observed between the measured values ​​of conjugates 5 and 6 and the comparative oligonucleic acid, but it was found that the PEG-nucleic acid caused stronger complement activation than the oligonucleic acid.

[0112] Example 3: Preparation of antibody conjugate 0.4 g of ammonium bicarbonate was dissolved in 80 mL of ultrapure water, and formic acid was added appropriately until the pH reached 7.1. Ultrapure water was then added to the solution to make a 100 mL volume, producing conjugate buffer C. 10 mg of IgG antibody (derived from normal human, Fujifilm Wako Pure Chemical Corporation) was dissolved in 1 mL of conjugate buffer C, and TCEP (tris(2-carboxyethyl)phosphine) hydrochloride was added to a final concentration of 5 mM. The reaction mixture was incubated at room temperature for 30 minutes to produce a reduced antibody bearing thiol groups. Polymer 1 prepared in Preparation Example 1 was added to the reduced antibody in an amount of 5 molar equivalents (polymer 1:antibody = 1:3.33 (mass ratio)) and incubated at 25°C for 1 hour to obtain a reaction product. After incubation, the reaction product was concentrated using an ultrafiltration unit (Amicon Ultra 50K device, Merck), diluted with PBS, and concentrated again using the ultrafiltration unit. This procedure was repeated three times to remove unreacted polymer 1, yielding polymer-reduced antibody conjugate 7 (PGLMMA-Mal-IgG). Using the same method as above, polymer 3 prepared in Production Example 3 was also used to obtain IgG antibody conjugate 8 (PGLMMA-Mal-IgG).

[0113] The conjugates 1 to 8 produced in the above Examples 1 to 3 are summarized in Table 1. In the right column of Examples 1, 2, and 3, -Mal- represents a bond containing the structure of general formula (1) above, and -Suc- in Example 1 represents a bond containing the structure of general formula (3) above.

[0114] [Table 1]

[0115] [Cytotoxicity testing of terminally functionalized compounds (polymers) and conjugates using cultured cells] Mouse-derived fibroblast L929 cells (DS Pharma Biomedical) were cultured in DMEM medium (Nacalai Tesque) supplemented with fetal bovine serum (FBS) (DS Pharma Biomedical) at a final concentration of 10 w / v%. 3 cells / cm 2The cells were seeded onto a 100 mm cell culture dish (BD Falcon) so that the cell count was 2.5 × 10 cells per well, and cultured at 37°C under 5% CO2 conditions. L929 cells cultured in a 100 mm cell culture dish until they were 70% confluent were treated with 0.25 w / v% trypsin / 50 mM EDTA solution, and the serum-supplemented DMEM medium described above was added to stop the trypsin reaction, yielding an L929 cell suspension. The number of cells in the L929 cell suspension was measured using 0.4 w / v% trypan blue solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The cell suspension was diluted to a cell count of 2.5 × 10 cells per well. 3 Cells were seeded into a 96-well plate (Thermo Fisher Scientific Inc.) and cultured for 24 hours at 37°C and 5% CO2. After 24 hours, 50 μL of medium was removed from each well. Then, 50 μL of a polymer solution prepared by dissolving the terminal functionalized compounds (polymers) prepared in Preparation Examples 1 and 2 or the conjugates (PGLMA-Mal-BSA, PGLMA-Suc-BSA, PGLMA-Mal-nucleic acid, and PGLMA-Mal-IgG) prepared in Examples 1 to 3 in PBS at a concentration of 2 w / v% was added to each well. The cells were then incubated at 37°C and 5% CO2 for 24 hours. After incubation, 51 μL of Cell Proliferation Kit II (XTT) (Merck) reagent was added to each well and the cells were incubated at 37°C and 5% CO2 for 3 hours. The absorbance was then measured using a plate reader SH-9000 (Corona Electric Co., Ltd.). The measurement protocol followed the instructions provided with the kit. Based on the measured values ​​of the wells to which PBS was added instead of the polymer solution and the measured values ​​of the wells to which each sample was added, the viability of L929 cells was calculated using the following formula.

[0116] (Viability) [%] = (measurement value of wells to which each sample was added) ÷ (measurement value of wells to which PBS was added) × 100 As a result, as shown in FIG. 6, none of the polymer and conjugate solutions had any significant effect on the viability of L929 cells, and no significant cytotoxicity was observed.

[0117] This application is based on Japanese Patent Application No. 2021-202991, filed on December 15, 2021, the disclosure of which is incorporated by reference in its entirety.

Claims

1. A conjugate compound comprising: a polymer (A) having a structural unit derived from a monomer (a) having two or more hydroxyl groups and having 2 to 10 carbon atoms constituting a side chain among the carbon atoms of the structural unit; and a component (B) containing at least one member selected from the group consisting of amino acids, polypeptides, proteins, nucleosides, nucleotides, and nucleic acids, the polymer (A) and the component (B) are bonded via a divalent bonding group, The bond is at least one of the bonds represented by general formulas (1) to (4), 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 (In Formulas 1 to 4, the dashed lines indicate bonding to the polymer (A) and the component (B), respectively.) The structural unit derived from the monomer (a) is represented by the general formula (5): 【Transformation 5】 (in formula (5), R 1 represents a hydrogen atom or a methyl group, and X represents —C(═O)—O—, —C(═O)—NH—, —O—, —CH 2 O—, or —CH 2 CH 2 O—), The conjugate compound, wherein the polymer (A) has a number average molecular weight (Mn) of 1,000 to 50,000.

2. The conjugate compound according to claim 1 , wherein the content of the structural unit derived from the monomer (a) in 100 parts by mass of the polymer (A) is 5 parts by mass or more.

3. A conjugate compound described in claim 1 or 2, wherein the polymer (A) has a maleimide structure or a succinimide structure at its terminal, and the component (B) has at least one selected from the group consisting of a thiol group and an amino group.

4. The conjugate compound according to claim 1 or 2, wherein the polydispersity index Mw / Mn of the polymer (A) is 1.00 or more and 2.00 or less.

5. 3. The conjugate compound according to claim 1, wherein the mass ratio of the polymer (A) to the component (B), polymer (A) / component (B), is 1 / 999 to 499 / 1.

6. A composition comprising the conjugate compound of claim 1.

7. A pharmaceutical additive comprising the conjugate compound of claim 1 or 2 or the composition of claim 6.

8. A method for producing a conjugate compound, comprising the step of reacting a polymer (A) having a structural unit derived from a monomer (a) which has two or more hydroxyl groups and in which the number of carbon atoms constituting a side chain of the structural unit is 2 to 10, with a component (B) containing at least one member selected from the group consisting of amino acids, polypeptides, proteins, nucleosides, nucleotides, and nucleic acids, The reaction bonds the polymer (A) and the component (B) via a divalent bonding group, The bond is at least one of the bonds represented by general formulas (1) to (4), 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 (In Formulas 1 to 4, the dashed lines indicate bonding to the polymer (A) and the component (B), respectively.) The structural unit derived from the monomer (a) is represented by the general formula (5): 【Chemistry 10】 (in formula (5), R 1 represents a hydrogen atom or a methyl group, and X represents —C(═O)—O—, —C(═O)—NH—, —O—, —CH 2 O—, or —CH 2 CH 2 O—), The number average molecular weight (Mn) of the polymer (A) is 1,000 to 50,000.

9. The method for producing a conjugate compound according to claim 8, wherein the polymer (A) has a maleimide structure or a succinimide structure at a terminal thereof, the component (B) has at least one selected from the group consisting of a thiol group and an amino group, and the maleimide structure or the succinimide structure is reacted with the at least one selected from the group consisting of a thiol group and an amino group.

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